Antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention relates to a bioactive substance conjugate as well as a preparation method and application thereof, and particularly discloses an antibody drug conjugate as shown in a formula XV, a preparation method thereof and application of the antibody drug conjugate in prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to prevention and / or treatment of tumor diseases. # imgabs0 #

Description

Antibody drug conjugates and preparation methods and uses thereof Technical Field

[0001] The present disclosure belongs to the field of medical technology and relates to various antibodies, antibody-drug conjugates and preparation methods thereof, as well as their use in preventing and / or treating diseases associated with abnormal cell activity, including but not limited to preventing and / or treating tumor diseases. Background Art

[0002] Cytotoxic chemotherapy was once the standard treatment for cancer, but highly potent cytotoxic molecules can kill normal cells and cause severe side effects. Targeted anti-tumor drugs, due to their combined targeting and anti-tumor activity, have become a hot topic in oncology research. However, these drugs often produce significant side effects due to their target selectivity, limiting their therapeutic efficacy. Biological macromolecule drugs, such as antibodies or antibody fragments, while highly targeted, have limited or no therapeutic effect against solid tumors. ADCs, conjugates of antibodies and small molecule drugs, combine the targeting properties of antibodies with the activity of bioactive molecules, creating a biological missile with highly promising efficacy and safety advantages. Antibodies guide ADCs to target cells, where they are then internalized. The small molecule drug is then released through enzymatic degradation by specific enzymes to treat the disease.

[0003] ADC drugs have developed rapidly in recent years, with 14 ADCs already on the market. Many ADCs targeting targets, such as NaPi2b, are currently in clinical development. For many targets, developing differentiated, higher-quality, and safer monoclonal antibodies or ADCs could provide cancer patients with a wider range of treatment options and hold broad market potential.

[0004] Summary of the Invention

[0005] In order to improve the therapeutic effect of antibody-drug conjugates (ADCs), reduce drug toxicity and side effects, and increase the therapeutic window, the present disclosure provides an antibody-drug conjugate represented by formula (XV) or a pharmaceutically acceptable salt or solvate thereof, which contains a bioactive molecule (drug molecule), a linker, and a targeting moiety, wherein the targeting moiety is connected to the linker via an active group (e.g., a thiol group) to form an antibody-drug conjugate. The present disclosure also develops a variable light chain domain (VL) and / or variable heavy chain (VH) domain comprising anti-Napi2b, wherein the variable light chain domain and / or variable heavy chain domain antibody is derived from a human antibody.

[0006] To this end, in the first aspect of the present disclosure, the present disclosure provides an antibody drug conjugate represented by formula XV,

[0007] or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof,

[0008] in:

[0009] Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof;

[0010] q is the drug-antibody coupling ratio;

[0011] D is a bioactive molecular fragment;

[0012] L1 is the extension unit;

[0013] L2 does not exist or is a connecting unit;

[0014] L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues;

[0015] L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0016] In addition, it should be noted that regarding "position 1 of L1 is connected to Tb via an S atom", those skilled in the art will understand that position 1 of L1 is connected to the sulfhydryl group contained in Tb (such as an antibody) after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing the reducing agent TCEP to generate a sulfhydryl group -SH). In other words, the -S- between L1 and Tb is not an additional external sulfur atom. For example, In the example, -S- is not an additional external sulfur atom, but the thiol group contained in Tb itself after the disulfide bond is opened and the thiol group of L1 is connected. -S- is formed by connecting the 1 bits of

[0017] The Stretcher unit is a component of an antibody-drug conjugate, a drug-linker conjugate, or a linker that connects the remaining portion of the antibody-drug conjugate or the remaining portion of the linker that binds to the target. The Stretcher unit can connect the Tb unit to L2 (if present) or L3. Specific examples include, but are not limited to, (where position 1 is connected to the target-binding antibody portion and position 2 is connected to L2 or L3):

[0018] In some embodiments, L1 is selected from:

[0019] Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl group, a 5-10 membered heteroaryl group, an amide group, a sulfonamide group, an imine group, and CF2;

[0020] Rx and Ry are independently selected from H and C1-4 alkyl;

[0021] Each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;

[0022] y1, y2, y3 and y4 are independently selected from any integer between 0 and 20;

[0023] Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0024] The Linker is a component of an antibody-drug conjugate, drug-linker conjugate, or linker that is used to bind the Stretcher to an amino acid residue, or a fragment or short peptide consisting of 1-10 amino acid residues. When present, the Linker can connect L1 to L3. Specific examples include, but are not limited to, (wherein position 1 is connected to the Stretcher and position 2 is connected to L3):

[0025] In some embodiments, L2 is absent or present, and when present, L2 is selected from:

[0026] y1, y2, y3 and y4 are independently selected from any integer between 0 and 20, the 1st position is connected to L1, and the 2nd position is connected to L3.

[0027] In some embodiments, L1 is

[0028] In some embodiments, L1 is selected from Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aromatic group, a 5-10 membered heteroaromatic group and an amide group (preferably selected from a direct bond, a carbon-carbon triple bond and a carbon-carbon double bond); Rx and Ry are independently selected from H and a C1-4 alkyl group; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1 is independently selected from any integer between 1 and 6 (such as 4, 5, 6); each y2 is independently selected from any integer between 0 and 15 (such as 6-15); each y3 is independently selected from 1, 2 and 3; each y4 is independently selected from 0 and 1; position 1 is connected to Tb through an S atom, and position 2 is connected to L2 or L3.

[0029] In some embodiments, L1 is selected from m is selected from 2, 3, and 4, y1 is selected from any integer between 1 and 6 (such as 4, 5, and 6), each y2 is independently selected from any integer between 0 and 10 (such as 6 and 10), each y3 is independently selected from 1 or 2, position 1 is connected to Tb through an S atom, and position 2 is connected to L2 or L3.

[0030] In some embodiments, L1 is selected from

[0031] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0032] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, position 1 is connected to L1, and position 2 is connected to L3.

[0033] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0034] In some embodiments, L2 is absent.

[0035] In some embodiments, L2 is selected from

[0036] In some embodiments, L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or an amino acid residue represented by AA1 or a stereoisomer thereof.

[0037] In some embodiments, L3 is selected from the group consisting of amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA 1 or 2-10 selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AA 1 A short peptide composed of amino acid residues.

[0038] In some embodiments, L3 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AA 1 , Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA 1 、Ala-AA 1 、Gly-AA 1 AA 1 -Gly, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Asp, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gl y-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys and Lys-Ala-Asn.

[0039] In some embodiments, L3 is selected from AA 1 AA 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn and Gly-Gly-Phe-Gly.

[0040] In some embodiments, L3 is selected from AA 1 and Val-AA 1 -Gly.

[0041] In some embodiments, L3 is selected from Val-AA 1 -Gly.

[0042] In some embodiments, L3 is selected from X - Selected from halogen ions, carboxylate ions, sulfate ions, hydrogen sulfate ions and OH - , bit 1 is connected to L1 or L2, bit 2 is connected to L4 or D.

[0043] In some embodiments, L3 is selected from Bit 1 is connected to L1 or L2, and bit 2 is connected to L4 or D.

[0044] In some embodiments, L3 is selected from Bit 1 is connected to L1 or L2, and bit 2 is connected to L4 or D.

[0045] In some embodiments, AA 1 The structures of the indicated amino acid residues are shown below,

[0046] in:

[0047] R a 、R b Each independently selected from H, And R a 、R b Not at the same time H;

[0048] Or, R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 replaced by;

[0049] r、r 1 are each independently selected from any integer from 0 to 20;

[0050] R m1 、R n1 Each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and -COOR x1 ;

[0051] R x1 Selected from C1-6 alkyl;

[0052] Or, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0’ replaced by;

[0053] R z Selected from C1-6 alkyl;

[0054] R 0 、R 0’ Each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 and a 4-10 membered heterocyclic group optionally substituted by a C1-6 alkyl group;

[0055] R m2 、R n2 Each is independently selected from H and C1-6 alkyl.

[0056] In some embodiments, R a 、R b Among them, any one is H, and the other is selected from

[0057] In some embodiments, R a 、R b Among them, any one is H, and the other is selected from

[0058] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form the 0 Substituted 5-6 membered heterocycle.

[0059] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form the 0 a substituted piperidine ring or a piperazine ring.

[0060] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form the 0 Substituted piperidine ring.

[0061] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form Carbon atom No. 1 is R a and R b carbon atoms that are linked together.

[0062] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form Carbon atom No. 1 is R a and R b carbon atoms that are linked together.

[0063] In some embodiments, r, r 1 Each is independently selected from 0, 1, 2, 3, 4 and 5.

[0064] In some embodiments, r, r 1 are each independently selected from 0 and 4.

[0065] In some embodiments, r, r 1 If one of them is 0, the other one is 4.

[0066] In some embodiments, R m1 、R n1 Each is independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3.

[0067] In some embodiments, R m1 、R n1 Each is independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and tert-butyloxycarbonyl.

[0068] In some embodiments, R m1 、R n1 Each is independently selected from H and C1-6 alkyl.

[0069] In some embodiments, R m1 、R n1 Each is independently selected from H, methyl, ethyl and n-propyl.

[0070] In some embodiments, r, r 1 In the example, r is 4, r 1 When R is 0, m1 、R n1 Each is independently selected from H, C1-6 alkyl (such as H, methyl); r is 0, r 1 When R is 4, m1 、R n1 Each is independently selected from C1-6 alkyl (such as methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (such as ethyl, n-propyl).

[0071] In some embodiments, R m1 With R n1 Together with the nitrogen atom to which they are commonly attached, they form an optionally R 0’ Substituted 5-6 membered heterocycle.

[0072] In some embodiments, R m1 With R n1 Together with the nitrogen atom to which they are commonly attached, they form an optionally R 0’ a substituted piperidine ring or a piperazine ring.

[0073] In some embodiments, R m1 With R n1Together with the nitrogen atoms they are connected to, they form Nitrogen atom No. 1 is R m1 and R n1 Commonly connected nitrogen atoms.

[0074] In some embodiments, R z It is a methyl group.

[0075] In some embodiments, R 0 、R 0’ Each independently selected from C1-6 alkyl, -NR m2 R n2 and a 5-6 membered heterocyclic group optionally substituted by a C1-6 alkyl group.

[0076] In some embodiments, R 0 is selected from C1-6 alkyl and a 5-6 membered heterocyclic group substituted by a C1-6 alkyl group, wherein the 5-6 membered heterocyclic group is selected from piperidinyl and piperazinyl.

[0077] In some embodiments, R 0 is selected from methyl, ethyl and a 5-6 membered heterocyclic group substituted by a methyl group, wherein the 5-6 membered heterocyclic group is a piperidinyl group.

[0078] In some embodiments, R 0 is selected from methyl and a 5-6 membered heterocyclic group substituted by methyl, wherein the 5-6 membered heterocyclic group is piperidinyl.

[0079] In some embodiments, R 0 Selected from methyl, ethyl and

[0080] In some embodiments, R 0 Selected from methyl and

[0081] In some embodiments, R 0’ Selected from C1-6 alkyl and -NR m2 R n2 .

[0082] In some embodiments, R 0’ Selected from methyl and -NR m2 R n2 .

[0083] In some embodiments, R m2 、R n2 It is a methyl group.

[0084] In some embodiments, AA 1 The amino acid residues shown are selected from

[0085] In some embodiments, AA 1 The amino acid residues shown are selected from

[0086] In some embodiments, AA 1 The amino acid residues shown are selected from

[0087] In some embodiments, L4 is absent or present, and when L4 is present, L4 is Bit 1 is connected to L3 and bit 2 is connected to D.

[0088] In some embodiments, L4 is absent.

[0089] In some embodiments, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0090] In some embodiments, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0091] In some embodiments, The structure is selected from the following structural fragments:

[0092] In some embodiments, q is selected from any integer between 0.1 and 16.0; in a preferred embodiment, q is selected from any integer between 0.1 and 16.0.

[0093] In some embodiments, q is selected from any integer between 0.1 and 8.0. In a preferred embodiment, q is selected from any integer between 0.1 and 8.0.

[0094] In some embodiments, q is selected from any value between 2-8.

[0095] In some embodiments, q is selected from any value between 3-8.

[0096] In some embodiments, q is selected from any value between 4-8.

[0097] In some embodiments, q is selected from any value between 6-8.

[0098] In some embodiments, q is selected from any integer between 2-8.

[0099] In some embodiments, q is selected from any integer between 3-8.

[0100] In some embodiments, q is selected from any integer between 4-8.

[0101] In some embodiments, q is selected from any integer between 6-8.

[0102] In some embodiments, q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0103] In some embodiments, q is selected from 2, 4, 6, and 8.

[0104] In this disclosure, the bioactive molecular fragments are known in the art, and are part (fragment or group) of antibody-drug conjugates (or antibody-drug conjugates (ADCs)), which can form biologically active drugs (such as small molecule cytotoxic drugs, including groups after losing an atom or atomic group) or their derivatives (such as precursors) after the linker cleavage / degradation / enzymatic cleavage between tumor tissues or within tumor cells. For the avoidance of doubt, "drugs" do not only refer to "drugs" that have been approved by medical regulatory authorities, but also include any molecules with potential therapeutic biological activity in clinical practice, or in R&D and academic research.

[0105] In some embodiments, D is a molecular fragment having anti-tumor biological activity.

[0106] In some embodiments, D is a fragment of a molecule with anti-tumor biological activity, wherein the biologically active molecule is selected from a cytotoxic agent or a derivative thereof, such as a DNA topoisomerase inhibitor (e.g., a camptothecin-type biologically active molecule, such as camptothecin, DXD, a substituent-modified camptothecin, or a substituent-modified DXD) or a microtubule inhibitor (e.g., MMAF-type microtubule inhibitor, MMAE-type microtubule inhibitor).

[0107] In some embodiments, the antibody drug conjugate has the structure of Formula I:

[0108] in,

[0109] R1 and R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or,

[0110] R1 and R2 together with the carbon atoms to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, wherein the heterocyclic ring contains one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof;

[0111] R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or,

[0112] R3 and X together with the carbon atom to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring containing one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof, or

[0113] R3 and R2 together with the carbon atoms to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, wherein the heterocyclic ring contains one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof;

[0114] W is absent or present. When W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, bit 2 is connected to L4 or L3;

[0115] X is selected from a direct bond, an optionally substituted -O-(CH2) n3 -、-N(R4)-(CH2) n3 -、-S-(CH2) n3 -, carbonyl-(CH2) n3 、-SO2-(CH2) n3 -、 -(CH2) n1 -, C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1 position is connected to the parent ring, and the 2 position is connected to W or L4; the substituent is selected from one or more C1-4 alkyl, C3-6 cycloalkyl, or multiple C1-4 alkyl and the carbon atoms to which they are simultaneously connected together form a C3-6 cycloalkyl;

[0116] Each M is independently selected from a direct bond and -CR 5a R 5b -;

[0117] R4, R5, R 5a 、R 5b , R6, R7 are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3-6 cycloalkyl;

[0118] n, n', n1, n2, n3 are each independently selected from any integer between 0 and 6;

[0119] L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3, and bit 2 is connected to W or X.

[0120] Tb, L1, L2, L3 and q have the meanings provided above and in any of the embodiments specifically described herein.

[0121] In some embodiments, R1 and R2 are each independently selected from H, halogen, and C1-4 alkyl.

[0122] In some embodiments, R1 and R2, together with the carbon atom to which they are attached, form a 5-6 membered heterocyclic ring containing 1, 2 or 3 O, S or N or any combination thereof.

[0123] In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl.

[0124] In some embodiments, R1 and R2 and the carbon atom to which they are attached form The dotted line indicates the position where the heterocyclic ring is fused to the benzene ring.

[0125] In some embodiments, R1 is H or F, and R2 is H or methyl.

[0126] In some embodiments, R1 is F, R2 is methyl, or R1 and R2 together with the carbon atom to which they are attached form

[0127] In some embodiments, R1 is F and R2 is methyl.

[0128] In some embodiments, R1 and R2 together with the carbon atom to which they are attached form

[0129] In some embodiments, R3 is selected from H and C1-4 alkyl.

[0130] In some embodiments, R3 and X, together with the carbon atom to which they are attached, form a 5-6 membered carbocyclic ring.

[0131] In some embodiments, R3 is H or R3 and X together with the carbon atom to which they are attached form The dotted lines indicate where the carbocyclic ring is fused to the benzene and pyridine rings.

[0132] In some embodiments, R3 is H.

[0133] In some embodiments, W is absent or present, and when W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, and bit 2 is connected to L4 or L3.

[0134] In some embodiments, W is absent or present, and when W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, and bit 2 is connected to L4 or L3.

[0135] In some embodiments, W is selected from -O-, -NR4- and Bit 1 is connected to X, and bit 2 is connected to L4 or L3.

[0136] In some embodiments, W is selected from -O- and -NR4-, position 1 is connected to X, and position 2 is connected to L4 or L3.

[0137] In some embodiments, X is selected from optionally substituted -(CH2) n1 -、 C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1 position is connected to the parent ring, and the 2 position is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms to which they are simultaneously connected form a C3-6 cycloalkyl group.

[0138] In some embodiments, X is selected from optionally substituted Position 1 is connected to the parent ring, and position 2 is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl) and the carbon atoms to which they are simultaneously connected form a C3-6 cycloalkyl group (such as cyclopropyl).

[0139] In some embodiments, X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W or L4.

[0140] In some embodiments, X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W.

[0141] In some embodiments, when W is absent, X is selected from 1 is connected to the parent ring, 2 is connected to L4; when W exists, X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W.

[0142] In some embodiments, W is selected from -O-, -NR4- and Position 1 is connected to X, position 2 is connected to L4 or L3; X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W.

[0143] In some embodiments, R4 and R5 are each independently selected from H, C1-4 alkyl and C3-6 cycloalkyl.

[0144] In some embodiments, each R4 is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, and R5 is H.

[0145] In some embodiments, each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl, and cyclopropyl, and R5 is H.

[0146] In some embodiments, R 5a 、R 5b Each is independently selected from H and C1-4 alkyl.

[0147] In some embodiments, R 5a 、R 5b are each independently selected from H and methyl.

[0148] In some embodiments, each R7 is independently selected from H and C1-4 alkyl.

[0149] In some embodiments, R7 is H.

[0150] In some embodiments, n is selected from 1, 2, and 3.

[0151] In some embodiments, n is 1.

[0152] In some embodiments, n1 is selected from 1, 2, 3, and 4.

[0153] In some embodiments, n2 is 1.

[0154] In some embodiments, n3 is 0.

[0155] In some embodiments, L3 is selected from X - Selected from halogen ions, carboxylate ions, sulfate ions, hydrogen sulfate ions and OH - , bit 1 is connected to L1 or L2, bit 2 is connected to L4 or W.

[0156] In some embodiments, L3 is selected from Bit 1 is connected to L1 or L2, and bit 2 is connected to L4 or W.

[0157] In some embodiments, L3 is selected from Bit 1 is connected to L1 or L2, and bit 2 is connected to L4 or W.

[0158] In some embodiments, L4 is absent or present, and when L4 is present, L4 is Bit 1 is connected to L3, and bit 2 is connected to W or X.

[0159] In some embodiments, L4 is absent.

[0160] In some embodiments, L4 is selected from Bit 1 is connected to L3, and bit 2 is connected to W or X.

[0161] In some embodiments, L4 is selected from Bit 1 is connected to L3, and bit 2 is connected to W or X.

[0162] It should be noted that, as described above, W is absent or present. Therefore, when W is absent, position 1 of L4 is connected to L3 and position 2 is connected to X. When W is present, position 1 of L4 is connected to L3 and position 2 is connected to W. The following connection relationship of L4 can be understood with reference to the above content.

[0163] In some embodiments, The structure is selected from the following structural fragments:

[0164] Among them, bit 1 is connected to Tb and bit 2 is connected to W.

[0165] In some embodiments, D is The structural fragment shown; position 1 is connected to L3 or L4; for example

[0166] In some embodiments, The structure is selected from the following structural fragments:

[0167] Among them, bit 1 is connected to L4; when L4 does not exist, bit 1 is connected to L3.

[0168] In some embodiments,

[0169] W is absent or present. When W is present, W is selected from -O-, -S-, -NR4-, For example, there is no, -O-, -NR4- or R4 and R5 are each independently selected from H and C1-4 alkyl; n is independently selected from 0, 1, 2, 3 and 4;

[0170] X is selected from

[0171] R1 is selected from H, halogen, R2 is selected from H, C1-4 alkyl, or R1 and R2 and the carbon atom to which they are connected form The dotted line indicates the position where the heterocyclic ring is fused to the benzene ring;

[0172] R3 is selected from H and C1-4 alkyl, or R3 and X together with the carbon atom to which they are attached form a 5-6 membered carbocyclic ring;

[0173] Preferably, W is absent or present, and when W is present, W is selected from -O-, -NR4- (eg, -NH-, -N(CH3)-, -N(C2H5)-), R4 is independently selected from H, methyl, ethyl, isopropyl, n-propyl, tert-butyl and cyclopropyl;

[0174] When W does not exist, X is selected from 1 is connected to the parent ring; when W exists, X is selected from

[0175] AA1 wherein r is selected from 0, 1, 2, 3, 4 and 5;

[0176] R a 、R b Among them, any one is H, and the other is selected from Or, R a With R b Together with the carbon atoms they are connected to, they form 0 substituted 5-6 membered heterocycle;

[0177] R1 is selected from H, halogen, R2 is selected from H, C1-4 alkyl, or R1 and R2 and the carbon atom to which they are connected form

[0178] R3 is selected from H and C1-4 alkyl, or R3 and X together with the carbon atom to which they are attached form R3 and X together with the carbon atom to which they are attached form

[0179] More preferably, W is selected from -O- and -NR4-, position 1 is connected to X, and position 2 is connected to L4 or L3;

[0180] X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W;

[0181] for For example

[0182] R1 is F,

[0183] R2 is methyl or R1 and R2 together with the carbon atom to which they are attached form

[0184] R3 is H.

[0185] In some embodiments, the antibody drug conjugate has the structure shown in Formula I-1:

[0186] wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, R4 and q have the meanings provided above and in any of the embodiments specifically described herein.

[0187] In some embodiments, the antibody drug conjugate has the structure of Formula I-1A or I-1B:

[0188] wherein Tb, L2, L3, L4, X, R1, R2, R3, R4 and q have the meanings provided above and in any of the embodiments specifically described herein.

[0189] In some embodiments, the antibody drug conjugate has the structure shown in Formula I-2:

[0190] wherein Tb, L1, L2, L3, L4, X, R1, R2, R3 and q have the meanings provided above and in any embodiment specifically described herein.

[0191] In some embodiments, the antibody drug conjugate has the structure shown in Formula I-2A or I-2B:

[0192] wherein Tb, L2, L3, L4, X, R1, R2, R3 and q have the meanings provided above and in any of the embodiments specifically described herein.

[0193] In some embodiments, the antibody drug conjugate has the structure shown in Formula I-3:

[0194] wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, R5, n, and q have the meanings provided above and in any of the embodiments specifically described herein.

[0195] In some embodiments, the antibody drug conjugate has the structure shown in Formula I-3A or I-3B:

[0196] wherein Tb, L2, L3, L4, X, R1, R2, R3, R4 and q have the meanings provided above and in any of the embodiments specifically described herein.

[0197] In some embodiments, the antibody drug conjugate has the structure shown in Formula IA:

[0198] Among them, Tb, X, R1, R2, R3, R a 、R b and q have the meanings provided above and in any embodiments specifically described herein.

[0199] In some embodiments, the antibody drug conjugate has the structure shown in Formula IB:

[0200] Among them, Tb, X, R1, R2, R3, R a 、R b and q have the meanings provided above and in any embodiments specifically described herein.

[0201] In some embodiments, the antibody drug conjugate is selected from the group consisting of:

[0202] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof.

[0203] In some embodiments, the antibodies or antigen-binding fragments thereof and monoclonal antibodies or antigen-binding fragments thereof include: Fab, Fab', F(ab')2, Fd, Fv (e.g., scFv), dAb, complementarity determining region fragments, non-human antibodies, humanized antibodies, chimeric antibodies, fully human antibodies, probodies, monoclonal antibodies, bispecific antibodies or multispecific antibodies.

[0204] In some embodiments, Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof that is endocytic, has no endocytic activity, or is weakly endocytic.

[0205] In some embodiments, Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof having endocytic activity.

[0206] In some embodiments, Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof that has no endocytosis activity or weak endocytosis activity.

[0207] In some embodiments, Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof.

[0208] In some embodiments, the anti-Napi2b antibody is a non-human antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

[0209] In some embodiments, the anti-Napi2b antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0210] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0211] In some embodiments, the anti-NaPi2b antibody or antigen-binding fragment thereof is selected from: lifastuzumab or an antigen-binding fragment thereof, upifitamab or an antigen-binding fragment thereof, antibody C1.

[0212] In some preferred embodiments, the anti-NaPi2b antibody is selected from: 16G5B3, 60A12B3, 22H9C4, 66C12D12 and 34F2A10 murine antibodies, or humanized antibodies thereof.

[0213] In some preferred embodiments, the anti-NaPi2b antibody is selected from the group consisting of: 60A12B3-hz1 antibody, 22H9C4-hz1 antibody, 66C12D12-hz1 antibody and 34F2A10-hz1 antibody.

[0214] The Tb antibodies or antigen-binding fragments thereof can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the disclosed antibodies can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the disclosed antibodies.

[0215] In some preferred embodiments, in the antibody-drug conjugate, Tb is the antibody Napi2b antibody or an antigen-binding fragment thereof described in the second aspect.

[0216] In a second aspect, the present disclosure provides an anti-Napi2b antibody or an antigen-binding fragment thereof.

[0217] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0218] HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33; and / or

[0219] LCDR1 or a variant thereof, LCDR2 or a variant thereof, and LCDR3 or a variant thereof contained in the light chain variable region (VL) shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51.

[0220] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 1 or 17; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 2 or 18.

[0221] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 19 or 34; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 20 or 35.

[0222] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 36 or 52; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 37 or 53.

[0223] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 54, 71 or 73; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 55 or 72.

[0224] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 74 or 33; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 75 or 51.

[0225] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 17; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 18.

[0226] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 34; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 35.

[0227] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 52; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 53.

[0228] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 71 or 73; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 72.

[0229] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the VH shown in SEQ ID NO: 33; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 51.

[0230] In certain preferred embodiments, the sequence variant is a CDR having one or several amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the CDR from which it is derived.

[0231] In certain preferred embodiments, the substitutions are conservative substitutions.

[0232] In certain embodiments, the CDRs are defined according to the AbM, Chothia, Kabat, or IMGT numbering systems.

[0233] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0234] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0235] (a) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 3 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 4 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 6 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0236] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 7 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SEQ ID NO: 9 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 10 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0237] (b) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 3 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 5 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 6 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0238] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 8 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SEQ ID NO: 9 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0239] (c) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 21 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 22 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 24 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0240] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 25 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SEQ ID NO: 26 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 27 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0241] (d) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 21 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 23 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 24 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0242] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 25 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SEQ ID NO: 26 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 27 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0243] (e) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 38 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 39 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 41 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0244] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 42 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of SEQ ID NO: 44 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 45 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0245] (f) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 38 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 40 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 41 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0246] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 42 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of SEQ ID NO: 44 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 45 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0247] (g) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 56 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 57 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 60 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0248] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 61 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of SEQ ID NO: 63 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 64 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

[0249] (h) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 56 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 58 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 60 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0250] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 62 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of SEQ ID NO: 63 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 64 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; or

[0251] (i) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 56 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 59 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 60 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0252] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 62 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of SEQ ID NO: 63 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 64 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

[0253] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0254] (a) the VH comprises: a HCDR1 of SEQ ID NO: 3, a HCDR2 of SEQ ID NO: 4, and a HCDR3 of SEQ ID NO: 6; and / or,

[0255] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 7, LCDR2 with a sequence of SEQ ID NO: 9, and LCDR3 with a sequence of SEQ ID NO: 10;

[0256] (b) the VH comprises: a HCDR1 of SEQ ID NO: 3, a HCDR2 of SEQ ID NO: 5, and a HCDR3 of SEQ ID NO: 6; and / or,

[0257] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 8, LCDR2 with a sequence of SEQ ID NO: 9, and LCDR3 with a sequence of SEQ ID NO: 10;

[0258] (c) the VH comprises: a HCDR1 of SEQ ID NO: 21, a HCDR2 of SEQ ID NO: 22, and a HCDR3 of SEQ ID NO: 24; and / or,

[0259] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 25, LCDR2 with a sequence of SEQ ID NO: 26, and LCDR3 with a sequence of SEQ ID NO: 27;

[0260] (d) the VH comprises: a HCDR1 of SEQ ID NO: 21, a HCDR2 of SEQ ID NO: 23, and a HCDR3 of SEQ ID NO: 24; and / or,

[0261] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 25, LCDR2 with a sequence of SEQ ID NO: 26, and LCDR3 with a sequence of SEQ ID NO: 27;

[0262] (e) the VH comprises: a HCDR1 of SEQ ID NO: 38, a HCDR2 of SEQ ID NO: 39, and a HCDR3 of SEQ ID NO: 41; and / or,

[0263] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 42, LCDR2 with a sequence of SEQ ID NO: 44, and LCDR3 with a sequence of SEQ ID NO: 45;

[0264] (f) the VH comprises: a HCDR1 of SEQ ID NO: 38, a HCDR2 of SEQ ID NO: 40, and a HCDR3 of SEQ ID NO: 41; and / or,

[0265] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 43, LCDR2 with a sequence of SEQ ID NO: 44, and LCDR3 with a sequence of SEQ ID NO: 45;

[0266] (g) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 57, and a HCDR3 of SEQ ID NO: 60; and / or,

[0267] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 61, LCDR2 with a sequence of SEQ ID NO: 63, and LCDR3 with a sequence of SEQ ID NO: 64;

[0268] (h) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 58, and a HCDR3 of SEQ ID NO: 60; and / or,

[0269] The VL comprises: LCDR1 of SEQ ID NO: 62, LCDR2 of SEQ ID NO: 63, and LCDR3 of SEQ ID NO: 64; or

[0270] (i) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 59, and a HCDR3 of SEQ ID NO: 60; and / or,

[0271] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 62, LCDR2 with a sequence of SEQ ID NO: 63, and LCDR3 with a sequence of SEQ ID NO: 64.

[0272] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0273] (a) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 11 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 12 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 14 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0274] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 15 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of WAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0275] (b) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 11 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 13 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 14 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0276] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 15 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of WAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0277] (c) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 28 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 29 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 31 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0278] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 32 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of YTS or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 27 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0279] (d) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 28 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 30 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 31 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0280] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 32 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of YTS or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 27 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0281] (e) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 46 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 47 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 49 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0282] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 50 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 45 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0283] (f) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 46 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 48 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 49 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0284] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 50 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of SAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 45 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0285] (g) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 65 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 66 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 68 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0286] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 69 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of WAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 64 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom;

[0287] (h) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 65 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, a HCDR2 having a sequence of SEQ ID NO: 67 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto, a HCDR3 having a sequence of SEQ ID NO: 68 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto; and / or,

[0288] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 69 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR2 having a sequence of WAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, LCDR3 having a sequence of SEQ ID NO: 64 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; or

[0289] (i) a heavy chain variable region (VH) comprising the following three CDRs: a HCDR1 having a sequence of SEQ ID NO: 76 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR2 having a sequence of SEQ ID NO: 77 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a HCDR3 having a sequence of SEQ ID NO: 78 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; and / or,

[0290] A light chain variable region (VL) comprising the following three CDRs: LCDR1 having a sequence of SEQ ID NO: 79 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR2 having a sequence of HTS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, LCDR3 having a sequence of SEQ ID NO: 16 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

[0291] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0292] (a) the VH comprises: a HCDR1 of SEQ ID NO: 11, a HCDR2 of SEQ ID NO: 12, and a HCDR3 of SEQ ID NO: 14; and / or,

[0293] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 10;

[0294] (b) the VH comprises: a HCDR1 of SEQ ID NO: 11, a HCDR2 of SEQ ID NO: 13, and a HCDR3 of SEQ ID NO: 14; and / or,

[0295] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 10;

[0296] (c) the VH comprises: a HCDR1 of SEQ ID NO: 28, a HCDR2 of SEQ ID NO: 29, and a HCDR3 of SEQ ID NO: 31; and / or,

[0297] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 32, LCDR2 with a sequence of YTS, and LCDR3 with a sequence of SEQ ID NO: 27;

[0298] (d) the VH comprises: a HCDR1 of SEQ ID NO: 28, a HCDR2 of SEQ ID NO: 30, and a HCDR3 of SEQ ID NO: 31; and / or,

[0299] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 32, LCDR2 with a sequence of YTS, and LCDR3 with a sequence of SEQ ID NO: 27;

[0300] (e) the VH comprises: a HCDR1 of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 47, and a HCDR3 of SEQ ID NO: 49; and / or,

[0301] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 50, LCDR2 with a sequence of SAS, and LCDR3 with a sequence of SEQ ID NO: 45;

[0302] (f) the VH comprises: a HCDR1 of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 48, and a HCDR3 of SEQ ID NO: 49; and / or,

[0303] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 50, LCDR2 with a sequence of SAS, and LCDR3 with a sequence of SEQ ID NO: 45;

[0304] (g) the VH comprises: a HCDR1 of SEQ ID NO: 65, a HCDR2 of SEQ ID NO: 66, and a HCDR3 of SEQ ID NO: 68; and / or,

[0305] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 69, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 64;

[0306] (h) the VH comprises: a HCDR1 of SEQ ID NO: 65, a HCDR2 of SEQ ID NO: 67, and a HCDR3 of SEQ ID NO: 68; and / or,

[0307] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 69, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 64;

[0308] or

[0309] (i) the VH comprises: a HCDR1 of SEQ ID NO: 76, a HCDR2 of SEQ ID NO: 77, and a HCDR3 of SEQ ID NO: 78; and / or,

[0310] The VL comprises: LCDR1 with a sequence of SEQ ID NO: 79, LCDR2 with a sequence of HTS, and LCDR3 with a sequence of SEQ ID NO: 16.

[0311] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof disclosed herein comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein, compared with the CDR defined by the aforementioned Kabat numbering system or IMGT numbering system, at least one CDR in the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions or any combination thereof), and still has Napi2b binding activity.

[0312] In certain preferred embodiments, the substitutions described herein are conservative substitutions.

[0313] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof binds to human Napi2b, monkey Napi2b, and / or rat Napi2b.

[0314] In certain embodiments, the VH of the anti-Napi2b antibody or its antigen-binding fragment of the present disclosure comprises a framework region (FR) derived from the heavy chain variable region (VH) of a human immunoglobulin, and / or the VL of the antibody or its antigen-binding fragment comprises a framework region (FR) derived from the light chain variable region (VL) of a human immunoglobulin. Therefore, in certain embodiments, the antibody or its antigen-binding fragment of the present disclosure is fully human. In certain embodiments, the antibody or its antigen-binding fragment of the present disclosure is humanized.

[0315] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0316] (a) a heavy chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which the variant is derived; and / or

[0317] (b) a light chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the amino acid sequence encoded by the germline antibody gene from which the variant is derived (e.g., up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids).

[0318] In certain embodiments, the degree of humanization of the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0319] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0320] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence selected from the group consisting of:

[0321] (i) the sequence shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33;

[0322] (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence shown in SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or

[0323] (iii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence set forth in SEQ ID 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33;

[0324] and / or

[0325] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence selected from the group consisting of:

[0326] (iv) the sequence shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51;

[0327] (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence shown in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or

[0328] (vi) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence set forth in SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51.

[0329] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the VH shown in SEQ ID NO: 1 or 17, and / or the VL shown in SEQ ID NO: 2 or 18.

[0330] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the VH set forth in SEQ ID NO: 19 or 34, and / or the VL set forth in SEQ ID NO: 20 or 35.

[0331] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the VH set forth in SEQ ID NO: 36 or 52, and / or the VL set forth in SEQ ID NO: 37 or 53.

[0332] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the VH set forth in SEQ ID NO: 54, 71, or 73, and / or the VL set forth in SEQ ID NO: 55 or 72.

[0333] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the VH set forth in SEQ ID NO: 74 or 33, and / or the VL set forth in SEQ ID NO: 75 or 51.

[0334] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VH of any of the five groups of schemes above; and / or, a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VL of the group; or

[0335] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof disclosed herein comprise a VH having one or several amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof, compared with the VH of any group in the above-mentioned five scheme groups; and / or, a VL having one or several amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof, compared with the VL of the group; in certain preferred embodiments, the substitutions are conservative substitutions.

[0336] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0337] (a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2;

[0338] (b) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 18;

[0339] (c) VH of SEQ ID NO: 19 and VL of SEQ ID NO: 20;

[0340] (d) VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35;

[0341] (e) VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37;

[0342] (f) VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53;

[0343] (g) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55;

[0344] (h) VH of SEQ ID NO: 71 and VL of SEQ ID NO: 72;

[0345] (i) VH of SEQ ID NO: 73 and VL of SEQ ID NO: 72;

[0346] (j) VH of SEQ ID NO: 74 and VL of SEQ ID NO: 75;

[0347] (k) VH of SEQ ID NO: 33 and VL of SEQ ID NO: 51;

[0348] (l) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) independently have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VH and VL described in any one of groups (a) to (k), respectively; or

[0349] (m) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) independently have one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof) compared to the VH and VL described in any one of groups (a) to (k), respectively. In certain preferred embodiments, the substitutions are conservative substitutions.

[0350] In certain embodiments, the anti-Napi2b antibodies of the present disclosure are chimeric antibodies, humanized antibodies or fully human antibodies. In certain embodiments, the antibodies of the present disclosure or their antigen-binding fragments are selected from Fab, Fab', (Fab')2, Fv fragments such as scFv or disulfide-linked Fv (dsFv), diabodies and multispecific antibodies. In certain embodiments, the anti-Napi2b antibodies of the present disclosure are scFv.

[0351] In certain embodiments, the heavy chain of an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having up to 50 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids). In certain embodiments, the light chain of the anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having up to 50 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids).

[0352] In some embodiments, the constant region is altered, for example, mutated, to modify the properties of the anti-Napi2b antibody molecule (e.g., to alter one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, by changing the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering the effector function (e.g., reducing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis (ADCP), CDC, and the like.

[0353] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure have a heavy chain constant region (CH) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly selected from the heavy chain constant region of IgG1 (e.g., human IgG1). In some embodiments, the human IgG1 heavy chain constant region is as shown in SEQ ID NO: 70. In some embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure have a light chain constant region selected from, for example, a κ or λ light chain constant region, preferably a κ light chain constant region (e.g., a human κ light chain constant region). In some embodiments, the light chain constant region has a sequence as shown in SEQ ID NO: 80.

[0354] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises CH as shown in SEQ ID NO:70 or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to SEQ ID NO:70 (e.g., up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids), or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO:70.

[0355] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises a light chain constant region (CL) as shown in SEQ ID NO: 80 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids compared to SEQ ID NO: 80 (e.g., a conservative substitution of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), or having a sequence identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% compared to SEQ ID NO: 80.

[0356] In some embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO:70 and a light chain constant region (CL) shown in SEQ ID NO:80.

[0357] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0358] (a) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of:

[0359] (i) a sequence comprising the VH sequence shown in SEQ ID NO: 71 and the CH sequence shown in SEQ ID NO: 70;

[0360] (ii) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0361] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in (i); and

[0362] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0363] (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 72 and the CL sequence shown in SEQ ID NO: 80;

[0364] (v) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0365] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence shown in (iv).

[0366] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0367] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0368] (a) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of:

[0369] (i) a sequence comprising the VH sequence shown in SEQ ID NO: 73 and the CH sequence shown in SEQ ID NO: 70;

[0370] (ii) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0371] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in (i); and

[0372] (b) a light chain comprising or consisting of an amino acid sequence selected from the group consisting of:

[0373] (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 72 and the CL sequence shown in SEQ ID NO: 80;

[0374] (v) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0375] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence shown in (iv).

[0376] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0377] In certain embodiments, an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0378] (a) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of:

[0379] (i) a sequence comprising the VH sequence shown in SEQ ID NO: 17 and the CH sequence shown in SEQ ID NO: 70;

[0380] (ii) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0381] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in (i); and

[0382] (b) a light chain comprising or consisting of an amino acid sequence selected from the group consisting of:

[0383] (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 18 and the CL sequence shown in SEQ ID NO: 80;

[0384] (v) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0385] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the sequence shown in (iv).

[0386] In certain embodiments, the substitution described in (ii) or (v) is a conservative substitution.

[0387] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain,

[0388] The heavy chain comprises or consists of the following sequence:

[0389] (i) the sequence shown in SEQ ID NO: 81;

[0390] (ii) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0391] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (i); and

[0392] The light chain comprises or consists of the following sequence:

[0393] (iv) the sequence shown in SEQ ID NO:82;

[0394] (v) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0395] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in (iv);

[0396] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0397] In certain embodiments, the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise a heavy chain and a light chain,

[0398] The heavy chain comprises or consists of the following sequence:

[0399] (i) the sequence shown in SEQ ID NO: 83;

[0400] (ii) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0401] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (i); and

[0402] The light chain comprises or consists of the following sequence:

[0403] (iv) the sequence shown in SEQ ID NO:84;

[0404] (v) a sequence having one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10 or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or

[0405] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in (iv);

[0406] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0407] In certain embodiments, the anti-Napi2b antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain and a light chain selected from the following groups:

[0408] (a) a heavy chain comprising the VH shown in SEQ ID NO: 71 and the CH shown in SEQ ID NO: 70, and a light chain comprising the VL shown in SEQ ID NO: 72 and the CL shown in SEQ ID NO: 80;

[0409] (b) a heavy chain comprising the VH shown in SEQ ID NO: 73 and the CH shown in SEQ ID NO: 70, and a light chain comprising the VL shown in SEQ ID NO: 72 and the CL shown in SEQ ID NO: 80; preferably comprising the heavy chain shown in SEQ ID NO: 81 and the light chain shown in SEQ ID NO: 82;

[0410] (c) a heavy chain comprising the VH of SEQ ID NO: 17 and the CH of SEQ ID NO: 70, and a light chain comprising the VL of SEQ ID NO: 18 and the CL of SEQ ID NO: 80. Preferably, it comprises the heavy chain of SEQ ID NO: 83 and the light chain of SEQ ID NO: 84;

[0411] In certain embodiments, the antibody provided herein is a multispecific antibody that binds to Napi2b and one or more other antigens. In certain preferred embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody. In some embodiments, the multispecific antibody of the present disclosure comprises the anti-Napi2b antibody or its antigen-binding fragment described above, and another antibody or its fragment or antibody analog.

[0412] Antibody derivatives disclosed herein

[0413] The anti-Napi2b antibodies or antigen-binding fragments thereof disclosed herein can be derivatized, for example, connected to another molecule (e.g., another polypeptide or protein). Typically, the derivatization (e.g., labeling) of an antibody or its antigen-binding fragment will not adversely affect its binding to Napi2b. Therefore, the anti-Napi2b antibodies or antigen-binding fragments thereof disclosed herein are also intended to include such derivatized forms. For example, the anti-Napi2b antibodies or antigen-binding fragments thereof disclosed herein can be connected (by chemical coupling, gene fusion, non-covalent connection or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that can mediate the binding of an antibody or its antigen-binding fragment to another molecule.

[0414] One type of derivatized antibody (e.g., bispecific antibody) is produced by cross-linking two or more antibodies (of the same type or different types). Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking, cell engineering (hybridoma method), or genetic engineering.

[0415] Another type of derivatized antibody is a labeled antibody. For example, the antibody of the present disclosure or its antigen-binding fragment can be connected to a detectable label. The detectable label described in the present disclosure can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). Detectable labels such as those described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments thereof of the present disclosure via linkers of varying lengths to reduce potential steric hindrance.

[0416] In addition, the antibodies or antigen-binding fragments thereof of the present disclosure can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or glycosyl groups. These groups can be used to improve the biological properties of the antibodies, such as increasing serum half-life.

[0417] Antibody preparation

[0418] The antibodies disclosed herein can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies disclosed herein can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies disclosed herein.

[0419] The antigen-binding fragments disclosed herein can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0420] In a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the second aspect of the present disclosure, or a heavy chain variable region and / or a light chain variable region thereof, or one or more CDRs thereof; in certain embodiments, the nucleotide sequence is replaceable based on codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0421] In certain embodiments, the isolated nucleic acid molecules of the present disclosure comprise: (i) a first nucleic acid and a second nucleic acid encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the second aspect of the disclosure, respectively, or (ii) a first nucleic acid encoding the heavy chain variable region and the heavy chain constant region of the antibody or antigen-binding fragment thereof of the second aspect of the disclosure, respectively, and a second nucleic acid encoding the light chain variable region and the light chain constant region, respectively, or (iii) a first nucleic acid and a second nucleic acid encoding the heavy chain and the light chain of the antibody or antigen-binding fragment thereof of the second aspect of the disclosure, respectively. In certain embodiments, the first nucleic acid and the second nucleic acid comprise a degenerate sequence or a substantially identical sequence to any of the first and second nucleic acids in (i)-(iii) above. In certain embodiments, the degenerate sequence or substantially identical sequence refers to a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity or a sequence having one or more nucleotide substitutions, or a sequence that differs by no more than 3, 6, 15, 30 or 45 nucleotides compared to the nucleic acid molecules in (i)-(iii).

[0422] In a fourth aspect, a vector (e.g., a cloning vector or an expression vector) is provided, comprising the isolated nucleic acid molecule of the third aspect of the present disclosure. In certain embodiments, the vector of the present disclosure is a cloning vector or an expression vector. In certain embodiments, the vector of the present disclosure is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present disclosure in a subject (e.g., a mammal, such as a human).

[0423] In a fifth aspect, a host cell is provided, which comprises the isolated nucleic acid molecule of the third aspect of the present disclosure or the fourth aspect vector of the present disclosure. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include but are not limited to NSO cells, SP2 / 0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include but are not limited to Sf9 cells. In certain embodiments, the host cell of the present disclosure is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0424] In a sixth aspect, a method for preparing the antibody or antigen-binding fragment thereof of the second aspect of the present disclosure, or the multispecific antibody of the present disclosure is provided, comprising culturing the host cell of the present disclosure under conditions allowing expression of the antibody or antigen-binding fragment thereof, or the multispecific antibody, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0425] Antibody-drug conjugates

[0426] In the seventh aspect, an anti-Napi2b antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate is provided, wherein the antibody is the above-mentioned antibody that binds to Napi2b or its antigen-binding fragment.

[0427] In some embodiments, the antibody drug conjugate is selected from:

[0428] in,

[0429] Tb1 is an anti-Napi2b antibody or an antigen-binding fragment thereof, such as: lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2 and 16G5B3-hz1 antibodies.

[0430] q is selected from any value between 0.1 and 16.0, preferably any value between 2 and 8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0431] Antibody-drug conjugate preparation

[0432] In an eighth aspect of the present disclosure, the present disclosure provides a method for preparing the aforementioned antibody-drug conjugate, comprising:

[0433] Conjugate Tb with the drug linker shown in formula III

[0434] Carry out the coupling reaction in a suitable solvent and conditions;

[0435] in:

[0436] Tb has the meaning provided above and in any embodiment specifically described herein;

[0437] R1, R2, R3, X, W, L1, L2, L3, and L4 have the meanings provided above and in any of the embodiments specifically described herein.

[0438] Lg is a leaving group, which is selected from halogen, sulfone, tertiary amine salt (Me3N + 、Et3N + ), diazonium salt, -OMs, MeSO2-, CF3SO3-.

[0439] In some embodiments, Lg is selected from F, Cl, MeSO2-.

[0440] In some embodiments, Lg is selected from F, MeSO2-.

[0441] In some embodiments, the method comprises conjugating Tb with a drug linker of formula III The step of forming a C-S bond by coupling reaction is carried out in a suitable solvent and conditions.

[0442] In some embodiments, the ratio of the amount of Tb to the amount of the drug-linker conjugate is 1:(1-20), such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20) or 1:(18-20).

[0443] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0444] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (such as methanol, ethanol) or any combination thereof.

[0445] In some embodiments, the method further comprises the step of purifying the coupling product.

[0446] In some embodiments, the coupled product is purified by chromatography.

[0447] In some embodiments, the chromatography method comprises one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0448] In the ninth aspect of the present disclosure, the present disclosure provides a group of antibody drug conjugates, comprising the antibody drug conjugates described above, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts or their pharmaceutically acceptable solvates, wherein the antibody drug conjugates have one, two or more q values.

[0449] In some embodiments, when the antibody drug conjugates in the population have a q value, the q value and the DAR value are equal.

[0450] In some embodiments, when the antibody drug conjugates in the population have two or more q values, the proportion of antibody drug conjugates with a particular q value among all antibody drug conjugates in the population is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0451] In some embodiments, the drug to antibody ratio (DAR) in the antibody drug conjugates in the population is an integer or decimal selected from 1-10.

[0452] In some embodiments, the drug to antibody ratio (DAR) of the population is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5;

[0453] In some embodiments, the drug to antibody ratio (DAR) of the population is selected from about 2.0, 4.0, 6.0, and 8.0;

[0454] In some embodiments, the drug to antibody ratio (DAR) in the antibody drug conjugates in the population is selected from the group consisting of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0455] In some embodiments, the population contains ADCs with a distribution of DARs of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 drug loading species). It is worth noting that degradation products can be produced such that the population also includes DARs of 1, 3, 5, and 7. In addition, the population may also have a DAR greater than 8. The antibody drug conjugate is produced by reducing the interchain disulfide followed by conjugation. In some embodiments, the antibody drug conjugate comprises both: an antibody drug conjugate having a DAR of 4 or less (i.e., a drug loading species of 4 or less) and an antibody drug conjugate having a DAR of 6 or more (i.e., a drug loading species of 6 or more).

[0456] In the tenth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the first or seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the anti-Napi2b antibody or antigen-binding fragment thereof of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the antibody-drug conjugate group of the ninth aspect, and optionally one or more pharmaceutical excipients.

[0457] In certain embodiments, the pharmaceutical composition comprises the above-mentioned antibody-drug conjugate of the first aspect or seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the anti-Napi2b antibody or antigen-binding fragment thereof of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the antibody-drug conjugate group of the ninth aspect, all in therapeutically effective amounts.

[0458] In certain embodiments, the pharmaceutical composition comprises the antibody-drug conjugate of the first or seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, and a pharmaceutically acceptable carrier and / or excipient.

[0459] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the antibody-drug conjugate population of the ninth aspect and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises the above-mentioned antibody-drug conjugate population, or comprises the antibody-drug conjugate population and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0460] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an anti-Napi2b antibody or an antigen-binding fragment thereof of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient. In certain preferred embodiments, the pharmaceutical composition of the present disclosure comprises an anti-Napi2b antibody or an antigen-binding fragment thereof, and a buffer. In certain further preferred embodiments, the pharmaceutical composition of the present disclosure further comprises an excipient and / or a surfactant.

[0461] In some embodiments, the buffer is a histidine buffer. In certain preferred embodiments, the buffer is a 20 mM histidine buffer at a pH of 6.0.

[0462] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the isolated nucleic acid molecule or vector as described above.

[0463] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise the multispecific antibodies of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0464] In some embodiments, the drug to antibody ratio (DAR) in the pharmaceutical composition is an integer or decimal selected from 1-10.

[0465] In some embodiments, the drug to antibody ratio (DAR) in the pharmaceutical composition is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5;

[0466] In some embodiments, the drug to antibody ratio (DAR) in the pharmaceutical composition is selected from about 2.0, 4.0, 6.0, and 8.0;

[0467] In some embodiments, the drug to antibody ratio (DAR) in the antibody drug conjugate in the pharmaceutical composition is selected from the group consisting of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.

[0468] In an eleventh aspect, the present disclosure provides a use of an antibody or antigen-binding fragment thereof in the preparation of a kit for detecting the presence or level of Napi2b in a sample. In another aspect, the present disclosure provides a diagnostic or therapeutic kit comprising one or more of the following: an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, an antibody-drug conjugate, a population of antibody-drug conjugates, or a pharmaceutical composition as described herein. Optionally, the diagnostic or therapeutic kit further comprises instructions for use.

[0469] In a twelfth aspect of the present disclosure, the present disclosure provides the use of the aforementioned antibody-drug conjugate composition or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity (e.g., a cancer disease). The antibody-drug conjugate composition or the aforementioned pharmaceutical composition can be in a therapeutically effective amount.

[0470] In some embodiments, provided is the use of the anti-Napi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the preparation of a medicament for regulating (inhibiting or blocking) the activity of Napi2b.

[0471] In some embodiments, provided is a use of the anti-Napi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure in the preparation of a medicament for treating or preventing a disease associated with the activity of Napi2b.

[0472] In some embodiments, provided are uses of the anti-Napi2b antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, antibody-drug conjugates, or multispecific antibodies disclosed herein in the preparation of drugs for treating or preventing tumors associated with the activity of Napi2b.

[0473] In some embodiments, the antibody-drug conjugate of the first or seventh aspect of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the anti-Napi2b antibody or its antigen-binding fragment of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of a drug is provided, and the drug is used to treat or prevent tumors.

[0474] In some embodiments, the antibody-drug conjugate of the first or seventh aspect of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect is provided for use in preparing a drug for treating or preventing tumors associated with Tb targets.

[0475] In some embodiments, the antibody-drug conjugate of the first or seventh aspect of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect is provided for use in the preparation of a drug for treating or preventing tumors associated with NaPi2b.

[0476] In some preferred embodiments, provided are the antibody-drug conjugate of the first or seventh aspect of the present disclosure, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, the anti-Napi2b antibody or its antigen-binding fragment of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody-drug conjugate group of the ninth aspect, and the use of the pharmaceutical composition of the tenth aspect in the preparation of a drug, wherein the drug is used to treat or prevent tumors associated with Napi2b.

[0477] In the thirteenth aspect of the present disclosure, the present disclosure provides a method for treating and / or preventing diseases associated with abnormal cell activity (such as tumors) by using the antibody-drug conjugate of the first or seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts or pharmaceutically acceptable solvates, the anti-Napi2b antibody or its antigen-binding fragment of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0478] In the above-mentioned twelfth and thirteenth aspects, the tumor is selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colon adenocarcinoma), rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumor (such as glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0479] In some embodiments, the tumor is a NaPi2b-associated tumor.

[0480] In some embodiments, the tumor is a Napi2b-associated tumor.

[0481] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0482] As used herein, examples of the term "pharmaceutically acceptable salts" are organic acid addition salts formed with organic acids that form pharmaceutically acceptable anions, including but not limited to formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarate, α-glycerophosphates, alkylsulfonates, or arylsulfonates; preferably, the alkylsulfonate is methylsulfonate or ethylsulfonate; and the arylsulfonate is benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates, carbonates, sulfates, or phosphates.

[0483] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.

[0484] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0485] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These substances, in addition to the active ingredients, have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, dissolution enhancement, and sustained-release control. They are important components that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be categorized as natural, semi-synthetic, and fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0486] The pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the compound of the present disclosure or a pharmaceutically acceptable salt or conjugate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0487] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, sterile powder for injection, and concentrated solution for injection. Among them, the carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils such as monoglycerides or diglycerides can also be used as solvents or suspending media.

[0488] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0489] In the present disclosure, the term "individual" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0490] As used herein, the term "effective dose" refers to that amount of a compound which, when administered, will relieve to some extent one or more of the symptoms of the condition being treated.

[0491] In the present disclosure, the term "bioactive substance", "bioactive molecule" or "drug molecule" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. In some embodiments of the present disclosure, the bioactive substance, bioactive molecule or drug molecule in the conjugate is a molecule with anti-tumor biological activity. For example: radioactive isotopes, such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212and radioactive isotopes of Lu; metal complexes, such as metal platinum complexes, metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin and bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, etc.; drugs that act on structural proteins, such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, polyoxazine Citaxel, Cabazitaxel, etc.; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; also including proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; growth inhibitors; drug moieties. The term "toxin" refers to a substance that can have a deleterious effect on cell growth or proliferation.

[0492] In the present disclosure, the term "linker" refers to a fragment that connects a biologically active molecule (drug molecule) to a targeting moiety.

[0493] In this disclosure, the term "targeting moiety" refers to a portion of a conjugate that is capable of specifically binding to a target (or portion of a target) on the cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.

[0494] In the present disclosure, antibodies or antigen-binding fragments thereof include derivatized antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments thereof having sulfhydryl groups, wherein the derivatization provides the antibody with a group or ability to react with a drug-linker conjugate. The sulfhydryl group (SH) can be derivatized by opening a disulfide bond (e.g., by reduction with the reducing agent TCEP).

[0495] As used herein, the terms "cancer" and "tumor" are used synonymously.

[0496] The term "gene" used herein includes not only DNA but also its mRNA, its cDNA, and its cRNA.

[0497] As used herein, the term "polynucleotide" is used with the same meaning as nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers.

[0498] As used herein, the terms "polypeptide" and "protein" are used indiscriminately.

[0499] The term "cell" as used herein also includes cells in an animal individual and cultured cells.

[0500] The Napi2b involved in the anti-Napi2b antibody described in the present disclosure may be conventional Napi2b in the art, and of course also refers to Napi2b variants.

[0501] The term "NaPi2b" is encoded by the SLC34A2 gene and consists of 690 amino acids. It has eight transmembrane spans and four extracellular loops. Sodium-dependent phosphate transporter 2B (NaPi2b) selectively transports divalent Pi (HPO4 2- ) to maintain Pi homeostasis. Human NaPi2b shares 85.3% homology with rat and 95.8% with cynomolgus monkey. NaPi2b shares high amino acid sequence similarity with NaPi2a / 2c in the same family (51.29% homology with NaPi2a and 44.64% homology with NaPi2c), particularly in the transmembrane region, where homology exceeds 80%.

[0502] KD refers to the dissociation constant obtained from the ratio of Kd (the off-rate of a particular binding molecule-target protein interaction) to Ka (the on-rate of a particular binding molecule-target protein interaction) (or Kd / Ka, expressed in molar concentration (M)). KD values ​​can be determined using methods well established in the art. A preferred method for determining the KD of a binding molecule is by using surface plasmon resonance, for example, a biosensor system such as the Biacore™ (GE Healthcare Life Sciences) system.

[0503] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity of a sequence between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparisons and determination of percent identity between sequences can be performed using methods generally known in the art, and such sequence comparisons and determination of percent identity can be performed using mathematical algorithms. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci. 4: 11-17 (incorporated into the ALIGN program (version 2.0)) can be used to determine percent identity between amino acid sequences and / or between nucleotide sequences. In addition, the GAP program in the GCG software package available online from Accelrys (using its default parameters) can be used to determine percent identity between amino acid sequences or between nucleotide sequences. In one embodiment, the two sequences are of equal length.

[0504] The term "epitope" refers to a portion of an antigenic polypeptide or protein that has antigenic or immunogenic activity in an animal, preferably a mammal. The epitope of the antibodies or antigen-binding fragments thereof disclosed herein can be determined by existing technologies, such as synthetic peptide methods, immunoinformatics predictions, determination of polypeptide activity, epitope peptide scanning methods, phage display technology, X-ray diffraction and nuclear magnetic resonance analysis, and antibody homology modeling protein docking prediction methods. The phrase "antibodies that bind to the same epitope" as used herein refers to different antibodies that bind to a common epitope. If a second antibody binds to a portion of the peptide or a portion of the tertiary structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope.

[0505] In the present disclosure, the term "antibody" is interpreted in the broadest sense, including complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (such as bispecific antibodies) formed by at least two complete antibodies, as long as they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" can be used interchangeably. As described herein, "antibody molecule" or "antibody" refers to immunoglobulin molecules and immunoactive parts of immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to antigens. Therefore, the term antibody broadly encompasses not only complete antibody molecules, but also fragments of the antibodies and variants (including derivatives) of the antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as antigen-binding fragment, "antibody molecule" or "antibody" refers to complete antibody molecules or full-length antibodies. The term antibody molecule in this specification, for example, includes but is not limited to single-chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fv (sdFv), Fv, and complete antibodies or full-length antibodies. The term "single-chain Fv" or "scFv" refers to a polypeptide comprising the VL domain of an antibody connected to the VH domain of an antibody. For example, an antibody that immunospecifically binds to Napi2b may cross-react with other antigens. Preferably, the antibody that immunospecifically binds to Napi2b does not cross-react with other antigens. Immunospecific binding can be identified, for example, by immunoassays or other methods known to those skilled in the art. A "complete" antibody or "full-length" antibody refers to a protein comprising two heavy chains (H) and two light chains (L), the heavy chains and light chains being interconnected by disulfide bonds, the protein comprising: (1) in terms of the heavy chain, a variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains CH1, CH2, and CH3; and (2) in terms of the light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising one domain CL. The antibodies disclosed herein include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the disclosed antibodies), and epitope-binding fragments of any of the above antibodies. The immunoglobulin molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin. Preferably, the antibodies disclosed herein comprise or consist of a VH domain, VH CDR (herein often referred to as HCDR), VL domain, or VL CDR (herein often referred to as LCDR) having any of the amino acid sequences described in the Sequence and Specific Information Tables, or a fragment or variant thereof.

[0506] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. Monoclonal antibodies have high specificity for a single determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized without contamination by other antibodies. The modifier "monoclonal" herein indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by a specific method.

[0507] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0508] The term "antigen-binding fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region fragments, diabodies, linear antibodies and single-chain antibody molecules. The term "antigen-binding fragment" as used herein refers to a partial fragment of an antibody with antigen-binding activity, wherein the fragment has all or part of the function of an antibody, including but not limited to single-chain Fv (scFv), Fab, Fab', F(ab')2, disulfide-linked Fv (sdFv), Fv, di-scFv, etc. The term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, the term is not limited to these molecules, as long as the fragment has binding affinity to the antigen. In addition, these functional fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0509] The term "Fab'" as used herein refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, the Fab' disclosed herein also includes Fab' produced using genetically modified antibody genes.

[0510] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are linked by a linker or directly (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0511] As used herein, the antibody or antigen-binding fragment thereof still has the activity of binding to the antigen even if it contains variants, amino acid substitutions, deletions or additions.

[0512] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm. The conjugate retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0513] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0514] The term "intact antibody" or "full-length antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

[0515] The term "probody" refers to a modified antibody, including an antibody or an antibody fragment, that can specifically bind to its target and can be coupled to a masking group, wherein the masking group refers to a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, 1000 times, or 10,000 times greater than the cleavage constant for the binding ability of the antibody or antibody fragment to its target without the coupled masking group.

[0516] In the present disclosure, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also include residues that are not present in the recipient antibody or the donor antibody. These modifications are to further optimize the performance of the antibody. Humanized antibodies generally include at least one, typically two, variable regions in which all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, while the FRs are entirely or almost entirely human immunoglobulin sequences. The humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

[0517] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0518] In this article, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably identified by the Kabat, Chothia or, AbM or IMGT numbering systems.

[0519] As used herein, the term "framework residue region" or "FR residues" refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0520] As used herein, the term "germline antibody gene" refers to a gene encoding an immunoglobulin expressed by non-lymphocytes that has not undergone the genetic rearrangement and maturation process that results in the expression of a specific immunoglobulin. One advantage provided by various embodiments of the present disclosure stems from the recognition that the amino acid sequences encoded by germline antibody genes retain more of the important amino acid sequence structure characteristic of individual animal species than the amino acid sequences encoded by mature antibody genes. Therefore, when therapeutically applied to that species, the amino acid sequences are less likely to be recognized as foreign by that species.

[0521] As amino acid substitutions, in this specification, conservative amino acid substitutions are preferred. Conservative amino acid substitutions refer to substitutions that occur within a set of amino acids related to the amino acid side chain. Preferred amino acid sets are as follows: acidic set (aspartic acid and glutamic acid); basic set (lysine, arginine and histidine); non-polar set (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan); and uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine). More preferred amino acid sets are as follows: aliphatic hydroxyl (serine and threonine); amide-containing set (asparagine and glutamine); aliphatic set (alanine, valine, leucine and isoleucine); and aromatic set (phenylalanine, tryptophan and tyrosine). Such amino acid substitutions are preferably carried out within a set that does not impair the performance of the substance having the original amino acid sequence.

[0522] In addition, it is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells are deleted, and the proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody.

[0523] The compilation of the twenty conventional amino acids involved herein follows conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; and glutamine can be represented by Q or Gln.

[0524] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., tumor and infectious disease) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0525] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human. In certain embodiments, the subject (e.g., human) has a tumor and an infectious disease, or is at risk of having the above-mentioned disease.

[0526] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., tumors and infectious diseases) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., tumors and infectious diseases); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0527] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody, and varies with the antibody isotype.

[0528] The term "pharmaceutically acceptable" means that when the molecule itself, molecule fragment or composition is appropriately administered to an animal or human, it does not produce adverse, allergic or other untoward reactions. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0529] The term "antibody drug conjugate population" refers to a group or population of antibody-drug conjugates of the present invention, their stereoisomers, prodrugs, pharmaceutically acceptable salts, tautomers, or pharmaceutically acceptable solvates thereof, wherein the q of the antibody drug conjugates may be the same or different. Alternatively, this may be referred to as an "antibody drug conjugate mixture."

[0530] The in vivo therapeutic effects of antibodies and antibody-drug conjugates against cancer can be determined using experimental animals, for example, by administering the antibody to nude mice implanted with a tumor cell line expressing Napi2b and measuring any changes in the cancer cells.

[0531] Although amino acid substitutions in antibodies are generally made with L-amino acids in the present disclosure, this is not limiting. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0532] Monoclonal antibodies used in the present disclosure can be produced by many methods. For example, monoclonal antibodies for the present disclosure can be obtained by hybridoma methods using cells from many species (including mice, hamsters, rats and people) (see, for example, Kohler et al., 1975, Nature, 256:495), or obtained by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0533] Herein, unless otherwise expressly stated, the descriptions “each independently selected from” and “each independently selected from” used herein are interchangeable and should be understood in a broad sense, which may mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, or that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0534] Herein, the term "direct bond" means that the groups on both sides are directly connected. For example, the compound shown in formula II If X is a direct bond, then its structural formula is The remaining direct keys can be understood by referring to the above content.

[0535] Herein, the term "absence" means that the group does not exist. For example, the compound shown in formula II If W does not exist, the structural formula is

[0536] Compound represented by formula II In the equation, R1 and R2 form a carbon atom connected to them. The "dashed" bond indicates the position where the heterocycle is fused to the benzene ring, e.g., to form

[0537] Herein, the drug-linker conjugate shown in Formula III In the L4 When the numbers 1 and 2 represent the connection positions of L4 and the other groups, specifically, position 1 is connected to L3, position 2 is connected to the drug molecule, i.e. When connected, it forms The remaining numbers 1 and 2 can be understood by referring to the above content.

[0538] Herein, the compound shown in formula II In the formula, R3 and X together with the carbon atom to which they are attached form The dotted line indicates where the carbon ring is fused to the benzene ring and the pyridine ring to form

[0539] Herein, the definition of X is, for example, "X is selected from the group consisting of optionally substituted The substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl groups), and X can be, for example Other similar definitions of X can be understood with reference to the above content.

[0540] Herein, the definition of X is, for example, "X is selected from the group consisting of optionally substituted The substituent is selected from two C1-4 alkyl groups (such as methyl) and the carbon atoms to which they are simultaneously connected together to form a C3-6 cycloalkyl group (such as cyclopropyl). Then X can be, for example Other similar definitions of X can be understood with reference to the above content.

[0541] AA 1 The structures of the indicated amino acid residues If r is 0, it will be understood by those skilled in the art that AA 1 The structure of the indicated amino acid residues will become

[0542] AA 1 The structures of the indicated amino acid residues In, if R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 substituted, wherein the term "the 4-10 membered heterocyclic ring is optionally substituted by one or more R 0 "substituted" means that the 4-10 membered heterocyclic ring may be unsubstituted or may be replaced by one or more R 0 substituted, and the plurality of R 0 In each R 0 The definitions of can be the same or different. Other similar definitions can be understood with reference to the above content.

[0543] Herein, for example, when L3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala-Asn, “the distal amino group of lysine (Lys) is optionally substituted with 1, 2 or 3 substituents selected from tert-butyloxycarbonyl, C1-6 alkyl (preferably methyl), and O” means that the distal amino group of Lys in each option of L3 is optionally substituted with 1, 2 or 3 substituents selected from tert-butyloxycarbonyl, C1-6 alkyl (preferably methyl), and O. Wherein, “the distal amino group of Lys” refers to the lysine residue The exposed amino group -NH2 in the lysine (Lys) is optionally substituted by one, two or three substituents selected from tert-butyloxycarbonyl, C1-6 alkyl (preferably methyl), and O. The expression "the distal amino group of the lysine (Lys) is optionally substituted by one, two or three substituents selected from tert-butyloxycarbonyl, C1-6 alkyl (preferably methyl), and O" means that the distal amino group of the lysine (Lys) may be unsubstituted or substituted by one, two or three substituents selected from tert-butyloxycarbonyl, C1-6 alkyl (preferably methyl), and O. For example, it may be substituted by one tert-butyloxycarbonyl, i.e., it becomes Alternatively, it can be substituted by two methyl groups, i.e. Or it can be replaced by 2 methyl groups and 1 O at the same time, which becomes It should be noted that "the distal amino group of the lysine (Lys) is substituted by O" means that the distal amino group of the lysine (Lys) is oxidized, i.e., becomes If the distal amino group is further substituted by two methyl groups, it becomes

[0544] Throughout this specification, substituents of the disclosed compounds are disclosed by group class or range. It is specifically noted that the disclosure includes every independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0545] As used herein, the term "C1-6 alkyl" refers to a linear or branched alkyl group containing 1 to 6 carbon atoms, including, for example, "C1-3 alkyl" or "C1-4 alkyl", methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0546] In this article, the term “C 1-4 "Alkyl" means a straight or branched chain alkyl group containing 1 to 4 carbon atoms, including for example "C 1-3Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0547] In this article, the term “C 2-6 "Alkenyl" refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0548] In this article, the term “C 2-6 "Alkynyl" refers to a straight or branched chain alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0549] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0550] In this context, the term "3-6 membered cycloalkyl" or "C 3-6 The term "cycloalkyl" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms, including cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexyl.

[0551] As used herein, the term "3-7 membered carbocycloalkyl" or "C 3-7 "Cycloalkyl" refers to a saturated cyclic alkyl group containing 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0552] As used herein, the term "C1-6 alkoxy" refers to an alkyl group as defined above that is attached to the parent molecular moiety via an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0553] In this article, the term “C 1-4"Alkoxy" refers to an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0554] In this article, the term "4-10 membered heterocyclic radical" refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "4-6 membered heterocyclic radical" refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed. "4-8 membered heterocyclic radical" includes, for example, "4-8 membered nitrogen-containing heterocyclic radical", "4-8 membered oxygen-containing heterocyclic radical", "4-7 membered heterocyclic radical", "4-7 membered oxygen-containing heterocyclic radical", "4-7 membered heterocyclic radical", "4-6 membered heterocyclic radical", "5-7 membered heterocyclic radical", "5-6 membered heterocyclic radical", "5-6 membered nitrogen-containing heterocyclic radical", including but not limited to oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.

[0555] As used herein, the term "4-10 membered heterocycle" refers to a ring containing 4-10 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom). The term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran and the like.

[0556] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromatic properties, such as a 6-10 membered aryl group, a 5-8 membered aryl group, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl. The "6-10 membered aryl" refers to an aryl group containing 6-10 ring atoms. The "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0557] In this article, term " heteroaryl " refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulphur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atom or sulphur atom) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-6 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3, 4-oxadiazolyl, pyridinyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0558] Bonds in structural formulae represented herein by a wavy line "~~" are intended to indicate that the structure represents either a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

[0559] The term "drug to antibody ratio" or "DAR" refers to the ratio of drug to antibody in a population (or mixture) or composition or ADC molecule, for example, a small molecule toxin attached to the antibody of an ADC. The DAR of an ADC can range from 1 to 16, but higher loadings (e.g., 20) are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. It should be understood that the latter is generally referred to as the average DAR. In the process of determining the DAR value by mass spectrometry, the antibody has been reduced to separated heavy and light chains, and DAR1 represents a conjugate comprising a light chain or heavy chain coupled to 1 toxin molecule; DAR2 represents a conjugate comprising a light chain or heavy chain coupled to 2 toxin molecules; and DAR3 represents a conjugate comprising a light chain or heavy chain coupled to 3 toxin molecules.

[0560] Advantageous Effects of the Invention

[0561] This disclosure, through extensive research on multiple target antibody-drug conjugates (ADCs), achieves enrichment of the tumor microenvironment, unique in vivo enzymatic cleavage properties of the linker, and coupling methods with the targeting moiety, combined with extensive in vivo and in vitro efficacy screening and verification, to obtain a novel class of antibody-based bioactive molecule conjugates. The conjugates obtained using this method can achieve a variety of surprising technical effects as follows:

[0562] The conjugate obtained by the above method has better solubility and excellent chemical stability. For example, the reversible Michael addition reaction caused by the maleimide linkage in traditional ADCs does not occur, so a high drug-antibody ratio can be obtained. In some embodiments, the DAR value of the conjugate can reach 6-8.

[0563] Having extremely high coupling efficiency. In some embodiments, the coupling efficiency can reach or exceed 90%;

[0564] Through extensive research, a class of linkers has been discovered that have high plasma stability but can be cleaved in the tumor microenvironment (both inside and outside tumor cells). Therefore, they can produce good anti-tumor effects in tumors with low or no antigen expression.

[0565] The conjugate (ADC) obtained by the above method improves the exposure of the entire ADC molecule to the relatively acidic tumor environment by adjusting the physicochemical properties of the linker and the entire ADC molecule. As a result, the ADC has better tumor tissue targeting, that is, the ability to be enriched in the tumor microenvironment, increases the ratio of the bioactive molecule concentration in the tumor to that in the blood, and reduces the mechanism-related toxicity of the ADC molecule (toxicity caused by the ADC binding to cell surface antigens in non-tumor tissues and internalization, also known as "on-target toxicity"), thereby having a higher therapeutic index;

[0566] The conjugate obtained according to the above method has high stability in the body circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of toxins in non-target tissues;

[0567] The bioactive molecule of the conjugate has higher anti-tumor cell activity and thus has an excellent bystander effect. The ADC can more effectively kill tumor cells with high antigen expression as well as tumor cells with low or no antigen expression in tumor tissues.

[0568] The toxin-linker disclosed herein can be combined with antibodies without endocytosis ability to form antibody-drug conjugates, utilizing its extracellular cleavage ability in the tumor microenvironment. Such antibody-drug conjugates still have high anti-tumor activity.

[0569] The toxin-linker disclosed herein, utilizing its extracellular cleavage ability and enrichment ability in the tumor microenvironment, can be combined with antibodies that lack endocytosis ability and antibodies that lack tumor extracellular antigen binding ability to form antibody-drug conjugates. Such antibody-drug conjugates still have high anti-tumor activity.

[0570] The anti-Napi2b antibodies provided by the present disclosure have a very high degree of humanization or are fully human antibodies, and thus can be safely administered to human subjects without triggering an immunogenic response.

[0571] In summary, the ADC of the present invention has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0572] Figure 1. Flow cytometry affinity evaluation of humanized anti-Napi2b antibodies

[0573] Figure 2. Endocytic activity detection of anti-Napi2b humanized antibody

[0574] Figure 3A. Affinity detection of 66C12D12-hz1 antibody in serum

[0575] Figure 3B. Affinity detection of 16G5B3-hz2 antibody in serum

[0576] Figure 4. In vivo efficacy testing of anti-human Napi2b ADC in the HT29-h.Napi2b CDX model

[0577] Figure 5. Changes in mouse body weight during administration of anti-human Napi2b ADC in the HT29-h.Napi2b CDX model

[0578] Figure 6. In vivo efficacy testing of anti-human Napi2b ADC in the IGR-OV1CDX model

[0579] Figure 7. Changes in mouse body weight during administration of anti-human Napi2b ADC in the IGR-OV1CDX model DETAILED DESCRIPTION

[0580] The present disclosure will be further described below by way of specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0581] The abbreviations in this disclosure have the following meanings:

[0582] Sequence and its specific information:

[0583] The present disclosure will now be described with reference to the following examples which are intended to illustrate the present disclosure rather than to limit it.

[0584] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present disclosure are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the present disclosure by way of example and are not intended to limit the scope of protection claimed in the present disclosure.

[0585] Preparation plan

[0586] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0587] Nuclear magnetic resonance (NMR) 1 H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0588] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0589] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0590] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0591] 1. Synthesis of Bioactive Molecules and Intermediates Used in the Synthesis of Drug-Linker Compounds

[0592] A. Synthesis of Bioactive Molecules

[0593] Example A1.1: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1)

[0594] Step 1: 4-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (4.6 g) was dissolved in anhydrous tetrahydrofuran (50 ml). The solution was cooled to -78°C with dry ice-acetone under nitrogen protection. Then, n-butyl lithium (12 ml, 2 M) was added dropwise. The reaction solution was stirred at -78°C for 20 minutes, and then methyl 2-amino-4-fluoro-5-methylbenzoate (1.83 g) was added. After the addition was complete, the reaction mixture was naturally warmed to room temperature and stirred for 5 hours. The reaction mixture was quenched with methanol (3 ml) and ethyl acetate (200 ml) was added. The solution was washed with water (100 ml x 3). The organic phase was dried and the organic solvent was removed. The target product (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone was separated by silica gel column chromatography. ESI-MS (m / z): 304 [M+H] + .

[0595] Step 2: (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2.63 g), (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone (3.03 g), and p-toluenesulfonic acid (1.74 g) were dissolved in dichloromethane (50 ml), and the solvent was removed. The mixture was heated to 120°C under nitrogen protection for 4 hours. The mixture was dissolved in ethyl acetate (300 ml), the organic phase was washed with water (100 ml x 2), dried, and the organic solvent removed. The residue was separated by silica gel column chromatography to obtain the target product (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.1). ESI-MS (m / z): 447 [M+H] + .

[0596] Example A1.2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]dioxo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2)

[0597] Using the same method and reaction conditions as in Example A1.1, replacing methyl 2-amino-4-fluoro-5-methylbenzoate with methyl 6-aminobenzo[d][1,3]dioxolane-5-carboxylate, the target product (S)-7-ethyl-7-hydroxy-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]dioxo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.2) was obtained. ESI-MS (m / z): 459 [M+H] + .

[0598] Example A1.3: Synthesis of (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3)

[0599] Compound (A1.3-A) (1.4 g), compound (A1.3-B) (2.2 g), Pd2(DBA)3 (300 mg), tricyclohexylphosphine (300 mg), and potassium acetate (1.1 g) were added sequentially to a mixed solvent of dioxane (30 ml) and water (5 ml). The mixture was heated and stirred at 100°C under nitrogen for 12 hours. After cooling, ethyl acetate (200 ml) was added, the mixture was washed with water (100 ml), dried, and separated by silica gel column chromatography to obtain the target product, (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.3). ESI-MS (m / z): 484 [M+H] + .

[0600] Example A1.4: Synthesis of (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.4)

[0601] Compound (A1.4-A) (1.4 g), compound (A1.4-B) (2.2 g), Pd2(DBA)3 (300 mg), tricyclohexylphosphine (300 mg), and potassium acetate (1.1 g) were added sequentially to a mixed solvent of dioxane (30 ml) and water (5 ml). The mixture was heated and stirred at 100°C under nitrogen for 12 hours. After cooling, ethyl acetate (200 ml) was added, the mixture was washed with water (100 ml), dried, and separated by silica gel column chromatography to obtain the target product, (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.4).

[0602] ESI-MS (m / z): 484 [M+H] + ;

[0603] 1H NMR (400MHz, DMSO) δ7.56(s,1H),7.34(d,J=8.0Hz,2H),7.27(s,1H),7.14(s,1H),6.89(d,J= 7.8Hz,2H),6.26(s,2H),5.40(s,2H),5.05(s,2H),1.96–1.78(m,2H),0.88(t,J=7.2Hz,3H).

[0604] Example A1.5: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.5)

[0605] Step 1: Synthesis of (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0606] To a 75% sulfuric acid solution (5 mL) of compound (S) 7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indeno[1,2-b]quinoline-8,11(7H)-dione (A1.5-A, 500 mg) was added ferrous sulfate heptahydrate (570 mg dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) under ice-cooling conditions. The reaction mixture was stirred for three minutes, followed by the dropwise addition of hydrogen peroxide (29%, 2.5 mL). The reaction mixture was stirred at 0°C for 5 minutes, then warmed to room temperature and stirred for 3 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (80 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5%-60%) to give the title compound (yellow solid, 400 mg, yield: 67%).

[0607] LCMS (ESI) [M+H] + :468.9;

[0608] 1H NMR(400MHz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.42(s, 2H), 5.26 (s, 2H), 3.81 (d, J = 5.9Hz, 2H), 3.22 (s, 2H), 1.98 (d, J = 6.7Hz, 4H), 0.88 (t, J = 7.2Hz, 3H).

[0609] Step 2: Synthesis of (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0610] To a solution (3 mL) of compound (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (200 mg) in N,N-dimethylformamide was added sodium azide (284 mg), and the reaction mixture was stirred at 100°C for 1 hour. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (60 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the target compound (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (yellow solid, 180 mg, yield: 88%).

[0611] LCMS (ESI) [M+H] + :476;

[0612] 1 H NMR(400MHz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.48(s,1H),6.29(s,2H),5.42(s, 2H), 5.25 (s, 2H), 3.53–3.49 (m, 2H), 3.16–3.12 (m, 2H), 1.93–1.80 (m, 4H), 0.88 (t, J = 7.2Hz, 3H).

[0613] Step 3: Synthesis of (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione

[0614] To a mixture of (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (130 mg, 0.274 mmol) in tetrahydrofuran (3 mL) and water (1 mL) was added triphenylphosphine (108 mg, 0.411 mmol) and the mixture was allowed to react at 55°C for 16 hours. LCMS indicated the reaction was complete. Water (5 mL) was added to the reaction mixture, which was acidified with 2N hydrochloric acid (3 mL) and extracted with ethyl acetate (10 mL). The aqueous phase was purified by HPLC (acetonitrile / 0.05% formic acid in water) to give the target compound (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.5, yellow solid, 15 mg, yield: 12%).

[0615] LCMS (ESI) [M+H] + :449.9;

[0616] 1 H NMR(400MHz,DMSO-d6)δ7.72(s,3H),7.53(s,1H),7.25(s,1H),6.50(s,1H),6.30(s,2H),5.43(s,2H) ,5.24(s,2H),3.15(d,J=6.4Hz,2H),3.03(t,J=6.9Hz,2H),1.96–1.81(m,4H),0.88(t,J=7.3Hz,3H).

[0617] Example A1.6: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

[0618] Step 1: Preparation of (S)-2-(ethyl(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)amino)-2-oxoethyl acetate (A1.6-B):

[0619] Compound (S)-7-ethyl-14-(2-(ethylamino)ethyl)-7-hydroxy-10,13-dihydro-11H-[1,3]dioxetine[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-8,11(7H)-dione (A1.6-A, 50 mg), acetoxyacetyl chloride (73 mg), and triethylamine (50 mg) were added sequentially to dichloromethane (5 mL) and stirred at room temperature for 1 hour. The reaction solution was evaporated to remove the solvent to obtain 75 mg of the crude oily compound.

[0620] LCMS (ESI) [M+H] + :564.2.

[0621] Step 2: Synthesis of (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6)

[0622] The crude compound (S)-2-(ethyl(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-14-yl)ethyl)amino)-2-oxoethyl acetate (75 mg, 0.13 mmol) was dissolved in a mixed solution of concentrated hydrochloric acid and anhydrous ethanol (1 / 2 by volume, 3 mL), and the reaction solution was refluxed at 85°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative chromatography (0.01% trifluoroacetic acid in water, acetonitrile) to give the target compound (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6) (15 mg, yield: 26%) as a white solid.

[0623] LCMS (ESI) [M+H] + :522.0;

[0624] 1 H NMR(400MHz,DMSO)δ7.92(s,1H),7.53(m,1H),7.25(s,1H),6.50(s,1H),6.31(s,2 H),5.43(s,2H),5.34(m,2H),4.65(m,1H),4.07(m,2H),3.52(m,2H),3.41(m,2H), 2.00(m,2H),1.88(m,2H),1.16–1.04(m,3H),0.89–0.83(m,3H).

[0625] Example A1.7: Synthesis of (S)-N-methyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.7)

[0626] Step 1: Preparation of compound methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxetyl-5-yl)-3-oxopropyl) carbamate

[0627] Compound 1-(6-nitrobenzo[d][1,3]dioxin-5-yl)ethan-1-one (A1.7-A, 500 mg), methylamine hydrochloride (1.6 g), and paraformaldehyde (714 mg) were dissolved in ethanol (8 mL) and reacted in a sealed container at 100°C for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (80 mL), and the organic phase was extracted with water (50 mL x 3). The resulting aqueous phase was adjusted to pH 9 with sodium bicarbonate, followed by the addition of benzyl chloroformate (513 mg, 3.0 mmol), and the reaction was continued at room temperature for 16 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The crude product was separated and purified by C18 (acetonitrile / water containing 0.05% formic acid = 5-95%) to give the target compound (A1.7-B, 180 mg, yield 23%).

[0628] LCMS (ESI) [M+H] + :387.1.

[0629] 1H NMR (400MHz, CDCl3) δ7.56(s,1H),7.35(m,5H),7.32(m,1H),6.17(s,2H),5.13(s,2H),3.71(m,2H),3.03(m,3H),2.99–2.86(m,2H).

[0630] Step 2: Preparation of compound benzyl (3-(6-aminobenzo [d] [1,3] dioxin-5-yl) -3-oxypropyl) (methyl) carbamate (A1.7-C)

[0631] Compound A1.7-B, methylbenzyl(3-(6-nitrobenz[d][1,3]dioxetyl-5-yl)-3-oxopropyl)carbamate (180 mg, 0.47 mmol) was dissolved in a mixture of saturated aqueous ammonium chloride (8 mL) and ethanol (8 mL). Iron powder (130 mg) was then added to the reaction mixture, and the reaction was stirred at 80°C for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by TLC (petroleum ether:ethyl acetate = 2 / 1) to obtain the target compound A1.7-C (76 mg).

[0632] LCMS (ESI) [M+H] + :357.0.

[0633] Step 3: Preparation of benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxetane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(methyl)carbamate (A1.7-D)

[0634] At room temperature, compound A1.7-C benzyl(3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(methyl)carbamate (38 mg), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (29 mg), and p-toluenesulfonic acid (23 mg) were dissolved in dichloromethane (5 mL). The solution was clarified and mixed, then concentrated under reduced pressure and evacuated to a vacuum using an oil pump. The reaction was incubated at 120°C under vacuum for 2 hours. The reaction solution was cooled to room temperature, water (30 mL) was added, and the mixture was extracted with dichloromethane (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound A1.7-D (50 mg).

[0635] LCMS (ESI) [M+H] +=584.0.

[0636] Step 4: Preparation of (S)7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxetine[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-8,11(7H)-dione (A1.7-E)

[0637] Compound A1.7-D benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxetane[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-14-yl)ethyl)(methyl)carbamate (50 mg) was dissolved in dichloromethane (5 mL) at room temperature. Trimethylsilyl iodide (51 mg, 0.26 mmol) was added at 0°C and reacted for 3 hours. The reaction solution was concentrated to remove the solvent and purified by HPLC (acetonitrile / water containing 0.05% formic acid) to give compound A1.7-E (15 mg) as a brown solid.

[0638] LCMS (ESI) [M+H] + :450.0.

[0639] Step 5: Preparation of compound (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-14-yl)ethyl)(methyl)amino)-2-oxoethyl acetate (A1.7-F)

[0640] Compound A1.7-E(S)7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxetine[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (15 mg) and triethylamine (15 mg) were dissolved in dichloromethane (5 mL). Acetoxyacetyl chloride (20 mg) was added and reacted at 0°C for 1 hour. The reaction solution was concentrated to remove the solvent to obtain a crude product. The resulting solid was used directly in the next step without further purification.

[0641] LCMS (ESI) [M5+H] + =550.1.

[0642] Step 6: Preparation of compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxetane[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-methylacetamide (A1.7)

[0643] Compound A1.7-F(S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(methyl)amino)-2-oxoethyl acetate (15 mg) was dissolved in ethanol (5 mL), concentrated hydrochloric acid (1.5 mL) was added, and the mixture was reacted at 80°C for 2 hours. The reaction solution was concentrated to remove the solvent and purified by high performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) to give compound A1.7 (1.6 mg) as a white solid.

[0644] LCMS (ESI) [M+H] + :508.2.

[0645] Example A1.8: Synthesis of (S)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.8)

[0646] Step 1: Preparation of isopropylbenzyl (3-(6-nitrobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)carbamate (A1.8-B)

[0647] To a solution of compound (A1.8-A) 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (900 mg) in dichloromethane (10 mL) were added triethylamine (1.8 g) and benzyloxycarbonyl chloride (734 mg, 4.3 mmol) in sequence. The mixture was allowed to react at room temperature for 2 hours. The reaction solution was diluted with water (50 mL) and extracted with dichloromethane (50 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 3 / 1) to obtain the target compound (A1.8-B) (600 mg).

[0648] LCMS (ESI) [M+H] + :414.9;

[0649] 1 H NMR(400MHz,DMSO-d6)δ7.72(s,1H),7.34(br s,6H),6.31(s,2H),5.08(s,2H),4.14–4.10(m,1H),3.48(d,J=7.9Hz,2H),3.03(s,2H),1.13-1.11(m,6H).

[0650] Step 2: Preparation of compound benzyl (3-(6-aminobenzo [d] [1,3] dioxin-5-yl) -3-oxypropyl) (isopropyl) carbamate (A1.8-C)

[0651] Compound (A1.8-B) isopropylbenzyl (3-(6-nitrobenz[d][1,3]dioxin-5-yl)-3-oxopropyl)carbamate (200 mg, 0.48 mmol) was dissolved in a mixture of saturated ammonium chloride (3 mL) and ethanol (3 mL). Iron powder (135 mg) was then added to the reaction mixture, and the reaction was stirred at room temperature for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 3 / 1) to obtain the target compound (A1.8-C) (65 mg).

[0652] LCMS (ESI) [M+H] + :395.2.

[0653] Step 3: Preparation of (S)7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxetine[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-8,11(7H)-dione (A1.8-D)

[0654] At room temperature, compound (A1.8-C) benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(isopropyl) carbamate (65 mg), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (45 mg) and p-toluenesulfonic acid (33 mg, 0.17 mmol) were dissolved in dichloromethane (10 mL) solution. After the solution was clarified and mixed, it was concentrated under reduced pressure and evacuated to vacuum with an oil pump. The reaction was carried out at 120°C under vacuum for 2 hours. The reaction solution was cooled to room temperature, and water (50 mL) was added. The mixture was extracted with dichloromethane (30 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane / methanol = 10 / 1) to obtain the target compound (A1.8-D) (40 mg).

[0655] LCMS (ESI) [M+H] + :478.0;

[0656] 1 H NMR(400MHz,DMSO-d6)δ8.46(s,1H),7.67(s,1H),7.57(s,1H),7.27(s,1H),6.52(s,1H),6.33(s,2H),5.44(s,2H),5.35(s,2H),3.41(br s,2H),3.23(br s,3H),1.94–1.78(m,2H),1.25(d,J=6.4Hz,6H),0.88(t,J=7.3Hz,3H).

[0657] Step 4: Preparation of compound ((S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-14-yl)ethyl)(isopropyl)amino)-2-oxoacetic acid ethyl ester (A1.8-E)

[0658] To a dichloromethane solution (2 mL) of (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.8-D) (40 mg) was added triethylamine (35 mg) and 2-chloro-2-oxoethyl acetate (57 mg) under ice-cooling. The mixture was reacted at zero degrees Celsius for 30 minutes. The reaction mixture was concentrated under reduced pressure to afford compound A1.8-E, which was used directly in the next step.

[0659] LCMS (ESI) [M+H] + :578.0.

[0660] Step 5: Preparation of compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxo[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-isopropylacetamide (A1.8)

[0661] To a solution (3 mL) of compound (S)-ethyl 2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-14-yl)ethyl)(isopropyl)amino)-2-oxoacetate (A1.8-E, 50 mg) in ethanol was added concentrated hydrochloric acid (0.5 mL), and the mixture was reacted at 70°C for 1 hour. The reaction solution was concentrated to obtain a crude product. The product was then purified by HPLC (acetonitrile / 0.05% formic acid in water) to obtain the title compound (A1.8, 3 mg).

[0662] LCMS (ESI) [M+H] + :464.0;

[0663] 1 H NMR(400MHz,DMSO-d6)δ7.99(s,1H),7.53(s,1H),7.26(s,1H),6.31(s,2H),5.43(s,2H),5.36(s,2H),4.22(s,2H),3.99 –3.94(m,1H),3.44(dd,J=16.7,7.8Hz,2H),3.33–3.21(m,2H),1.95–1.79(m,2H),1.19(dd,J=16.4,5.8Hz,6H),0.88(t, J=7.2Hz,3H).

[0664] Example A1.9: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9)

[0665] Compound (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) and reacted at 110°C for 48 hours. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL x 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Purification by HPLC preparative method (acetonitrile / water containing 0.05% formic acid) gave (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.9, 1.78 mg).

[0666] LCMS (ESI) [M+H] + :451.0;

[0667] 1 H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.47–5.37( m,2H),5.32–5.19(m,2H),3.51–3.46(m,2H),3.17–3.13(m,2H),1.92–1.76(m,4H),0.90–0.84(m,3H).

[0668] Example A1.10: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.10)

[0669] Step 1:

[0670] To a solution of compound A1.10-A (10 g) in 1,2-dichloroethane (200 mL) at 0°C, 1 mol / L boron trichloride (96 mL) and 4-chlorobutyronitrile (9.9 g) were added dropwise. The mixture was stirred at 80°C for 2 hours. The reaction mixture was cooled to room temperature, 2 mol / L hydrochloric acid (90 mL) was added, and the mixture was refluxed at 80°C with stirring for 0.5 hours. The reaction mixture was cooled to room temperature, diluted with a small amount of water, and extracted with dichloromethane (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A1.10-B (4 g).

[0671] LCMS (ESI) [M+H] + :230.0.

[0672] Step 2:

[0673] To compound A1.10-B (50 mg) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (35 mg) and p-toluenesulfonic acid monohydrate (41.4 mg). The mixture was dissolved in dichloromethane (30 mL). The solution was clarified and mixed, then concentrated under reduced pressure and evacuated to a vacuum using an oil pump. The reaction was incubated at 120°C under vacuum for 3 hours. LCMS indicated completion of the reaction. The reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with dichloromethane (20 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography (dichloromethane to methanol = 20:1) to yield the target compound A1.10-C (80 mg) as a white solid.

[0674] LCMS (ESI) [M+H] + :457.0.

[0675] Step 3:

[0676] Compound A1.10-C (75 mg) was dissolved in hexamethylphosphoric triamide, and pure water (0.8 mL) was added. The reaction mixture was stirred at 100°C for 72 hours. LCMS confirmed the reaction was complete. Preparative chromatography (0.01% TFA in water, MeCN) gave the title compound (10 mg).

[0677] LCMS (ESI) [M+H] + :439.2;

[0678] 1H NMR (400MHz, DMSO-d6) δ8.22(d,J=8.4Hz,1H),7.87(d,J=10.9Hz,1H),7.31(s,1H),6.50(s,1H),5.43(s,2H),5.30(s,2 H),4.67(t,J=4.9Hz,1H),3.55–3.47(m,2H),3.28–3.20(m,2H),2.51(s,3H),1.93–1.81(m,4H),0.88(t,J=7.3Hz,3H).

[0679] Example A1.11: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-aminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.11)

[0680] Step 1:

[0681] Sodium azide (432 mg) was added to a solution of A1.10-C (395 mg) in N'N-dimethylformamide (10 mL). The reaction mixture was reacted at 80°C for 16 hours, then cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (80 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by suction to obtain the target product A1.11-A (290 mg).

[0682] LCMS (ESI) [M+H] + :464.0.

[0683] Step 2:

[0684] Compound A1.11-A (93 mg) was dissolved in tetrahydrofuran (5 mL), and triphenylphosphine (78 mg) was added. The mixture was allowed to react at room temperature for 4 hours. Hydrochloric acid (4 M, 1 mL) was then added to the reaction solution, and the temperature was raised to 55°C for 16 hours. The reaction was monitored for completion by LCMS. The reaction solution was directly concentrated, and the crude product was purified by reverse-phase column chromatography (mobile phase A: 0.05% formic acid in water, mobile phase B: acetonitrile) to yield the desired product A1.11 (28 mg, yield: 36%) as a white solid.

[0685] LCMS (ESI) [M+H] + :438.4;

[0686] 1H NMR (400MHz, CD3OD) δ8.52(s,1H),8.16(d,J=7.8Hz,1H),7.76(d,J=10.7Hz,1H),7.63(s,1H),5.49(ABq,J=78.9,16.3Hz,2H),5.31(br s,2H),3.35–3.32(m,2H),3.22–3.10(m,2H),2.55(s,3H),2.13–2.11(m,2H),1.96–1.94(m,2H),1.01(t,J=7.4Hz,3H).

[0687] Example A1.12: Synthesis of (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12)

[0688] Step 1:

[0689] Compound A1.12-A (200 mg, 0.39 mmol), compound A1.12-B (112 mg, 0.39 mmol), cesium fluoride (152 mg, 0.975 mmol), and tetrakistriphenylphosphine palladium (45 mg, 0.039 mmol) were added to a 1,4-dioxane solution (8 mL). Microwave reaction was carried out at 120°C under a nitrogen atmosphere for 0.5 h. LCMS indicated the reaction was complete. A mixture of dichloromethane (20 mL) and methanol (10 mL) was added to the reaction solution, which was diluted and filtered. The filtrate was concentrated, and the crude product was purified by preparative TLC (dichloromethane:methanol = 30:1) to obtain the target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl acetate (A1.12-C, 60 mg, yield: 26%) as a brown solid. LCMS (ESI) [M+H] + =590.3;

[0690] 1H NMR (400MHz, DMSO-d6) δ7.63(s,1H),7.49(s,1H),7.35(s,1H),7.09(d,J=16.7Hz,1H),6.93(s, 1H),6.45(d,J=16.6Hz,1H),6.30(s,2H),5.47(s,2H),5.34–5.24(m,2H),3.94(s,2H),2.21(br s,3H),2.03–1.96(m,2H),1.45(s,9H),0.91(t,J=6.7Hz,3H).

[0691] Step 2:

[0692] To a methanol solution (15 mL) of (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl acetate (A1.12-C, 50 mg, 0.085 mmol) was added sodium methoxide (9.2 mg, 0.17 mmol), and the mixture was stirred at 50° C. for 2 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated to give the target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12-D, 50 mg) as a crude brown solid. LCMS (ESI) [M+H] + =548;

[0693] Step 3:

[0694] To a solution of (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12-D, 50 mg, 0.091 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (1 mL), and the reaction was stirred at room temperature for 30 minutes. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by HPLC (acetonitrile / 0.05% formic acid in water) to give the target compound (S,E)-14-(3-amino-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.12) (8.1 mg, yield 21%) as a brown solid.

[0695] LCMS (ESI) [M+H] + =448.3;

[0696] 1 H NMR (400MHz, DMSO-d6) δ8.20(s,2H),7.72(s,1H),7.54(s,1H),7.40(d,J=16.5Hz,1 H),7.27(s,1H),6.52–6.46(m,2H),6.31(s,2H),5.42(s,2H),5.27(s,2H),3.86(br s,2H),1.90–1.83(m,2H),0.88(t,J=7.1Hz,3H).

[0697] Example A1.13: Synthesis of (S,E)-14-(3-hydroxy-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13)

[0698] Step 1:

[0699] Compound A1.9 (200 mg, 0.444 mmol) was dissolved in dimethyl sulfoxide (2 mL), and IBX (311 mg, 1.11 mmol) was added. The mixture was stirred at room temperature for 2 hours. Then, IBX (186 mg, 0.666 mmol) was added to the reaction solution. Tetrahydropyrrole (6.3 mg, 0.089 mmol) and acetonitrile (3 mL) were added to the reaction solution, and the reaction solution was stirred at room temperature overnight. The reaction was complete by LCMS, and the product was extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried to give a crude product. The crude product was purified on a silica gel column (DCM:MeOH = 50:1 to 10:1) to obtain the target compound (S,E)-14-(3-oxo-1-propen-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.13-A, 100 mg, purity 50.0%, yield 25.0%) as a yellow solid. LCMS (ESI) [M+H] + =447.1.

[0700] Step 2:

[0701] Compound A1.13-A (40 mg, 0.09 mmol) was dissolved in tetrahydrofuran (1 mL). Sodium cyanoborohydride (28 mg, 0.448 mmol) was then added to the reaction mixture and stirred overnight at room temperature. LCMS indicated the reaction was complete. The reaction mixture was concentrated to obtain a crude product, which was then purified by prep-HPLC (HCl in water / MeCN) to yield the title compound (3.56 mg, 93.6% purity, 9.0% yield) as a pale yellow solid.

[0702] LCMS (ESI) [M+H] + =449.2;

[0703] 1H NMR(400MHz,DMSO-d6)δ7.61(s,1H),7.50(s,1H),7.24(s,1H),7.20(d,J=16.4Hz,1 H),6.69–6.59(m,1H),6.49(s,1H),6.30(s,2H),5.42(s,2H),5.26(s,2H),5.16(br s,1H),4.34(br s,2H),1.93–1.81(m,2H),0.88(t,J=7.3Hz,3H).

[0704] Example A1.14: Synthesis of (S,E)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14)

[0705] Step 1:

[0706] To a 25 mL single-necked bottle was added compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.10, 100 mg, 0.228 mmol), DMSO (0.5 mL) and acetonitrile (0.75 mL), followed by IBX (160 mg, 0.571 mmol) and (R)-diphenyl(pyrrolidin-2-yl)methanol (12 mg, 0.047 mmol), and the reaction was continued with stirring at room temperature for 16 hours. A mixed solvent of methanol and dichloromethane (200 mL) (DCM:MeOH=10:1) was added to the reaction solution, and the mixture was extracted with saturated brine (300 mL). The organic phases were combined, washed with water, dried, and filtered. The filtrate was evaporated under reduced pressure to dryness to obtain a crude product, which was purified by flash chromatography (DCM:MeOH=10:1) to obtain the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-oxopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.14-A, 40 mg, yield 40%).

[0707] LCMS (ESI) [M+H] + =435.1.

[0708] Step 2:

[0709] To a 50 mL three-necked flask, add compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-oxopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.14-A, 40 mg, 0.228 mmol) and anhydrous THF (5 mL). The temperature was lowered to -78 °C under N2 protection, and a THF solution of lithium tri-sec-butylborohydride (0.11 mL, 1N) was slowly added dropwise. The temperature was maintained at -78 °C and stirring was continued for 1 hour. After completion of the reaction, saturated aqueous ammonium chloride solution was added to quench the reaction. The temperature was raised to room temperature, diluted with saturated brine (50 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with water, dried, and filtered. The solvent was evaporated under reduced pressure to give (S,E)-4-ethyl-8-fluoro-4-hydroxy-11-(3-hydroxy-1-propen-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.14, 28 mg) as an off-white solid.

[0710] LCMS (ESI) [M+H] + =437.1.

[0711] Example A1.15: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-(n-propylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1.15a) and (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N-propylacetamide (A1.15b)

[0712] Step 1:

[0713] Compound A1.15-A (5.0 g, 23.9 mmol), propylamine hydrochloride (22.8 g, 239 mmol), and paraformaldehyde (7.18 g, 239 mmol) were dissolved in ethanol (100 mL) and allowed to react at 110°C in a sealed canister for 12 hours. LCMS confirmed the reaction was complete. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Dichloromethane (100 mL) was added to the solid, which was then washed with water (80 mL x 3). The aqueous phase was adjusted to pH 9 with sodium bicarbonate. Benzyloxycarbonyl chloride (3.7 g, 21.8 mmol) was added to the aqueous solution and allowed to react at room temperature for 2 hours. LCMS confirmed the reaction was complete. The reaction mixture was extracted with dichloromethane (100 mL x 3), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A1.15-B (1.5 g, yield 20.1%) as a yellow oily liquid.

[0714] LCMS (ESI) [M+H] + =415.1;

[0715] 1H NMR(400MHz, CDCl3)δ7.56(s,1H),7.35(m,6H),6.18(s,2H),5.17–5.12(m,2H),3 .69–3.67(m,2H),3.32–3.30(m,2H),3.09–2.90(m,2H),1.59(m,2H),0.90(m,3H).

[0716] Step 2:

[0717] Compound A1.15-B (1.5 g, 3.63 mmol) was dissolved in a mixture of saturated ammonium chloride (20 mL) and ethanol (20 mL). Iron powder (1.02 g, 18.1 mmol) was then added to the reaction mixture, and the reaction was stirred at 80°C for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was purified by reverse phase separation on a C18 column (acetonitrile / 0.05% FA in water: 5% to 55%) to obtain the target compound A1.15-C (600 mg, 43.0% yield) as a yellow solid.

[0718] LCMS (ESI) [M+H] + =385.2,t R =1.329min.

[0719] Step 3:

[0720] At room temperature, compound A1.15-C (350 mg, 0.91 mmol), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (A1.15-D, 218 mg, 0.83 mmol), and p-toluenesulfonic acid (170 mg, 0.87 mmol) were dissolved in dichloromethane (5 mL). The solution was clarified and mixed, then concentrated under reduced pressure and evacuated to a vacuum using an oil pump. The reaction was incubated at 120°C under vacuum for 2 hours. LCMS indicated completion of the reaction. The reaction mixture was cooled to room temperature, water (50 mL) was added, and the mixture was extracted with dichloromethane (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford compound A1.15-E (390 mg, 76% yield) as a brown solid.

[0721] LCMS (ESI) [M+H] + =612.0.

[0722] Step 4:

[0723] Compound A1.15-E (390 mg, 0.638 mmol) was dissolved in dichloromethane (5 mL) at room temperature. Iodotrimethylsilane (510 mg, 2.55 mmol) was added dropwise at 0°C under nitrogen, and the reaction system was stirred at room temperature for 2 hours. Ether (2 mL) and concentrated hydrochloric acid (4 mL) were added to the reaction solution, and the mixture was stirred for 30 minutes. The reaction mixture was adjusted to H = 9 with saturated sodium bicarbonate and extracted with dichloromethane (50 mL x 5). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by HPLC (acetonitrile / water containing 0.05% formic acid) afforded compound A1.15a (200 mg, 66.1% yield) as an off-white solid.

[0724] LCMS (ESI) [M+H] + =478.2;

[0725] 1H NMR(400MHz,DMSO-d6)δ8.30(s,1H,HCO2H),7.67(s,1H),7.51(s,1H),7.23(s,1H),6.62–6.41(m,1H),6.29(s,2H),5.42(s,2H) ,5.28(s,2H),3.27–3.25(m,2H),2.91–2.79(m,2H),2.58–2.56(m,2H),1.91–1.79(m,2H),1.44–1.42(m,2H),0.90–0.84(m,6H).

[0726] Step 5:

[0727] Compound A1.15a (120 mg, 0.251 mmol) was dissolved in dichloromethane (5 mL) at room temperature. Acetoxyacetyl chloride (167 mg, 1.25 mmol) and triethylamine (133 mg, 1.25 mmol) were added dropwise at 0°C. The reaction was stirred at 0°C for 30 minutes. LCMS confirmed the reaction was complete. The reaction solution was concentrated under reduced pressure to afford crude compound A1.15-F (120 mg, 82.8% yield) as a yellow solid. LCMS (ESI) [M+H] + =578.3.

[0728] Step 6:

[0729] Compound A1.15-F (120 mg, 0.207 mmol) was dissolved in ethanol (4 mL) at room temperature. Concentrated hydrochloric acid (2 mL) was then added to the reaction mixture, and the reaction was stirred at 70°C for 1 hour. LCMS confirmed the reaction was complete. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (30 mL x 5). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by HPLC (acetonitrile / water containing 0.05% formic acid) afforded compound A1.15b (20 mg, 18.1% yield) as an off-white solid.

[0730] LCMS (ESI) [M+H] + =536.2;

[0731] 1H NMR(400MHz,DMSO-d6)δ7.93(s,0.7H),7.69(s,0.3H),7.51(s,1H),7.24(s ,1H),6.49(s,1H),6.30(s,2H),5.42(s,2H),5.35(s,1.5H),5.29(s,0.5H), 4.65–4.62(m,1H),4.13-3.97(m,2H),3.51–3.49(m,2H),3.33–3.30(m,2H), 3.24–3.20(m,2H),1.86–1.84(m,2H),1.60–1.52(m,2H),0.89–0.86(m,6H).

[0732] Example A1.16: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.16)

[0733] To a DMF solution (10 mL) of A1.10-C (200 mg, 0.439 mmol) and isopropylamine (50 mg, 0.877 mmol) were added diisopropylethylamine (170 mg, 1.32 mmol) and sodium iodide (99 mg, 0.659 mmol). The reaction was allowed to proceed at 50°C under a sealed tube for 2 hours. Ethyl acetate was added to the reaction solution, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by pre-TLC (dichloromethane:methanol=10:1) to give a yellow solid. The compound was further purified by HPLC (acetonitrile / water containing 0.05% formic acid) to give the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.16, 2.03 mg).

[0734] LCMS (ESI) [M+H] + =480.2;

[0735] 1 H NMR (400MHz, DMSO-d6) δ8.28(d,J=8.0Hz,1H),8.27(s,1H,HCO2H)7.92(d,J=11.0Hz,1H),7.33(s,1H),6.53(s,1H),5.45(s,2H),5.30(s, 2H),33.30–3.22(m,3H),3.18–3.10(m,2H),2.54(s,3H),2.06–1.94(m,2H),1.93–1.80(m,2H),1.26–1.20(m,6H),0.88(t,J=7.3Hz,3H).

[0736] Example A1.17: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-cyclopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.17)

[0737] To a DMF solution (10 mL) of A1.10C (200 mg, 0.439 mmol) and cyclopropylamine (52 mg, 0.881 mmol) was added diisopropylethylamine (165 mg, 1.28 mmol) and sodium iodide (96 mg, 0.640 mmol). The mixture was allowed to react at 50°C under a sealed tube for 2 hours. LCMS monitored the reaction for completion. Ethyl acetate was added to the reaction solution, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the crude product. The crude product was separated and purified by TLC (dichloromethane:methanol=10:1) and then further purified by HPLC preparative purification (acetonitrile / water containing 0.05% formic acid) to obtain the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(3-cyclopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.17, 5.12 mg).

[0738] LCMS (ESI) [M+H] + =478.2;

[0739] 1 H NMR(400MHz,DMSO-d6)δ8.22(br s,2H,one is HCO2H),7.86(br s,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.28(s,2H),3.24–3.19(m,2H),2.75–2.68(m,2H),2.49(s,3 H),2.16–2.09(m,1H),1.95–1.76(m,4H),0.91–0.83(t,J=7.3Hz,3H),0.43–0.33(m,2H),0.31–0.21(m,2H).

[0740] Example A1.18: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18)

[0741] Step 1:

[0742] Under nitrogen, 3-fluoro-4-methylaniline (A1.18A, 2.0 g, 16.3 mmol) was dissolved in 1,2-dichloroethane (40 mL). Boron trichloride (19.2 mL, 19.2 mmol) was added dropwise at 0°C, followed by the slow addition of p-nitrobenzonitrile (2.8 g, 19.2 mmol). After the addition was complete, the reaction mixture was heated to 80°C and stirred overnight. The reaction mixture was cooled to room temperature, hydrochloric acid (40 mL, 2 M) was added, and the temperature was raised to 80°C and stirred for 30 minutes. After completion of the reaction as determined by LCMS, water (80 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation. The crude product was purified by silica gel column chromatography (PE:EA = 10:1) to obtain the target compound (A1.18B, 2.0 g, 46.5% yield).

[0743] LCMS (ESI) [M+H] + =275.1;

[0744] 1 H NMR (400MHz, DMSO) δ 8.34 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.39 (s, 2H), 7.11 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 12.4 Hz, 1H), 2.00 (s, 3H).

[0745] Step 2:

[0746] Compound A1.18B (620 mg, 2.28 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (600 mg, 2.28 mmol), and p-toluenesulfonic acid (560 mg, 2.96 mmol) were added to a reaction flask and uniformly dissolved with dichloromethane. The dichloromethane was spin-dried and evacuated. The mixture was heated to 120°C under vacuum and maintained at this temperature for six hours. After the reaction was completed as determined by LCMS, methanol (10 mL) was added to the system, followed by water (80 mL), to produce a large amount of precipitate. The precipitate was filtered and dried to give the target compound (A1.18C, 800 mg, yield 80.0%) as a yellow solid.

[0747] LCMS (ESI) [M+H] + =502.2;

[0748] 1H NMR (400MHz, DMSO) δ8.52(d,J=8.2Hz,2H),8.13–7.89(m,3H),7.64(d,J=8.4Hz,1H),7.37(d,J=1 3.4Hz,1H),6.54(s,1H),5.41(s,2H),5.06(m,2H),2.40(s,3H),1.94–1.84(m,2H),0.87(m,3H).

[0749] Step 3:

[0750] Raney nickel (609 mg, 10.5 mmol) was added to a mixture of compound A1.18C (1.0 g, 2.1 mmol) in methanol (25 mL) and tetrahydrofuran (50 mL). The reaction was stirred at room temperature under a hydrogen atmosphere for 5 hours. LCMS confirmed the reaction was complete. The reaction solution was filtered and the filtrate was dried to afford the target compound (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-aminophenyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1.18, 650 mg, 67% yield) as a yellow solid.

[0751] LCMS (ESI) [M+H] + =472.3;

[0752] 1 H NMR (400MHz, DMSO) δ7.93–7.81(m,2H),7.40–7.25(m,3H),6.81(d,J=8.2Hz,2H),6.51(s,1H), 5.62(s,2H),5.41(s,2H),5.10(s,2H),2.41(s,3H),1.93–1.80(m,2H),0.88(t,J=7.2Hz,3H).

[0753] B. Synthesis of intermediates containing bioactive molecular fragments

[0754] Example B1.1: Synthesis of (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)butoxy)methyl)acetamide (B1.1)

[0755] Compound (B1.1-A) (368 mg), compound (B1.1-B) (452 ​​mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 ml) for 20 hours, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution, respectively. The organic solvent was removed under reduced pressure, and the residue was dissolved in DMF (5 ml). Piperidine (1 ml) was added, and the compound was stirred for 20 minutes and dissolved in DMF under reduced pressure to remove most of the low-boiling components. The residue was separated by preparative HPLC to obtain the target product (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)butoxy)methyl)acetamide (B1.1).

[0756] ESI-MS (m / z): 539 [M+H] + .

[0757] Example B1.2: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.2)

[0758] Step 1:

[0759] Compound B1.2-A (274 mg), diisopropylethylamine (334 mg), and HBTU (369 mg) were added sequentially to N,N-dimethylformamide (10 mL), followed by compound B1.2-B (300 mg). The reaction mixture was stirred at room temperature for 2 hours. Ethyl acetate (50 mL) was added to the reaction mixture, which was washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1.2-C (300 mg).

[0760] LCMS (ESI) [M+H] + :830.2;

[0761] 1H NMR(400MHz,DMSO-d6)δ8.74(s,1H),7.91–7.84(m,2H),7.80–7.67(m,2H),7.65–7.54(m ,2H),7.53–7.46(m,1H),7.45–7.37(m,2H),7.36–7.27(m,2H),7.25–7.23(m,1H),6.57–6 .44(m,1H),6.29(s,2H),5.50–5.19(m,4H),4.71–4.57(m,2H),4.32–3.97(m,7H),3.80– 3.53(m,4H),3.20–3.14(m,2H),1.92–1.80(m,2H),1.28–1.22(m,3H),0.87–0.82(m,3H).

[0762] Step 2:

[0763] To a solution of compound B1.2-C (300 mg) in N,N-dimethylformamide (4 mL) was added piperidine (1 mL), and the reaction mixture was stirred at room temperature for 20 minutes. After removing low-boiling components from the reaction mixture, the desired product was obtained, which was used directly in the next step.

[0764] LCMS (ESI) [M+H] + =608.0.

[0765] Example B1.3: Synthesis of (S)-2-amino-N-((2-(((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.3)

[0766] Using the same method and reaction conditions as Example B1.1, replacing (B1.2-B) with compound (B1.3-A) to obtain the target product (S)-2-amino-N-((2-(((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.3).

[0767] ESI-MS (m / z): 554 [M+H] + .

[0768] Example B1.4: Synthesis of (S)-2-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (B1.4)

[0769] To a solution of compound (B1.4-A) (175 mg) and compound (B1.3-A) (409 mg) in DMF (5 ml) was added DIPEA (200 μl) and HBTU (420 mg). The mixture was stirred gently at room temperature for 20 hours. Ethyl acetate (100 ml) was added to the mixture and washed with water (100 ml x 3). The organic solvent was removed under reduced pressure, and 1:1 DCM / TFA (10 ml) was added to the residue, which was allowed to stand at room temperature for 20 minutes. Low-boiling components were removed under reduced pressure, and the residue was separated by preparative HPLC to yield the desired product, (S)-2-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (B1.4).

[0770] ESI-MS (m / z): 467 [M+H] + .

[0771] Example B1.5: Synthesis of (S)-2-amino-N-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (B1.4)

[0772] B1.5-A (200 mg) was dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (154.80 mg) and 2,5-dioxopyrrolidin-1-yl (tert-butyloxycarbonyl) glycine (157 mg) were added to the reaction solution in sequence. The reaction solution was stirred at 25°C for 60 minutes. Water (30 mL) was added to the reaction solution, filtered, and the filter cake was dried to obtain a white solid product (ESI-MS (m / z): 579.4 [M+H] +The white solid product was dissolved in a mixture of trifluoroacetic acid and dichloromethane (volume ratio 1:3; 4 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated to obtain a crude product, which was then purified using a C18 column with reverse phase separation (acetonitrile / 0.05% formic acid aqueous solution: 5% to 50%) to obtain the target product (S)-2-amino-N-((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)acetamide (B1.5, 110 mg).

[0773] ESI-MS (m / z): 479.3 [M+H] + .

[0774] Example B1.6: Synthesis of (S)-2-amino-N-((4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-1H-pyrazol-1-yl)methyl)acetamide (B1.6)

[0775] Using the same method and reaction conditions as Example B1.1, replacing compound (B1.1-B) with compound (B1.6-A) to obtain the target product (S)-2-amino-N-((4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-1H-pyrazol-1-yl)methyl)acetamide (B1.6).

[0776] ESI-MS (m / z): 545 [M+H] + .

[0777] Example B1.7: Synthesis of (S)-2-amino-N-((2-(((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7)

[0778] Using the same method and reaction conditions as Example B1.2, compound (B1.5-A) was used instead of compound (B1.2-B) to obtain a mixture containing the target product (S)-2-amino-N-((2-(((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7). The above mixture was directly used in the following synthesis reaction.

[0779] ESI-MS (m / z): 566 [M+H] + .

[0780] Example B1.8: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.8)

[0781] Using the same method and reaction conditions as Example B1.4, replacing compound (B1.3-A) with compound (A1.3) to obtain the target product (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.8).

[0782] ESI-MS (m / z): 541 [M+H] + .

[0783] Example B1.9: Synthesis of (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9)

[0784] Using the same method and reaction conditions as in Example B1.4, substituting compound (A1.4) for compound (B1.3-A), the target product (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9) was obtained. Alternatively, the target product was obtained using the following reaction conditions.

[0785] Compound A1.4 (60 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (3 mL). (tert-Butyloxycarbonyl)glycine (26 mg, 0.15 mmol), HATU (56 mg, 0.15 mmol), and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. The reaction was complete after TLC. TFA (1.0 mL) was then added directly to the reaction mixture. The mixture was stirred at room temperature for 1 hour. LCMS detected that the reaction was complete. The reaction solution was concentrated to remove trifluoroacetic acid to obtain a crude product. The crude product was purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to give the target product (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9) (26.3 mg, yield 38%) as a yellow solid.

[0786] ESI-MS (m / z): 541 [M+H] + ;

[0787] 1 H NMR (400MHz, DMSO) δ10.71 (s, 1H), 8.16 (s, 2H), 7.86 (d, J = 8.6Hz, 2H), 7.67-7.58 (m, 3H), 7.29 (s, 1H), 7.04 (s, 1H), 6.50 (s, 1H), 6.28 (s, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 3.87 (s, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0788] Example B1.10: Synthesis of (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10)

[0789] Step 1:

[0790] To a solution of compound A1.5 (200 mg) in N,N-dimethylformamide (5 mL) were added triethylamine (67 mg) and 2,5-dioxopyrrolidin-1-yl(tert-butyloxycarbonyl)glycine (182 mg) at room temperature. The mixture was allowed to react for 1 hour. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to obtain the target compound (B1.10-A, 100 mg).

[0791] LCMS (ESI) [M+H] + :607.

[0792] Step 2:

[0793] To a solution of compound B1.10-A (100 mg) in dichloromethane (4 mL) was added trifluoroacetic acid (2 mL), and the reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the crude product was added to N,N-dimethylformamide (3 mL) and further purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to obtain the target compound (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propyl)acetamide (B1.10, 80 mg).

[0794] LCMS (ESI) [M+H] + :507;

[0795] 1H NMR(400MHz,DMSO-d6)δ8.26(s,1H),7.64(s,1H),7.51(s,1H),7.24(s,1H),6.5 1(s,1H),6.29(s,2H),5.42(s,2H),5.23(s,2H),3.31(s,2H),3.30–3.27(m,2H), 3.13–3.07(m,2H),2.04–1.76(m,4H),0.87(t,J=7.3Hz,3H).

[0796] Example B1.11: Synthesis of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.11)

[0797] Step 1:

[0798] Compound 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazacyclo-11-carboxylic acid (242 mg), diisopropylethylamine (330 mg) and N,N,N`,N`-tetramethyluronium hexafluorophosphate (359 mg) were dissolved in N,N-dimethylformamide (10 mL). Compound (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-8,11(7H)-dione (300 mg) was then added and the reaction was allowed to proceed at room temperature for 2 hours. Ethyl acetate (50 mL) was added to the reaction solution, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to give the title compound, Compound B1.11B (220 mg).

[0799] LCMS (ESI) [M+H] + :844.0.

[0800] Step 2: Preparation of (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)acetamide

[0801] To a solution of compound B1.11B (220 mg, 0.261 mmol) in N,N-dimethylformamide (4 mL) was added piperidine (1 mL), and the reaction was stirred at room temperature for 20 minutes. LCMS confirmed the reaction was complete. The reaction solution was concentrated under reduced pressure to afford the title compound (220 mg) as a brown solid, which was used directly in the next step without purification.

[0802] LCMS (ESI) [M+H] + :622.1.

[0803] Example B1.12: Synthesis of (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propoxy)methyl)acetamide (B1.12)

[0804] Step 1:

[0805] Compound A1.10 (160 mg, 0.365 mmol) was dissolved in N,N-dimethylformamide (3 mL), and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (B1.1-A, 672 mg, 1.83 mmol) was added. Ethyl acetate hydrochloride (0.073 mL, 3 M) was then added to the reaction solution, and the reaction solution was stirred at room temperature overnight. LCMS confirmed the reaction was complete. The reaction solution was directly purified by reverse phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to obtain the title compound (80 mg, 29.0% yield) as a white solid.

[0806] LCMS (ESI) [M+H] + =747.4;

[0807] 1H NMR(400MHz, DMSO-d6)δ8.74(t,J=6.4Hz,1H),8.28–8.17(m,1H),7.99–7.88(m,3H),7.73(d,J=7.3H z,2H),7.63(t,J=5.7Hz,1H),7.44(t,J=7.4Hz,2H),7.35(t,J=7.2Hz,3H),6.58(s,1H),5.48(s,2H) ,5.29(s,2H),4.66(d,J=6.3Hz,2H),4.32(d,J=6.9Hz,2H),4.26(d,J=6.1Hz,1H),3.71(d,J=5.8Hz, 2H),3.57(t,J=5.7Hz,2H),3.29–3.20(m,2H),2.55(s,3H),2.00–1.86(m,4H),0.93(t,J=7.2Hz,3H).

[0808] Step 2: B1.12-A (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, the mixture was stirred for 20 minutes, low-boiling components were removed under reduced pressure, and the residue was used directly in the next step.

[0809] ESI-MS (m / z): 525.2 [M+H] + .

[0810] A small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to give the title compound.

[0811] ESI-MS (m / z): 525.1 [M+H] + ;

[0812] 1 H NMR (400MHz, DMSO) δ9.13(t,J=6.6Hz,1H),8.21(d,J=8.1Hz,1H),8.02(brs,2H),7.89(d,J=10.8Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H), 5.28(s,2H),4.66(d,J=6.5Hz,2H),3.64(s,2H),3.53(t,J=6.1Hz,2H),3 .25-3.18(m,2H),2.52(s,3H),1.98-1.84(m,4H),0.88(t,J=7.3Hz,3H).

[0813] Example B1.13: Synthesis of (S)-2-amino-N-(3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13)

[0814] Step 1:

[0815] To a solution of compound A1.11 (200 mg) in N,N-dimethylformamide (5 mL) was added triethylamine (67 mg) and 2,5-dioxopyrrolidin-1-yl(tert-butyloxycarbonyl)glycine (182 mg) at room temperature. The mixture was allowed to react for 1 hour. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford the crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to afford the target compound (B1.13-A, 104 mg).

[0816] LCMS (ESI) [M+H] + :595.

[0817] Step 2:

[0818] To a solution of compound B1.13-A (100 mg) in dichloromethane (4 mL) was added trifluoroacetic acid (2 mL), and the reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the crude product was added to N,N-dimethylformamide solution (3 mL). The product was further purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to obtain the target compound (S)-2-amino-N-(3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propyl)acetamide (B1.13, 88 mg).

[0819] LCMS (ESI) [M+H] + :495.

[0820] Example B1.14: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B1.14)

[0821] Step 1: Compound (B1.1-A) (368 mg), compound (A1.9) (440 mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 ml) for 20 hours, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution, respectively. The organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 10 / 1) to obtain the target compound Compound B1.14A (240 mg).

[0822] LCMS (ESI) [M+H] + :759.5.

[0823] Step 2: Dissolve B1.14-A (240 mg) in DMF (5 mL) and add piperidine (1 mL). Stir the mixture for 20 minutes, remove low-boiling components under reduced pressure, and use the residue directly in the next step. A small amount of the crude product was purified by reverse-phase chromatography (acetonitrile / 0.05% FA in water: 5% to 50%) to afford the title compound.

[0824] ESI-MS (m / z): 537.4 [M+H] + ;

[0825] 1 H NMR (400MHz, DMSO-d6) δ9.13(t,1H), 8.04(br,2H), 7.58(s, 1H), 7.51(s, 1H), 7.25(s, 1H), 6.29(s, 2H), 5.43 (S, 2H), 5.21 (s, 2H), 4.65 (d, 2H), 3.63 (m, 2H), 3.53 (m, 2H), 3.11 (m, 2H), 1.87 (m, 4H), 0.88 (t, 3H).

[0826] Example B1.15: (S,E)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)allyl)acetamide

[0827] Step 1:

[0828] To a solution of compound A1.12 (200 mg, 0.447 mmol) in N,N-dimethylformamide (5 mL) was added triethylamine (135 mg, 1.341 mmol) and N-hydroxy-2,5-dioxopyrrolidine-tert-butyloxycarbonylglycine ester (183 mg, 0.671 mmol) at room temperature. The mixture was allowed to react for 1 hour. LCMS indicated the reaction was complete. Water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford the target compound B1.15A (130 mg, 48% yield) as a brown solid.

[0829] LCMS (ESI) [M+H] + =605.6.

[0830] Step 2:

[0831] Compound B1.15-A (130 mg, 0.215 mmol) was dissolved in 1,4-dioxane hydrochloride (5 mL) and allowed to react at room temperature for 30 minutes. LCMS indicated the reaction was complete. The reaction solution was purified by reverse transpiration to afford the title compound (60 mg, 52% yield) as a brown solid.

[0832] LCMS (ESI) [M+H] + =505.2;

[0833] 1 H NMR (400MHz, DMSO-d6) δ8.85(s,1H),8.15(s,2H),7.70(s,1H),7.54(s,1H),7.26(s,1H),7.21(d,J=16.2Hz,1H),6. 51(d,J=16.2Hz,1H),6.31(s,2H),5.42(s,2H),5.28(s,2H),4.19(s,2H),1.86–1.82(m,4H),0.87(t,J=7.3Hz,3H).

[0834] Example B1.16: (S,E)-2-amino-N-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)allyl)oxy)methyl)acetamide

[0835] Step 1:

[0836] Under nitrogen, raw material A1.13 (150 mg, 0.335 mmol) was dissolved in toluene (3 mL). (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (B1.1-A, 308 mg, 0.84 mmol) was added. Zinc acetate (123 mg, 0.67 mmol) was then added to the reaction mixture. The reaction mixture was stirred at 100°C overnight. LCMS confirmed the reaction was complete. The reaction solution was directly purified by reverse phase chromatography (acetonitrile / 0.05% formic acid in water: 5% to 50%) to obtain the target compound (9H-fluoren-9-yl)methyl (S,E)-(2-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxetane[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-14-yl)allyl)oxy)methyl)amino)-2-oxoethyl)carbamate (B1.16-A, 80 mg, yield 31%) as a white solid.

[0837] LCMS (ESI) [M+H] + =757.4.

[0838] Step 2:

[0839] Compound B1.16-A (80 mg, 0.11 mmol) was dissolved in N,N-dimethylformamide (2 mL). Piperidine (0.05 mL) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was concentrated to obtain the crude product, which was then purified using a C18 column with reverse phase separation (acetonitrile / 0.05% ammonia solution: 5% to 50%) to yield the title compound (35 mg, 62% yield) as a brown solid.

[0840] LCMS(ESI)[M+H]+=535.2, tR=1.070min.

[0841] Example B1.17: (S,E)-2-amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide

[0842] Step 1:

[0843] Compound A1.14 (80 mg, 0.183 mmol) and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (B1.1-A, 135 mg, 0.367 mmol) were dissolved in toluene (4 mL). Zinc acetate (190 mg, 1.03 mmol) was added, and the reaction mixture was stirred at 100°C for 48 hours. After cooling to room temperature, water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by reverse phase chromatography (0.05% FA in water / acetonitrile: 5% to 100%) to give the target compound (S,E)-2-(Fmocamino)-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide (B1.17-A, 30 mg) as a white solid.

[0844] LCMS (ESI) [M+H] + =745.2;

[0845] 1 H NMR (400 MHz, DMSO) δ 8.82 (m 1H),8.21(m,1H),7.89(m,2H),7.84(d,J=7.8Hz,2H),7.68(m,1H),7.61(m,1H), 7.42(m,1H),7.37(m,1H),7.33–7.28(m,4H),6.72–6.55(m,1H),6.53(s,1H),5. 42(s,2H),5.28(s,2H),4.75(m,2H),4.34(m,2H),4.28–4.24(m,2H),4.20(m,1H ), 3.70 (d, J = 6.1Hz, 2H), 2.47 (s, 3H), 1.90–1.82 (m, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0846] Step 2:

[0847] Compound 4 (25 mg, 0.034 mmol) was dissolved in N,N-dimethylformamide (2 mL), and then diethylamine (0.2 mL) was added. After the addition was complete, the reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS, the reaction solution was directly evaporated to dryness under reduced pressure to obtain the crude product target compound (S,E)-2-amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide as a yellow viscous product.

[0848] LCMS (ESI) [M+H] + = 523.2;

[0849] LCMS (ESI) [M+H] + =523.1,t R =0.446min,1.311min.

[0850] 1 H NMR (400MHz, DMSO) δ9.26(t,J=6.5Hz,1H),8.23(d,J=8.2Hz,1H),8.06(s,2H),7.91(m,1H),7.40(d,J=16.3Hz,1H),7.34(s,1H),6.70–6.60(m,1H) ),6.54(s,1H),5.44(s,2H),5.35(s,2H),4.80(d,J=6.6Hz,2H),4.38(d, J=3.9Hz,2H),3.68(d,2H),2.52(s,3H),1.90–1.84(m,2H),0.88(m,3H).

[0851] C. Synthesis of molecular fragment intermediates containing coupling linkers

[0852] Example C1.1: N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valine)-L-lysine (Compound C1.1)

[0853] Step 1: tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate

[0854] To a solution of 26-azido-3,6,9,12,15,18,21,24-octaoxa-26-alkan-1-ol (4.39 g) in DMF (40 ml) was added NaH (0.6 g, 60%). The mixture was stirred for 30 minutes, followed by the addition of tert-butyl bromoacetate (2.4 g). The mixture was stirred under dry conditions for 20 hours. Ethyl acetate (200 ml) and water (200 ml, initially added slowly) were then added to the mixture. The organic phase was washed with water (100 ml x 3), dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure. The residue was purified by silica gel column chromatography to yield the desired product, tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate.

[0855] ESI-MS (m / z): 554 [M+H] + .

[0856] Step 2: tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate

[0857] To a solution of tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (1.16 g) in ethyl acetate (20 ml) was added palladium-carbon catalyst (Pd / C, 10%, 100 mg). The solution was stirred under a hydrogen atmosphere for 5 hours, then the palladium-carbon was filtered off and the solvent was removed under reduced pressure to obtain the desired product.

[0858] ESI-MS (m / z): 528 [M+H] + .

[0859] Step 3: 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid

[0860] 29-Amino-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid tert-butyl ester (527 mg) and 2-methylthio-pyrimidine-5-carboxylic acid (170 mg) were added to dry DMF (10 ml), and then DIPEA (0.2 ml) and HBTU (420 mg) were added to the above solution in sequence under ice cooling. The above mixture was stirred at room temperature for 20 hours. The mixture was then diluted with ethyl acetate (100 ml) and washed with water (100 ml x 4). The organic phase was dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure. The residue was dissolved in DCM (10 ml), and TFA (10 ml) was added. The mixture was stirred at room temperature for 1 hour, and then low-boiling components were removed under reduced pressure. The residue was separated by preparative HPLC to give 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid.

[0861] ESI-MS (m / z): 624 [M+H] + .

[0862] Step 4: N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valine)-L-lysine

[0863] 1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoic acid (623 mg) was dissolved in DMF (10 ml), and DIPEA (300 μl) and HBTU (420 mg) were added at 0°C. The mixture was stirred for 30 minutes, and then a valine-(Boc)lysine dipeptide compound (345 mg) was added. The reaction was stirred for 20 hours, and then ethyl acetate (100 ml) was added. The mixture was washed with dilute hydrochloric acid (20 ml x 3). The organic phase was dried and the organic solvent was removed under reduced pressure. The crude product was separated by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-L-valine)-L-lysine (Compound C1.1).

[0864] ESI-MS (m / z): 951 [M+H] + .

[0865] Example C1.2: N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valine)-L-lysine (Compound C1.2)

[0866] Step 1: tert-Butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate

[0867] tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (1.7 g) and 2-methylthio-5-ethynylpyrimidine (450 mg) were dissolved in DMSO-water (20 ml, 4:1), and cuprous bromide (50 mg) was added to the mixture. The reaction mixture was stirred at room temperature for 2 hours, then ethyl acetate (100 ml) was added, the mixture was washed with water (100 ml x 3), dried, and the organic solvent was removed under reduced pressure. The crude product was separated by silica gel column chromatography to obtain the desired product, tert-butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate.

[0868] MS (m / z): 704 [M+H] + .

[0869] Step 2: 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid

[0870] Tert-butyl 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoate (1 g) was dissolved in dichloromethane (10 ml), and TFA (5 ml) was added. The mixture was allowed to stand at room temperature for 1 hour, and then low-boiling components were removed under reduced pressure to obtain the desired product, 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid.

[0871] MS (m / z): 648 [M+H] + .

[0872] Step 3: N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valine)-L-lysine

[0873] 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoic acid (647 mg) was dissolved in DMF (10 ml), and DIPEA (300 ul) and HBTU (420 mg) were added at 0°C. The mixture was stirred for 30 minutes, and then a valine-lysine dipeptide compound (345 mg) was added. The reaction was stirred for 20 hours, and then ethyl acetate (100 ml) was added and washed with dilute hydrochloric acid (20 ml x 3). The organic phase was dried and the organic solvent was removed under reduced pressure. The crude product was separated by preparative HPLC to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)-L-valine)-L-lysine (Compound C1.2).

[0874] ESI-MS (m / z): 975 [M+H] + .

[0875] Example C1.3: (1-(2-(Methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-glycine-glycine-L-phenylalanine (Compound C1.3)

[0876] Using the same method and reaction conditions as step 4 of Example C1.1 and the corresponding starting materials, the target product (1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-alkanoyl)-glycine-glycine-L-phenylalanine (Compound C1.3) was obtained.

[0877] ESI-MS (m / z): 885 [M+H] + .

[0878] Example C1.4: (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexatricarboxylic-(35-yn)-acyl)glycine glycine phenylalanine (Compound C1.4)

[0879] Using the same method and reaction conditions as step 4 in Example C1.1 and the raw materials shown in the reaction formula, the target product (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-triacontahexadecan-(35-yn)-acyl)glycineglycinephenylalanine (compound C1.4) was obtained.

[0880] ESI-MS (m / z): 951 [M+H] + .

[0881] Example C1.5: N 6 -(tert-Butoxycarbonyl)-N 2 -((36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexatricarboxylic-(35-ynyl)-acyl)-L-valine)-L-lysine (C1.5)

[0882] The target product N was obtained by the same method and reaction conditions as in step 4 of Example C1.1, using the compound shown in the reaction formula as the raw material. 6 -(tert-Butoxycarbonyl)-N 2 -((36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-30-aza-hexatricarbox-(35-yn)-acyl)-L-valine)-L-lysine (Compound C1.5).

[0883] ESI-MS (m / z): 1017 [M+H] + .

[0884] Example C1.6: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)-hex-5-ynyl)-L-valine)-L-lysine (Compound C1.6)

[0885] 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynoic acid (920 mg) and N-hydroxysuccinimide (537 mg) were dissolved in dichloromethane (50 mL), and then dicyclohexylcarbodiimide (963 mg) was added. The reaction solution was stirred at room temperature for 1 hour, and then the dichloromethane was removed under reduced pressure. The residue was dissolved in N,N-dimethylformamide (50 mL), and then N 2 -L-valine-N 6 -(Boc)-L-lysine (1.5 g), the reaction solution was stirred at room temperature for 16 hours, the reaction solution was poured into 200 mL of water, the pH was adjusted to 11 with saturated sodium bicarbonate solution, the aqueous solution was extracted twice with ethyl acetate, the organic phase was discarded, the aqueous phase was adjusted to pH 4-5 with citric acid, extracted three times with ethyl acetate (100 mL x 3), the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain 2.8 g of crude product, which was separated and purified by flash chromatography (DCM:MeOH=30:1) to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylthio)pyrimidin-5-yl)-hex-5-ynyl)-L-valine)-L-lysine (1.0 g) (Compound C1.6).

[0886] ESI-MS (m / z): 564.3 [M+H] + .

[0887] 1 H NMR (400MHz, DMSO-d6) δ12.45(s,1H),8.68(s,2H),8.12(d,J=7.3Hz,1H),7.88(d,J=8.9H z,1H),6.76(s,1H),4.27–4.19(m,1H),4.11(d,J=4.9Hz,1H),2.88(d,J=7.9Hz,3H),2.73( s,1H),2.59(s,1H),2.52(s,4H),2.40–2.24(m,3H),2.03–1.88(m,2H),1.77(d,J=3.2Hz, 2H), 1.68 (d, J = 7.0Hz, 1H), 1.60–1.49 (m, 1H), 1.36 (s, 15H), 0.85 (dd, J = 14.8, 6.7Hz, 7H).

[0888] Example C1.7: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)-hex-5-ynyl)-L-valine)-L-lysine (Compound C1.7)

[0889] Potassium peroxymonosulfate (1.414 g) was added to a mixture of compound C1.6 (260 mg) in tetrahydrofuran (3 mL) and water (3 mL), and the mixture was stirred at room temperature for 1 hour. The reaction solution was filtered, and the filtrate was purified using a C18 column (acetonitrile / 0.05% formic acid in water: 5%-60%) to obtain the target compound N. 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(Methylsulfonyl)pyrimidin-5-yl)-hex-5-ynyl)-L-valine)-L-lysine (Compound C1.7) (120 mg).

[0890] LCMS (ESI) [M+H] + :596;

[0891] 1 H NMR (400MHz, DMSO-d6) δ12.48(s,1H),9.13(s,2H),8.14(d,J=7.0Hz,1H),7.90(d,J=9.1Hz,1H),6.77(s,1H),4.24(t,J=7.7Hz,1H),4.12(br s,1H),3.41(s,3H),2.89(d,J=6.0Hz,2H),2.43–2.29(m,2H),2.03–1.93(m,2H),1.82(br s,2H),1.63–1.60(m,4H),1.37(s,12H),0.88–0.83(m,6H).

[0892] Example C1.8: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valine)-L-lysine (Compound C1.8)

[0893] The target product N was obtained by the same method and reaction conditions as in Example C1.6, using the known compound shown in the reaction formula as the raw material. 6 -(tert-Butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valine)-L-lysine (Compound C1.8).

[0894] ESI-MS (m / z): 611 [M+H] + .

[0895] Example C1.9: N 6-(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine (Compound C1.9)

[0896] Step 1: 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid

[0897] At room temperature, 2-methylthio-5-ethynylpyrimidine (1.5 g) and methyl 6-azidohexanoate (1.71 g) were dissolved in a mixture of tert-butyl alcohol and water (20 mL / 25 mL). Sodium ascorbyl palmitate (5.7 g) and cuprous bromide (2.9 g) were added and stirred for 10 h. Ethyl acetate (200 ml) was added to the reaction mixture, which was washed with water (100 ml x 5). After drying and removing the organic solvent, silica gel column chromatography was performed to obtain methyl 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoate (2.8 g). ESI-MS (m / z): 321.9 [M+H] + The above product was dissolved in tetrahydrofuran (100 ml), and lithium hydroxide (1 M, 20 ml) was added. The mixture was stirred for 3 hours, and then hydrochloric acid was added to the reaction solution until the pH was 2. Ethyl acetate (200 ml) was then added, and the mixture was washed with water (100 ml x 3), dried, and the organic solvent was removed to obtain the target product, 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid. ESI-MS (m / z): 308.4 [M+H] + .

[0898] Step 2: N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine

[0899] The compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (370 mg) and N-hydroxysuccinimide (152 mg) were dissolved in dichloromethane (5 mL) and stirred, and then dicycloethylcarbodiimide (273 mg) was added, and the reaction solution was stirred at room temperature for 1 hour. 10 mL of water was added to the reaction solution, and it was extracted with ethyl acetate (20 mL x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. This crude product was dissolved in N, N-dimethylformamide (10 mL) and stirred, and compound N was added. 2 -L-valine-N6 -(Boc)-L-lysine, the reaction solution was reacted at room temperature for 16 hours, citric acid was slowly added to the reaction solution to adjust the pH to about 5, water was added, and then extracted with ethyl acetate (3x10mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate to obtain the target product N 6 -(tert-Butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine (Compound C1.9, 400 mg).

[0900] ESI-MS (m / z): 635.3 [M+H] + ;

[0901] 1 H NMR (400MHz, DMSO-d6) δ12.46(s,1H),9.06(s,2H),9.06(s,1H),8.70(s,1H),8.07(d,J=7.4H z,1H),7.77(d,J=9.0Hz,1H),6.76(t,J=5.6Hz,1H),4.41(t,J=7.0Hz,2H),4.19(dd,J=8.9,7. 0Hz,1H),4.14–4.05(m,1H),2.92–2.85(m,2H),2.56(s,3H),2.24–2.10(m,2H),1.97–1.81(m, 3H),1.71–1.64(m,1H),1.61–1.50(m,3H),1.36(s,9H),1.28–1.22(m,6H),0.88–0.76(m,6H).

[0902] Example C1.10: (6-(2-(Methylthio)pyrimidin-5-yl)hex-(5-ynyl)-acyl)glycine glycine-L-phenylalanine (Compound C1.10)

[0903] Using the same method and reaction conditions as in Example C1.6, the known starting materials shown in the above reaction scheme were used to obtain the target product (6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynyl)glycine-L-phenylalanine (Compound C1.10). ESI-MS (m / z): 498 [M+H] + .

[0904] The same method and reaction conditions as in Example C1.6 were used, but with different starting materials, to obtain the target products shown in the table below.

[0905] Example C1.13: (29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29-alkanoyl)glycine-L-phenylalanine (Compound C1.13)

[0906] Using the same method and reaction conditions as step 4 in Example C1.1 and known starting materials, the target product (29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonaoxa-29alkanoyl)glycine-L-phenylalanine (Compound C1.13) was obtained.

[0907] ESI-MS (m / z): 909 [M+H] + .

[0908] Example C1.14: N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazanonatriacont-38-ynyl)-L-valine)-L-lysine (C1.14)

[0909] Step 1:

[0910] Compound C1.14-A (336 mg) was dissolved in dichloromethane (5 mL), and N-hydroxysuccinimide (98 mg, 0.85 mmol) and dicyclohexylcarbodiimide (175 mg, 0.85 mmol) were added and stirred at room temperature for 1 hour. 2 -L-valine-N 6 -(Boc)-L-lysine (230 mg) was added to the reaction solution, and the reaction was stirred at room temperature overnight. The solvent was concentrated to obtain a crude product, which was then purified by reverse phase separation on a C18 column (acetonitrile / 0.01% FA in water: 5%-50%) to afford the title compound (C1.14B, 290 mg) as a colorless oil.

[0911] LCMS (ESI) [M+H] + =882.3.

[0912] Step 2:

[0913] Compound C1.14-B (300 mg) was dissolved in anhydrous methanol (5 mL), and Pd / C (10%, 60 mg) was added. The atmosphere was replaced with hydrogen three times. The reaction was stirred at room temperature overnight, filtered, and concentrated to obtain the target compound C1.14-C (293 mg) as a colorless oil.

[0914] LCMS (ESI) [M+H] + :856.1.

[0915] Step 3:

[0916] 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (84 mg) was dissolved in N,N-dimethylformamide (3 mL), and then N,N-diisopropylethylamine (61 mg) and HATU (108 mg) were added in sequence. The reaction solution was stirred at room temperature for 20 minutes, and then compound C1.14-C (196 mg) was added. The reaction was stirred at 40°C for 3 hours. The crude product was separated and purified by reverse phase separation on a C18 column (acetonitrile / 0.01% FA in water: 5%-65%) to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12,15,18,21,24,27,30-nonaoxa-6,33-diazanonatriacont-38-ynyl)-L-valine)-L-lysine (C1.14) (170 mg).

[0917] LCMS (ESI) [M+H] + :1074.0;

[0918] 1H NMR (400MHz, DMSO-d6) δ8.68(s,2H),8.23(t,J=5.7Hz,1H),8.13(d,J=8.9Hz,1H),7.99(t,J=5.6Hz ,1H),7.43(d,J=6.5Hz,1H),6.69(s,1H),4.09(dd,J=8.8,6.5Hz,1H),4.02(s,2H),3.98(d,J=2.9Hz ,2H),3.74–3.70(m,1H),3.50(s,30H),3.45(d,J=6.2Hz,2H),3.39(d,J=5.8Hz,2H),3.26(d,J=6.1 Hz,2H),3.20(q,J=5.9Hz,2H),2.81(t,J=6.6Hz,2H),2.52(s,3H),2.24(t,J=7.4Hz,2H),2.12–2.06 (m,1H),1.79–1.74(m,2H),1.67–1.59(m,1H),1.50(d,J=5.3Hz,1H),1.36(s,9H),1.24(s,2H),1.20–1.09(m,2H),0.85(t,J=6.3Hz,6H).

[0919] Example C1.15: N 6 -(tert-Butoxycarbonyl)-N 2 -((3,2-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontanoyl)-L-valine)-L-lysine (C1.15)

[0920] Compound C1.14-B (130 mg), 5-ethynyl-2-methylthiopyrimidine (44 mg), sodium ascorbyl sulfate (3 mg), and copper sulfate (5 mg) were added to a mixture of tert-butyl alcohol (2 mL) and water (2 mL) and reacted at room temperature under nitrogen for 3 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by TLC (dichloromethane:methanol = 10:1) to obtain the target compound N 6 -(tert-Butoxycarbonyl)-N 2-((3,2-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontanoyl)-L-valine)-L-lysine (C1.15) (90 mg).

[0921] LCMS (ESI) [M+H] + =1032.3.

[0922] Example C1.16: N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine

[0923] Step 1:

[0924] To compound C1.16-A (10.0 g) was added a solution of hydrogen chloride in dioxane (100 mL), and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give the target compound C1.16-B (8.0 g) as a white solid.

[0925] LCMS (ESI) [M+H] + :380.1.

[0926] 1 H NMR (400MHz, DMSO) δ8.18(d,J=7.4Hz,1H),7.40–7.30(m,5H),7.26(d,J=8.8Hz,1H),5.08–4.99(m,2H),4.23–4.11(m,1H),3 .94–3.88(m,1H),2.78–2.73(m,2H),2.03–1.94(m,1H),1.77–1.51(m,4H),1.44–1.31(m,2H),0.87(dd,J=17.3,6.6Hz,6H).

[0927] Step 2:

[0928] Compound C1.16-B (3.0 g) and sodium acetate (1.90 g) were dissolved in methanol (100 mL) and reacted at room temperature for 10 minutes. Paraformaldehyde (2.8 g) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (1.0 g) was then added to the reaction mixture, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered, and the filtrate was purified using a C18 reverse phase column (acetonitrile to 0.05% formic acid in water: 5% to 55%) to obtain the target compound C1.16-C (1.70 g).

[0929] LCMS (ESI) [M+H] + :408.1;

[0930] 1 H NMR (400MHz, DMSO) δ7.86 (d, J = 7.3Hz, 1H), 7.40–7.26 (m, 6H), 5.03 (s, 2H), 4.08–4.06 (m, 1H), 3.90–3.85 (m, 1H), 2.50–2.45 ( m,2H),2.35(s,6H),2.05–1.93(m,1H),1.73–1.55(m,2H),1.51–1.40(m,2H),1.33–1.22(m,2H),0.85(dd,J=16.5,6.8Hz,6H).

[0931] Step 3:

[0932] Compound C1.16-C (1.6 g) was dissolved in methanol (80 mL) at room temperature. Pd / C (10%, 0.16 g) was then added to the reaction mixture. The mixture was stirred under hydrogen at room temperature for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the target compound C1.16-D (680 mg).

[0933] LCMS (ESI) [M+H] + :274.2.

[0934] Step 4:

[0935] 6-(2-(Methylthio)pyrimidin-5-yl)hex-5-ynoic acid (944 mg) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of N,N-diisopropylethylamine (1.3 g) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1.8 g). The reaction mixture was stirred at room temperature for 20 minutes, followed by the addition of compound C1.16-D (1.1 g). The reaction was stirred at 40°C for 3 hours. The crude product was purified by reverse phase separation on a C18 column (acetonitrile / 0.01% formic acid in water: 5%-65%) to obtain the target compound N as a white solid. 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine C1.16 (1.2 g).

[0936] LCMS (ESI) [M+H] + :492.1;

[0937] 1H NMR (400MHz, DMSO) δ8.68(s,2H),8.00(d,J=7.6Hz,1H),7.91(d,J=9.0Hz,1H),4.20(t,1H),4.08(dd,J=12.7,7.7Hz,1H),2.47–2.40(m,4H),2. 37–2.31(m,2H),2.30(s,6H),2.01–1.92(m,1H),1.82–1.73(m,2H),1.7 1–1.56(m,2H),1.49–1.37(m,2H),1.33–1.23(m,2H),0.88–0.82(m,6H).

[0938] Example C1.17: N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine C1.17

[0939] 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg), compound C1.16-D (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 mL). 1-Hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were then added.

[0940] The reaction solution was stirred at room temperature for 16 hours, and the reaction solution was directly purified by C18 column reverse phase (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1.17, white solid, 327 mg).

[0941] LCMS (ESI) [M+H] + :524.4.

[0942] 1H NMR(400MHz,)δ9.13(s,2H),7.95(t,J=8.8Hz,2H),4.21(dd,J=8.8,6.9Hz, 1H),4.08–4.03(m,1H),3.41(s,3H),2.55(t,J=7.0Hz,2H),2.42–2.32(m,4H ),2.27(s,6H),1.98(dd,J=13.6,6.8Hz,1H),1.86–1.77(m,2H),1.74–1.55 (m,2H),1.47–1.37(m,2H),1.31–1.23(m,2H),0.85(dd,J=12.8,6.8Hz,6H).

[0943] Example C1.18: N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine

[0944] Step 1:

[0945] Compound C1.18-B (3 g) was dissolved in dichloromethane (30 ml), and DIPEA (4 ml) was added, followed by compound C1.18-A (3.48 g). The mixture was stirred at room temperature for 20 hours. Ethyl acetate (200 ml) was added to the reaction solution, and the mixture was washed sequentially with hydrochloric acid (0.1 M, 30 ml x 3) and water (30 ml x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield the desired compound C1.18-C (4.7 g).

[0946] Step 2:

[0947] Compound C1.18-C (4.7 g) was dissolved in methanol (80 mL) at room temperature. Pd / C (10%, 0.6 g) was then added to the reaction mixture, and the mixture was stirred at room temperature under hydrogen for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C1.18-D (3.4 g). LCMS (ESI) [M+H] + :346.2.

[0948] Step 3:

[0949] 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (2.68 g) was dissolved in N,N-dimethylformamide (50 mL), and triethylamine (3 ml) and HBTU (3.8 g) were added. The mixture was stirred at room temperature for 10 minutes, and then ethyl acetate (200 ml) was added. The mixture was washed with saturated sodium bicarbonate (20 ml x 2), hydrochloric acid (0.1 M, 50 ml x 2), and water (50 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude intermediate. The above crude product was dissolved in DMF (30 ml), and DIPEA (1.6 ml) was added, followed by the addition of C1.18-D (3.4 g). The reaction solution was stirred at room temperature for 3 hours, and then ethyl acetate (200 ml) was added. The mixture was washed with hydrochloric acid (0.1 M, 50 ml x 2), water (50 ml x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product, which was separated and purified by silica gel column chromatography to obtain the target compound N 2 -(tert-Butoxycarbonyl)-N 6 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1.18, white solid, 3.8 g).

[0950] LCMS (ESI) [M+H] + :596.4.

[0951] Example C1.19: N 6 ,N 6 -dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine

[0952] Step 1:

[0953] The compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (1.0 g, 3.3 mmol) was dissolved in a mixture of tetrahydrofuran and water (40 mL, 3:1), and potassium peroxymonosulfonate (10.0 g, 16.3 mmol) was added. The reaction was stirred at room temperature for 3 hours. LCMS confirmed the reaction was complete. The reaction mixture was filtered, the filter cake was washed with DMSO, and the filtrates were combined. Reverse phase purification (C18, acetonitrile:0.1% formic acid = 5%-55%) was performed to obtain 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (750 mg, 67.9% yield) as a white solid.

[0954] LCMS (ESI) [M+H] + =340.1;

[0955] 1 H NMR (400MHz, DMSO-d6) δ9.48 (s, 2H), 8.95 (s, 1H), 4.49 (t, J = 7.0Hz, 2H), 3.45 (s, 3 H), 2.22 (t, J = 7.3Hz, 2H), 1.95–1.84 (m, 2H), 1.61–1.50 (m, 2H), 1.36–1.26 (m, 2H).

[0956] Step 2:

[0957] The compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (300 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (6 mL), and HATU (337 mg, 0.88 mmol) and DIPEA (286 mg, 2.21 mmol) were added. The mixture was stirred at room temperature for 30 minutes, and then the dipeptide C1.16-D (243 mg, 0.88 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction was completed by LCMS detection, and the reaction solution was separated and purified by C18 column (acetonitrile / 0.01% FA aqueous solution: 5%-50%) to obtain the target compound N 6 ,N 6 -dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine (300 mg, yield 57%) as a white solid.

[0958] LCMS (ESI) [M+H] + =595.5,t R =2.024min.

[0959] 1H NMR (400MHz, DMSO-d6) δ9.49(s,2H),9.01(s,1H),7.85(br s,1H),7.84(d,J=8.9Hz,1H),4.48(t,J=6.8Hz,2H),4.17–4.15(m,1H),4.02(br s,1H),3.44(s,3H),2.42(br s,2H),2.30(s,6H),2.18–2.14(m,2H),1.99–1.96(m,1H),1.88(dd,J=14.6,7.1Hz,2H),1.67(br s,1H),1.59–1.53(m,2H),1.43(br s,2H),1.28–1.26(m,5H),0.83–0.89(m,6H).

[0960] Example C1.20: N 6 ,N 6 -Diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine

[0961] Step 1:

[0962] Compound C1.16-B (5.0 g, 12.05 mmol) was dissolved in dichloromethane (100 mL). Acetaldehyde (3.2 g, 72.3 mmol) was added to the reaction mixture and stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 reverse phase column (acetonitrile to 0.05% formic acid in water: 5% to 55%) to obtain the target compound C1.20-A (4.57 g, 82.0% yield) as a white solid.

[0963] LCMS (ESI) [M+H] + =436.4;

[0964] 1H NMR(400MHz,DMSO-d6)δ7.70(d,J=7.0Hz,1H),7.41(d,J=9.0Hz,1H),7.38–7.26(m ,5H),5.08–4.99(m,2H),4.00(dd,J=12.6,6.5Hz,1H),3.86(dd,J=8.6,6.8Hz,1H), 2.74(dd,J=14.0,6.9Hz,4H),2.64–2.54(m,2H),2.05–1.94(m,1H),1.72–1.52(m,2 H),1.52–1.38(m,2H),1.38–1.18(m,2H),1.04(t,J=7.1Hz,6H),0.87–0.81(m,6H).

[0965] Step 2:

[0966] Compound C1.20-A (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction mixture, and the mixture was stirred under hydrogen at room temperature for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C1.20-B (900 mg, 82% yield) as an off-white solid.

[0967] 1 H NMR(400MHz,DMSO-d6)δ8.04(br s,1H),4.02–3.99(m,1H),3.10(d,J=4.5Hz,1H),2.65(q,J=7.1Hz,4H),2.55–2.51(m,2H),2.06–1.93(m,1H),1.73–1 .54(m,2H),1.47–1.38(m,2H),1.30–1.21(m,2H),1.01(t,J=7.1Hz,6H),0.89(d,J=6.9Hz,3H),0.79(d,J=6.8Hz,3H).

[0968] Step 3:

[0969] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL). HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added sequentially. After stirring at room temperature for 20 minutes, compound C1.20-B (301 mg, 1 mmol) was added and stirring continued at room temperature for 30 minutes. After completion of the reaction, the reaction mixture was directly purified on a C18 reverse phase column (acetonitrile and 0.05% formic acid in water) to obtain the title compound (280 mg, 51% yield) as a white solid.

[0970] LCMS (ESI) [M+H] + =552.3;

[0971] 1 H NMR(400MHz,DMSO-d6)δ9.13(s,2H),7.95(d,J=8.9Hz,1H),7.86(d,J=7.2Hz,1H),4.18 (dd,J=8.8,6.8Hz,1H),4.02(dd,J=12.8,7.2Hz,1H),3.41(s,3H),2.74–2.69(m,4H),2 .62–2.52(m,4H),2.44–2.29(m,2H),2.04–1.94(m,1H),1.86–1.77(m,2H),1.72–1.54( m,2H),1.51–1.39(m,2H),1.33–1.23(m,2H),1.02(t,J=7.2Hz,6H),0.87–0.82(m,6H).

[0972] Example C1.21: N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine

[0973] Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (500 mg, 1.475 mmol) was dissolved in N,N-dimethylformamide (10 mL), and then HATU (560 mg, 1.475 mmol) and N,N-diisopropylethylamine (476 mg, 3.688 mmol) were added. The mixture was stirred for 30 minutes, and then compound C1.20-B (444 mg, 1.475 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS, the reaction solution was directly purified by C18 reverse phase column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -Diethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine (330 mg, yield 34%) was obtained as a white solid.

[0974] LCMS (ESI) [M+H] + =623.4;

[0975] 1 H NMR (400MHz, DMSO) δ9.49 (s, 2H), 9.04 (s, 1H), 7.84 (d, J = 8.9Hz, 1H), 7.80 (d, J = 7.1Hz, 1H), 4.47 (t, J=7.0Hz,2H),4.14(dd,J=8.8,6.6Hz,1H),4.06–3.95(m,1H),3.44(s,3H),2.67–2.64(m,4H),2.55( t,J=7.4Hz,2H),2.21–2.10(m,2H),2.02–1.94(m,1H),1.92–1.84(m,2H),1.72–1.63(m,1H),1.72–1 .63(m,3H),1.59–1.54(m,2H),1.32–1.21(m,4H),1.01(t,J=7.1Hz,6H),0.81(dd,J=9.6,6.8Hz,6H).

[0976] Example C1.22: N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine

[0977] Step 1:

[0978] Compound C1.16-B (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL). Propionaldehyde (4.2 g, 72.3 mmol) was added to the reaction mixture and stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 column with reversed phase separation (acetonitrile to 0.05% formic acid in water: 5% to 55%) to afford the target compound C1.22-A (4.57 g, 82.0% yield) as a white solid.

[0979] LCMS (ESI) [M+H] + =464.0;

[0980] 1 H NMR(400MHz, DMSO-d6)δ7.81(d,J=7.3Hz,1H),7.38–7.28(m,5H),5.04(d,J=1.7Hz,2H),4.12–4.02(m,1H),3.94–3.82(m,1H),2.6 5–2.52(m,6H),2.06–1.94(m,1H),1.76–1.64(m,1H),1.64–1.53(m,1H),1.52–1.40(m,6H),1.34–1.18(m,2H),0.92–0.80(m,12H).

[0981] Step 2:

[0982] Compound C1.22-A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction mixture, and the mixture was stirred at room temperature under hydrogen for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C1.22-B (1.2 g, 85.5% yield) as a white solid.

[0983] Step 3:

[0984] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), and then HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added. The system was stirred for 30 minutes, and then compound C1.22-B (122 mg, 0.371 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS detection, the reaction solution was directly purified by C18 reverse phase column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (50 mg, yield 28%) was a light yellow solid.

[0985] LCMS (ESI) [M+H] + =580.0;

[0986] 1 H NMR (400MHz, DMSO-d6) δ8.24(s,2H),7.98–7.93(m,2H),4.24–4.16(m,1H),4.10(d,J=5.2Hz,1H),3.41(s,3H),2.7 9–2.64(m,6H),2.55(t,J=7.1Hz,2H),2.45–2.26(m,2H),2.06–1.91(m,1H),1.89–1.78(m,2H),1.76–1.66(m,1H), 1.64–1.57(m,1H),1.57–1.42(m,6H),1.37–1.24(m,2H),0.93–0.78(m,12H).

[0987] Example C1.23: N 6 ,N 6 -Dipropyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine

[0988] Compound 6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (500 mg, 1.475 mmol) was dissolved in N,N-dimethylformamide (10 mL), and then HATU (560 mg, 1.475 mmol) and N,N-diisopropylethylamine (476 mg, 3.688 mmol) were added. The system was stirred for 30 minutes, and then compound C1.22-B (485 mg, 1.475 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS detection, the reaction solution was directly purified by C18 reverse phase column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valine)-L-lysine (320 mg, yield 33%) was obtained as a white solid.

[0989] LCMS (ESI) [M+H] + =651.5;

[0990] 1 H NMR (400MHz, DMSO) δ9.49(s,2H),9.00(s,1H),7.95(d,J=7.2Hz,1H),7.79(d,J= 8.9Hz,1H),4.47(t,J=6.9Hz,2H),4.21–4.11(m,1H),4.09–4.01(m,1H),3.44(s ,3H),2.45–2.35(m,6H),2.23–2.09(m,2H),1.97–1.86(m,3H),1.72–1.64(m,1H ),1.61–1.50(m,3H),1.43–1.34(m,6H),1.32–1.22(m,4H),0.86–0.77(m,12H).

[0991] Example C1.24: (S)-tert-Butyl(6-((4-(Hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamido)-6-oxohexyl)carbamate

[0992] Step 1:

[0993] Compound C1.24-A (3.70 g, 15.7 mmol), diisopropylethylamine (11.0 mL, 62.8 mmol), and HBTU (8.90 g, 23.6 mmol) were dissolved in N,N-dimethylformamide (30 mL) and the reaction was allowed to proceed at room temperature for 30 minutes. Compound Boc-protected lysine (3.86 g, 15.7 mmol) was then added and the reaction was allowed to proceed at room temperature for 2 hours. LCMS confirmed the reaction was complete. Aqueous citric acid (30 mL) was added to the reaction solution and adjusted to pH 5. The aqueous solution was extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the target compound C1.24-B (7.0 g, crude) as a yellow oil. LCMS (ESI) [M+H] + =465.1,t R =1.751min.

[0994] Step 2:

[0995] Compound C1.24-B (7.00 g, 5.60 mmol), diisopropylethylamine (10.7 mL, 60.4 mmol), and HBTU (8.60 g, 22.7 mmol) were dissolved in N,N-dimethylformamide (100 mL). p-Aminobenzyl alcohol (3.71 g, 30.2 mmol) was then added and the mixture was allowed to react at room temperature for 2 hours. LCMS confirmed the reaction was complete. Ethyl acetate (300 mL) was added to the reaction solution, washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to give the target compound (S)-tert-butyl (6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynamido)-6-oxohexyl)carbamate (6.00 g, yield: 69.9%) as a yellow oil.

[0996] LCMS (ESI) [M+H] + =570.1;

[0997] 1H NMR (400MHz, CDCl3) δ9.24(s,1H),8.46(s,2H),7.51–7.47(m,2H),7.31–7.23(m,3H),7.02–6.95(m,1H),4.83(br s,1H),4.63–4.60(m,2H),3.19–2.96(m,4H),2.57(s,3H),2.52–2.48(m,2H),2.47– 2.41(m,2H),1.99–1.92(m,2H),1.87–1.77(m,2H),1.76–1.63(m,2H),1.43(s,9H).

[0998] Example C1.25: tert-Butyl (S)-(6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate

[0999] Step 1:

[1000] Compound C1.25-A (1.60 g, 5.21 mmol), diisopropylethylamine (2.68 mL, 20.8 mmol), and HBTU (2.97 g, 7.82 mmol) were dissolved in N,N-dimethylformamide (30 mL) and reacted at room temperature for 30 minutes. Boc-protected lysine (1.28 g, 5.21 mmol) was then added and the reaction continued at room temperature for 2 hours. LCMS confirmed the reaction was complete. Aqueous citric acid (30 mL) was added to the reaction solution and adjusted to pH 5. The aqueous solution was extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the target compound C1.25-B (3.0 g, crude) as a yellow oil.

[1001] LCMS (ESI) [M+H] + =536.0;

[1002] Step 2:

[1003] Compound C1.25-B (3.00 g, 5.60 mmol), diisopropylethylamine (2.89 g, 22.4 mmol), and HBTU (3.19 g, 8.40 mmol) were dissolved in N,N-dimethylformamide (30 mL). p-Aminobenzyl alcohol (1.38 g, 11.2 mmol) was then added and the reaction was allowed to proceed at room temperature for 2 hours. LCMS confirmed the reaction was complete. Ethyl acetate (200 mL) was added to the reaction solution, washed with saturated brine (80 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to give the title compound (S)-tert-butyl (6-((4-(hydroxymethyl)phenyl)amino)-5-(6-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)-6-oxohexyl)carbamate (2.90 g) as a yellow oil.

[1004] LCMS (ESI) [M+H] + =641.1;

[1005] 1 H NMR (400MHz, CDCl3) δ9.13 (s, 1H), 8.95 (s, 2H), 7.94 (s, 1H), 7.47 (d, J = 8.4 Hz,2H),7.24(d,J=8.4Hz,2H),6.83–6.77(m,1H),4.93–4.80(m,1H),4.64–4 .58(m,2H),4.40–4.29(m,2H),3.12–2.97(m,4H),2.60(s,3H),2.31–2.23(m ,2H),1.96–1.83(m,4H),1.76–1.61(m,4H),1.41(s,9H),1.37–1.29(m,4H).

[1006] Example C1.26: N 6 ,N 6 -Dibutyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine

[1007] Step 1:

[1008] Compound C1.16-B (10.0 g) was dissolved in dichloromethane (200 mL). n-Butyraldehyde (10.4 g) was added to the reaction mixture and stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (25.6 g) was then added portionwise to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The mixture was then dried by evaporation. The filtrate was filtered and purified using a C18 column (acetonitrile to 0.05% aqueous formic acid: 5% to 55%) to afford the target compound C1.26-A (4.9 g) as a white solid.

[1009] LCMS (ESI) [M+H] + =492.7;

[1010] 1 H NMR (400MHz, CDCl3) δ7.35–7.30(m,5H),5.14–5.08(m,2H),4.33–4.30(m,1H),4.14–4.12(m,1H),2.94–2. 80(m,6H),2.13–2.11(m,1H),1.85–1.82(m,2H),1.58–1.55(m,4H),1.40–1.22(m,8H),0.98–0.87(m,12H).

[1011] Step 2:

[1012] Compound C1.26-A (5.0 g) was dissolved in methanol (100 mL) at room temperature. Pd / C (10%, 1 g) was then added to the reaction mixture, and the mixture was stirred under hydrogen for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to yield the target compound C1.26-B (3.68 g) as a white solid.

[1013] LCMS (ESI) [M+H] + =358.3.

[1014] Step 3:

[1015] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1.0 g) was dissolved in N,N-dimethylformamide (15 mL), and then N,N-diisopropylethylamine (1.2 g) and HATU (1.4 g) were added in sequence. The reaction was stirred for 30 minutes, and then compound C1.26-B (1.3 g) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target compound N 6 ,N 6 -Dibutyl-N2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1.26, 500 mg) was obtained as a yellow solid.

[1016] LCMS (ESI) [M+H] + =608.7;

[1017] 1H NMR (400MHz, DMSO) δ9.37 (s, 1H), 9.13 (s, 2H), 8.19 (d, J = 7.3Hz, 1H), 7.92 (d, J =8.8Hz,1H),4.25–4.20(m,1H),4.20–4.13(m,1H),3.42(s,3H),3.06–2.99(m, 6H),2.57–2.53(m,2H),2.41–2.30(m,2H),1.99–1.95(m,2H),1.87–1.79(m,2H ),1.78–1.71(m,1H),1.62–1.56(m,4H),1.34–1.32(m,8H),0.94–0.85(m,12H).

[1018] Example C1.27: (S)-1-Ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)piperidine-4-carboxylic acid

[1019] Step 1:

[1020] Compound C1.27-A (5.0 g, 20.49 mmol) was dissolved in DMF (100 mL). N-((benzyloxy)carbonyl)-L-valine 2,5-dioxopyrrolidine-N-hydroxy ester (7.13 g, 20.49 mmol) was added to the reaction mixture, and the mixture was stirred at 100°C for 48 hours. LCMS indicated the reaction was complete. The reaction mixture was then purified using a C18 column (acetonitrile to 0.05% aqueous formic acid: 5% to 55%) to afford the target compound C1.27-B (1.3 g, 13.3% yield) as a yellow solid.

[1021] LCMS(ESI)[M-boc] + =378.2;

[1022] 1H NMR (400MHz, DMSO) δ7.38–7.33(m,5H),7.32–7.27(m,1H),7.20(d,J=9.0Hz,1H),5.14–5.02(m,2H),3.97–3.87(m,1H),3.7 0–3.59(m,2H),3.08–2.95(m,2H),2.04–1.97(m,1H),1.95–1.87(m,2H),1.76–1.69(m,2H),1.40(s,9H),0.91–0.84(m,6H).

[1023] Step 2:

[1024] Compound C1.27-B (1.3 g, 2.71 mmol) was dissolved in ethyl acetate (10 mL) at room temperature. Dioxane-HCl (3 N, 10 mL) was then added to the reaction mixture and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to afford the target compound C1.27-C (1.06 g, 94.4% yield) as a yellow solid.

[1025] LCMS (ESI) [M+H] + =378.4.

[1026] Step 3:

[1027] Compound C1.27-C (1.06 g, 2.81 mmol) was dissolved in dichloromethane (10 mL). Acetaldehyde (0.75 g, 16.87 mmol) was added to the reaction mixture and stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (3 g, 14.05 mmol) was then added in batches and stirred at room temperature for 1 hour. LCMS indicated the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction mixture and stirred for one hour. The dichloromethane was removed by distillation under reduced pressure, and the remaining aqueous phase was separated by reverse phase separation (acetonitrile to 0.05% aqueous formic acid: 5% to 55%) to afford the target compound C1.27-D (1.13 g, 99.23% yield) as a white solid.

[1028] LCMS (ESI) [M+H] + =406.1;

[1029] 1H NMR(400MHz,MeOD)δ7.42–7.25(m,5H),5.22–5.06(m,2H),3.91(d,J=7.0 Hz,1H),3.52–3.36(m,2H),3.25–2.93(m,4H),2.66–2.54(m,1H),2.47–2.36(m, 1H),2.27–2.14(m,2H),2.13–2.06(m,1H),1.35–1.29(m,4H),0.98–0.90(m,6H).

[1030] Step 4:

[1031] Compound C1.27-D (1.13 g, 2.78 mmol) was dissolved in methanol (20 mL) at room temperature. Pd / C (0.5 g) was then added to the reaction mixture, and the mixture was stirred under hydrogen for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to afford the target compound C1.27-E (0.58 g, 76.18% yield) as a white solid. LCMS (ESI) [M+H] + =272.4.

[1032] Step 5:

[1033] Compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (1 g, 3.73 mmol) was dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (1.2 g, 9.32 mmol) and HATU (1.42 g, 3.73 mmol) were added sequentially. The reaction solution was stirred for 30 minutes, and then compound C1.27-E (1.01 g, 3.73 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target compound (S)-1-ethyl-4-(3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)butanamido)piperidine-4-carboxylic acid (C1.27, 500 mg, yield 28.7%) as a yellow solid.

[1034] LCMS (ESI) [M+H] + =522.5;

[1035] 1H NMR(400MHz, DMSO)δ9.12(s,2H),8.08(s,1H),7.88(d,J=9.0Hz,1H),4.28–4.25(m,1H),3.41(s,3H),2.89–2.78(m,2H),2.57–2.52(m,4H) ,2.45–2.30(m,4H),2.07–2.01(m,2H),1.99–1.96(m,1H),1.95–1.88(m,2H),1.85–1.79(m,2H),1.04(t,J=7.1Hz,3H),0.88–0.83(m,6H).

[1036] 2. Synthesis of Drug-Linker Compounds

[1037] Example 2.1: N-((S)-1-(((S)-6-amino-1-((2-((((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamide (DL-001)

[1038] Step 1:

[1039] Compound C1.6 (50 mg, 0.10 mol), compound B1.5 (71 mg, 0.12 mol), and N,N-diisopropylethylamine (39 mg, 0.30 mol) were dissolved in DMF (5 mL). 1-Hydroxybenzotriazole (16 mg, 0.12 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23 mg, 0.12 mmol) were then added sequentially. The reaction mixture was stirred at room temperature for 16 hours, diluted with 50 mL of ethyl acetate, and the organic phase was washed three times with water (25 mL x 3), dried over anhydrous sodium sulfate, and concentrated to remove the ethyl acetate to obtain a crude product. The crude product was then purified by flash silica gel column chromatography (DCM:MeOH = 100:0 to 90:10) to obtain the target compound 2.1A (62 mg, yellow solid, 58% yield).

[1040] LCMS (ESI) [M+H] + :1024.8.

[1041] Step 2:

[1042] Compound 2.1A (40 mg, 0.04 mmol) was dissolved in a mixture of tetrahydrofuran and water (1 / 1 by volume; 4 mL). Potassium peroxymonosulfate (246 mg, 0.4 mmol) was added, and the reaction mixture was stirred at room temperature for 5 hours. The mixture was diluted with 30 mL of ethyl acetate, and the organic phase was washed three times with water (25 mL x 3), dried over anhydrous sodium sulfate, and concentrated to remove the ethyl acetate to obtain the target compound 2.1B (30 mg, yellow solid, 74% yield).

[1043] LCMS (ESI) [M+H] + =1055.9.

[1044] Step 3:

[1045] Compound 2.1B (30 mg, 0.03 mmol) was dissolved in a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio of 1:3; 4 mL), and the reaction solution was stirred at room temperature for 1 hour. The mixture was then concentrated under reduced pressure to give a crude product, which was purified by preparative chromatography (0.01% TFA in water, MeCN) to give the target compound N-((S)-1-(((S)-6-amino-1-((2-((((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ynamide (9 mg, white solid, 33% yield).

[1046] LCMS (ESI) [M+H] + :955.8;

[1047] 1H NMR(400MHz,DMSO-d6)δ9.09(s,2H),8.54(s,1H),8.15(m,2H),7.84(m,1H),7.80(s,1H),7.64(br s,3H),7.53(s,1H),7.26(s,1H),6.51(s,1H),6.30(d,J=2.9Hz,2H),5.59–5.46(m,2H) ,5.43(s,2H),4.87–4.78(m,1H),4.70–4.60(m,1H),4.17–4.03(m,2H),3.82–3.58(m,2H ),3.41(s,3H),2.80–2.70(m,2H),2.40–2.22(m,2H),1.93–1.68(m,6H),1.65–1.57(m, 1H), 1.56–1.45 (m, 3H), 1.36–1.22 (m, 2H), 0.87 (t, J = 7.3Hz, 3H), 0.80 (d, J = 5.2Hz, 6H).

[1048] Using the same method and reaction conditions as Example 2.1, using different starting materials as shown, the target products shown in Examples 2.2 to 2.8 in the following table were obtained.

[1049] Example 2.9: (S)-6-Amino-N-(2-((((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)butanamido)hexanamide (DL-009)

[1050] Step 1:

[1051] Compound B1.5 (30 mg), compound C1.9 (48 mg), and N,N-diisopropylethylamine (23 mg) were dissolved in DMF (5 mL). 1-Hydroxybenzotriazole (9 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13 mg) were added to the above solution. The reaction was stirred at room temperature for 16 hours, then diluted with 50 mL of ethyl acetate. The organic phase was washed three times with water (25 mL x 3), dried over anhydrous sodium sulfate, and concentrated to remove the ethyl acetate to obtain a crude product. The crude product was separated and purified by flash chromatography (DCM:MeOH = 100:0 to 90:10) to obtain the target compound 2.9A (35 mg, light yellow solid, 43% yield).

[1052] LCMS (ESI) [M+H] + :1095.8

[1053] Step 2:

[1054] Compound 2.9A (35 mg) was dissolved in a mixture of tetrahydrofuran and water (1 / 1 by volume; 4 mL). Potassium peroxymonosulfate (184 mg) was added, and the reaction mixture was stirred at room temperature for 5 hours. The mixture was diluted with 30 mL of ethyl acetate, and the organic phase was washed three times with water (25 mL x 3), dried over anhydrous sodium sulfate, and concentrated to remove ethyl acetate to obtain the target compound 2.9B (25 mg, light yellow solid, yield 71%).

[1055] LCMS (ESI) [M+H] + :1126.9.

[1056] Step 3:

[1057] Compound 2.9B (25 mg) was dissolved in a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio of 1:3; 4 mL), and the reaction solution was stirred at room temperature for 1 hour. The resulting liquid was concentrated to give a crude product, which was purified by prep-HPLC (0.01% TFA in water, MeCN) to give the target compound (S)-6-amino-N-(2-((((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamido)butanamido)hexanamide trifluoroacetate (5.1 mg).

[1058] LCMS (ESI) [M+H] + :1026.9;

[1059] 1 H NMR (400MHz, DMSO) δ9.46(s,2H),8.91(s,1H),8.53(s,1H),8.18(s,1H),8.11–8.10(m,1H),7.79(s,1H),7.73–7.72(m,1H),7. 63(s,3H),7.53(s,1H),7.26(s,1H),6.52(s,1H),6.29(m,2H),5.61–5.46(m,2H),5.43(s,2H),4.85–4.83(m,1H),4.63–4.60(m 1H),4.44(s,2H),4.06–4.03(m,3H),2.75(s,2H),2.12–2.10(m,2H),1.89–1.85(m,6H),1.56–1.53(m,8H),1.24(br s,4H),0.87–0.84(m,3H),0.77(d,J=6.1Hz,6H).

[1060] Example 2.10: N-((7S,10S)-7-(4-aminobutyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-10-isopropyl-3,6,9,12,16-pentaoxo-14,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,17-pentaazatricarboxan-43-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-010)

[1061] Step 1:

[1062] Compound C1.14 (112 mg) was dissolved in N,N-dimethylformamide (2 mL). Triethylamine (32 mg, 0.31 mmol), 1-hydroxybenzotriazole (17 mg, 0.13 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (24 mg, 0.13 mmol) were added sequentially. The mixture was stirred for 20 minutes. Compound B1.5 (50 mg) was then added to the reaction mixture, and the mixture was stirred at 40°C overnight. 10 mL of water was added to the reaction mixture, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain the crude product. The crude product was purified by reverse phase separation on a C18 column (acetonitrile / 0.01% FA in water: 0%-55%) to afford the target compound 2.10A (75 mg, 46% yield) as a white solid.

[1063] LCMS (ESI) [M+H] + :1534.0.

[1064] Step 2:

[1065] Compound 2.10A (75 mg) was dissolved in tetrahydrofuran and water (8 mL, 1:1) and potassium peroxymonosulfate (211 mg) was added. The mixture was stirred at room temperature for 10 hours. 10 mL of dichloromethane was added to the reaction mixture, and the mixture was extracted. The mixture was separated, dried over anhydrous sodium sulfate, and filtered to dryness to obtain the desired product 2.10B (50 mg, 65% yield).

[1066] LCMS (ESI) [M+H] + :1566.2.

[1067] Step 3:

[1068] Compound 2.10B (50 mg) was dissolved in a mixture of trifluoroacetic acid and dichloromethane (volume ratio 1:2; 4 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to obtain a crude product, which was purified by preparative chromatography (0.01% TFAin The target compound N-((7S,10S)-7-(4-aminobutyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-10-isopropyl-3,6,9,12,16-pentaoxo-14,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,17-pentaazatricarboxan-43-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (10.5 mg, yield 21%) was obtained.

[1069] LCMS (ESI) [M+H] + :1466.9;

[1070] 1H NMR(400MHz,DMSO-d6)δ9.12(s,2H),8.62(t,J=5.7Hz,1H),8.26–8.11(m,2H) ,8.03(t,J=5.8Hz,1H),7.93(t,J=5.3Hz,1H),7.85(d,J=8.7Hz,1H),7.79(d, J=5.6Hz,1H),7.61(s,3H),7.54(s,1H),7.26(s,1H),6.50(s,1H),6.31(d,J=1.7Hz,2H),5.46(d,J=17.2Hz,4H),4.76(d t,J=14.8,8.7Hz,2H),4.20–4.15(m,4H),4.00(s,3H),3.95(d,J=5.5Hz,3H),3.72–3.67(m,2H),3.50(d,J=3.9Hz,30H), 3.41(s,3H),3.25–3.19(m,4H),2.76(d,J=5.6Hz,2H),2.56(t,J=4.9Hz,2H),2.27(t,J=7.3Hz,2H),1.90(dd,J=16.1,7. 4Hz,2H),1.84–1.78(m,2H),1.63(s,1H),1.53–1.48(m,2H),1.30(d,J=7.7Hz,2H),0.87–0.84(m,4H),0.78–0.75(m,6H).

[1071] Example 2.11: N-((S)-1-(((S)-6-amino-1-((2-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]diox...

Claims

1. The antibody drug conjugate of formula XV, or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, in: Tb is an anti-NaPi2b antibody or an antigen-binding fragment thereof; q is the drug-antibody coupling ratio; D is a bioactive molecular fragment; L1 is the extension unit; L2 does not exist or is a connecting unit; L3 is selected from Val-AA 1 -Gly, Val-AA 1 、Ala-AA 1 , Gly-AA 1 AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, AA 1 -Ala-Asn, AA 1 ; the AA 1 The structures of the indicated amino acid residues are shown below, Where: R a 、R b Each independently selected from H, And R a 、R b Not at the same time H; Or, R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 replaced by; r、r 1 are each independently selected from any integer from 0 to 20; R m1 、R n1 Each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and -COOR x1 ; R x1 Selected from C1-6 alkyl; Or, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0’ replaced by; R z Selected from C1-6 alkyl; R 0 、R 0’ Each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 and a 4-10 membered heterocyclic group optionally substituted by a C1-6 alkyl group; R m2 、R n2 Each independently selected from H and C1-6 alkyl; L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

2. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 1, wherein: It meets one or more of the following conditions: (1) Tb, the antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, pro-antibody, monoclonal antibody, bispecific antibody or multispecific antibody; (2) q is selected from any value between 0.1 and 16.0; (3) D is a molecular fragment with anti-tumor biological activity.

3. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 2, wherein: It meets one or more of the following conditions: (1) q is selected from 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and any value therebetween; (2) In the bioactive molecule fragment, the bioactive molecule is a DNA topoisomerase inhibitor or a microtubule inhibitor; (3) L1 is selected from Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl group, a 5-10 membered heteroaryl group, and an amide group; Rx and Ry are each independently selected from H and a C1-4 alkyl group; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is selected from any integer between 1 and 6; each y2 is independently selected from any integer between 0 and 15; each y3 is independently selected from 1, 2, and 3; each y4 is independently selected from 0 and 1; position 1 is connected to Tb through an S atom, and position 2 is connected to L2 or L3; (4) L2 is absent or present. When L2 is present, L2 is selected from y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 and 2, each y4 is independently selected from 0 and 1, position 1 is connected to L1, and position 2 is connected to L3; (5)AA 1 Among the amino acid residues, R a 、R b Among them, any one is H, and the other is selected from Or, AA 1 Among the amino acid residues, R a With R b Together with the carbon atoms they are connected to, they form the 0 substituted 5-6 membered heterocycle; (6)AA 1 Among the amino acid residues, r, r 1 Each independently selected from 0, 1, 2, 3, 4 and 5; (7)AA 1 Among the amino acid residues, R m1 、R n1 Each is independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 and -COOCH2CH2CH2CH3; or, R m1 With R n1 Together with the nitrogen atoms they are connected to, they form the 0’ substituted 5-6 membered heterocycle; (8)AA 1 Among the amino acid residues, R z is methyl; (9)AA 1 Among the amino acid residues, R 0 、R 0’ Each independently selected from C1-6 alkyl, -NR m2 R n2 and a 5-6 membered heterocyclic group optionally substituted by a C1-6 alkyl group.

4. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 3, wherein: It meets one or more of the following conditions: (1) q is selected from any value between 0.1, 1, 2, 3, 4, 5, 6, 7, 8 and the like; (2) When the bioactive molecule in the bioactive molecule fragment is a DNA topoisomerase inhibitor, the DNA topoisomerase inhibitor is a camptothecin bioactive molecule; (3) When the bioactive molecule in the bioactive molecule fragment is a tubulin inhibitor, the tubulin inhibitor is an MMAF type tubulin inhibitor or an MMAE type tubulin inhibitor; (4) In L1, each Z is independently selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond; (5) In L1, y1 is 4, 5, or 6; (6) In L1, each y2 is independently selected from any integer between 6 and 10; (7) L2 is absent or present. When L2 is present, L2 is selected from Bit 1 is connected to L1, bit 2 is connected to L3; (8)AA 1 Among the amino acid residues shown, R a 、R b Among them, any one is H, and the other is selected from or, R a With R b Together with the carbon atoms they are connected to, they form the 0 The substituted 5-6 membered heterocyclic ring is R 0 a substituted piperidine ring or piperazine ring; (9)AA 1 Among the amino acid residues, r, r 1 Each independently selected from 0 and 4; (10)AA 1 Among the amino acid residues, R m1 、R n1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and tert-butyloxycarbonyl; or, R m1 With R n1 Together with the nitrogen atoms they are connected to, they form the 0’ a substituted piperidine ring or piperazine ring; (11)AA 1 Among the amino acid residues, R 0 is selected from C1-6 alkyl and a 5-6 membered heterocyclic group substituted by a C1-6 alkyl, wherein the 5-6 membered heterocyclic group is selected from piperidinyl and piperazinyl; (12)AA 1 Among the amino acid residues, R 0’ Selected from C1-6 alkyl and -NR m2 R n2 ; (13)AA 1 Among the amino acid residues, R m2 、R n2 is methyl; (14) L4 does not exist or exists. When L4 exists, L4 is Bit 1 is connected to L3 and bit 2 is connected to D.

5. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 4, characterized in that: It meets one or more of the following conditions: (1) q is selected from any value among 2, 3, 4, 5, 6, 7, 8 and so on; (2) L1 is selected from m is selected from 2, 3 and 4, y1 is selected from any integer between 1 and 6, each y2 is independently selected from any integer between 0 and 10, each y3 is independently selected from 1 or 2, position 1 is connected to Tb through an S atom, and position 2 is connected to L2 or L3; (3) L3 choice AA 1 , A.A. 1 -Gly, Val-AA 1 -Gly, AA 1 -Ala-Asn; (4)AA 1 Among the amino acid residues shown, R a With R b Together with the carbon atoms they are connected to, they form the 0 The substituted 5-6 membered heterocyclic ring is R 0 substituted piperidine ring; (5)AA 1 Among the amino acid residues, r, r 1 In the equation, either one is 0 and the other is 4; (6)AA 1 Among the amino acid residues, R m1 、R n1 are each independently selected from H and C1-6 alkyl; or, R m1 With R n1 Together with the nitrogen atoms they are connected to, they form the 0’ substituted piperidine ring; (7)AA 1 Among the amino acid residues, R 0 is selected from methyl, ethyl and 5-6 membered heterocyclic groups substituted by methyl, wherein the 5-6 membered heterocyclic group is piperidine preferably, R 0 Selected from methyl, ethyl and (8)AA 1 Among the amino acid residues, R 0’ Selected from methyl and -NR m2 R n2 ; (9) When the bioactive molecule in the bioactive molecule fragment is a DNA topoisomerase inhibitor and the DNA topoisomerase inhibitor is a camptothecin bioactive molecule, the camptothecin bioactive molecule is camptothecin, DXD, camptothecin with modified substituents, or DXD with modified substituents.

6. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 5, characterized in that: It meets one or more of the following conditions: (1) q is selected from any value among 3, 4, 5, 6, 7, 8 and the like; preferably, q is selected from any value among 4, 5, 6, 7, 8 and the like; preferably, q is selected from any value among 6, 7, 8 and the like; (2) L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3; (3) L2 does not exist or (4) L3 is selected from AA 1 、Val-AA 1 -Gly; preferably, L3 is selected from Val-AA 1 -Gly; (5)AA 1 Among the amino acid residues shown, R a With R b Together with the carbon atoms they are connected to, they form Carbon atom No. 1 is R a and R b Commonly linked carbon atoms; (6)r、r 1 In the example, r is 4, r 1 When R is 0, m1 、R n1 Each independently selected from H and C1-6 alkyl; r is 0, r 1 When R is 4, m1 、R n1 Each independently selected from C1-6 alkyl, or, R m1 With R n1 Together with the nitrogen atoms they are connected to, they form Carbon atom No. 1 is R a and R b carbon atoms that are linked together.

7. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 6, characterized in that: AA 1 The amino acid residues shown are selected from Most preferably, AA 1 The amino acid residues shown are selected from 8. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 7, wherein: L3 is selected from X - Selected from halogen ions, carboxylate ions, sulfate ions, hydrogen sulfate ions and OH - , bit 1 is connected to L1 or L2, bit 2 is connected to L4 or D; Most preferably, L3 is selected from Bit 1 is connected to L1 or L2, and bit 2 is connected to L4 or D.

9. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 8, characterized in that: The structure is selected from the following structural fragments: Preferably, The structure is selected from the following: Among them, bit 1 is connected to Tb and bit 2 is connected to D.

10. The antibody drug conjugate or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 9, characterized in that: in, The antibody drug conjugate has the structure of Formula I: Wherein: Tb, L1, L2, L3, L4 and q are defined as in any one of claims 1 to 9; R1 and R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or, R1 and R2 together with the carbon atoms to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, wherein the heterocyclic ring contains one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof; R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or, R3 and X together with the carbon atom to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring containing one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof, or R3 and R2 together with the carbon atom to which they are attached form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, wherein the heterocyclic ring contains one or more O, S, N, carbonyl, sulfoxide or sulfone groups or any combination thereof; W is absent or present. When W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, bit 2 is connected to L4 or L3; X is selected from a direct bond, an optionally substituted -O-(CH2) n3 -、-NR4-(CH2) n3 -、-S-(CH2) n3 -, carbonyl-(CH2) n3 -, -SO2-(CH2) n3 -、-(CH2) n1 -、 C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, the 1 position is connected to the parent ring, and the 2 position is connected to W or L4; the substituent is selected from one or more C1-4 alkyl, C3-6 cycloalkyl, or multiple C1-4 alkyl groups and the carbon atoms to which they are simultaneously connected together form a C3-6 cycloalkyl; Each M is independently selected from a direct bond and -CR 5a R 5b -; R4, R5, R 5a 、R 5b , R6, R7 are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3-6 cycloalkyl; n, n', n1, n2, n3 are each independently selected from any integer between 0 and 6; Position 1 is connected to the camptothecin nucleus, and position 2 is connected to W or L4.

11. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 10, wherein: It meets one or more of the following conditions: (1) R1 and R2 are each independently selected from H, halogen, or C1-4 alkyl; or, R1 and R2, together with the carbon atom to which they are attached, form a 5-6 membered heterocyclic ring containing 1, 2, or 3 O, S, or N atoms or any combination thereof; (2) R3 is selected from H, C1-4 alkyl; or, R3 and X together with the carbon atom to which they are attached form a 5-6 membered carbocyclic ring; (3) W is absent or present. When W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, bit 2 is connected to L4 or L3; (4) X is selected from optionally substituted -(CH2) n1 -、 C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1 position is connected to the parent ring, and the 2 position is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms to which they are simultaneously connected together form a C3-6 cycloalkyl group; (5) R4 and R5 are each independently selected from H, C1-4 alkyl and C3-6 cycloalkyl; (6)R 5a 、R 5b Each independently selected from H and C1-4 alkyl; (7) each R7 is independently selected from H and C1-4 alkyl; (8) n is selected from 1, 2 and 3; (9) n1 is selected from 1, 2, 3 and 4; (10) n2 is 1; (10)n3 is 0.

12. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 10 or 11, wherein: It meets one or more of the following conditions: (1) R1 is selected from H and halogen, R2 is selected from H and C1-4 alkyl; or, R1 and R2 and the carbon atom to which they are attached form The dotted line indicates the position where the heterocyclic ring is fused to the benzene ring; (2) R3 is H; or, R3 and X together with the carbon atom to which they are attached form The dotted lines indicate where the carbon ring is fused to the benzene ring and the pyridine ring; (3) W is absent or present. When W is present, W is selected from -O-, -S-, -NR4-, Bit 1 is connected to X, bit 2 is connected to L4 or L3; (4) each R4 is independently selected from H, C1-4 alkyl and C3-6 cycloalkyl, and R5 is H; (5)R 5a 、R 5b are each independently selected from H and methyl; (6) R7 is H; (7)n is 1.

13. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 12, wherein: It meets one or more of the following conditions: (1) R1 is H or F, R2 is H or methyl; or, R1 and R2 together with the carbon atom to which they are attached form The dotted line indicates the position where the heterocyclic ring is fused to the benzene ring; (2) W is selected from -O-, -NR4- and Bit 1 is connected to X, bit 2 is connected to L4 or L3; (3) each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl and cyclopropyl, and R5 is H; (4) X is selected from optionally substituted Position 1 is connected to the parent ring, and position 2 is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms to which they are simultaneously connected form a C3-6 cycloalkyl group; the C1-4 alkyl group is such as methyl; the C3-6 cycloalkyl group is such as cyclopropyl.

14. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 13, wherein: (1) W is selected from -O- and -NR4-; (2)X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W or L4.

15. The antibody drug conjugate or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 14, wherein: When W does not exist, X is selected from 1 is connected to the parent ring, 2 is connected to L4; when W exists, X is selected from Position 1 is connected to the mother ring, and position 2 is connected to W.

16. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 15, wherein: The structure is selected from the following: Among them, bit 1 is connected to L4; when L4 does not exist, bit 1 is connected to L3.

17. The antibody drug conjugate or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 16, wherein: The antibody-drug conjugate has a structure represented by Formula I-1, Formula I-2 or Formula I-3: wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, R4 and q are as defined in any one of claims 1 to 16; wherein Tb, L1, L2, L3, L4, X, R1, R2, R3 and q are as defined in any one of claims 1 to 16; wherein Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, R5, n and q are as defined in any one of claims 1 to 16.

18. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 17, wherein: The antibody drug conjugate has a structure represented by Formula I-1A, Formula I-1B, Formula I-2A, Formula I-2B, Formula I-3A or Formula I-3B: wherein Tb, L2, L3, L4, X, R1, R2, R3, R4 and q are as defined in any one of claims 1 to 16; wherein Tb, L2, L3, L4, X, R1, R2, R3 and q are as defined in any one of claims 1 to 16; wherein Tb, L2, L3, L4, X, R1, R2, R3, R4 and q are as defined in any one of claims 1 to 16.

19. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 10 to 18, characterized in that: in, The antibody drug conjugate has a structure of Formula IA or Formula IB: Among them, Tb, X, R1, R2, R3, R a 、R b and q is as defined in any one of claims 1 to 16; Among them, Tb, X, R1, R2, R3, R a 、R b and q is as defined in any one of claims 1-16.

20. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 19, characterized in that: in, The antibody drug conjugate is selected from the following:

21. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 20, characterized in that: Tb is an anti-Napi2b antibody or an antigen-binding fragment thereof; preferably, the anti-Napi2b antibody is lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2 and 16G5B3-hz1 antibodies or antigen-binding fragments thereof.

22. An anti-Napi2b antibody or antigen-binding fragment thereof, comprising the following complementarity determining regions (CDRs): (a) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region VH represented by SEQ ID NO: 1, 17, 19, 34, 36, 52, 54, 71, 73, 74 or 33; and / or (b) LCDR1, LCDR2, and LCDR3 contained in the light chain variable region VL represented by SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75, or 51.

23. The antibody or antigen-binding fragment thereof according to claim 22, wherein The antibody or antigen-binding fragment thereof comprises: (1) VH and / or VL, where defined according to the IMGT numbering system: (a) the VH comprises: a HCDR1 of SEQ ID NO: 11, a HCDR2 of SEQ ID NO: 12, and a HCDR3 of SEQ ID NO: 14; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 10; (b) the VH comprises: a HCDR1 of SEQ ID NO: 11, a HCDR2 of SEQ ID NO: 13, and a HCDR3 of SEQ ID NO: 14; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 15, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 10; (c) the VH comprises: a HCDR1 of SEQ ID NO: 28, a HCDR2 of SEQ ID NO: 29, and a HCDR3 of SEQ ID NO: 31; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 32, LCDR2 with a sequence of YTS, and LCDR3 with a sequence of SEQ ID NO: 27; (d) the VH comprises: a HCDR1 of SEQ ID NO: 28, a HCDR2 of SEQ ID NO: 30, and a HCDR3 of SEQ ID NO: 31; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 32, LCDR2 with a sequence of YTS, and LCDR3 with a sequence of SEQ ID NO: 27; (e) the VH comprises: a HCDR1 of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 47, and a HCDR3 of SEQ ID NO: 49; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 50, LCDR2 with a sequence of SAS, and LCDR3 with a sequence of SEQ ID NO: 45; (f) the VH comprises: a HCDR1 of SEQ ID NO: 46, a HCDR2 of SEQ ID NO: 48, and a HCDR3 of SEQ ID NO: 49; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 50, LCDR2 with a sequence of SAS, and LCDR3 with a sequence of SEQ ID NO: 45; (g) the VH comprises: a HCDR1 of SEQ ID NO: 65, a HCDR2 of SEQ ID NO: 66, and a HCDR3 of SEQ ID NO: 68; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 69, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 64; (h) the VH comprises: a HCDR1 of SEQ ID NO: 65, a HCDR2 of SEQ ID NO: 67, and a HCDR3 of SEQ ID NO: 68; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 69, LCDR2 with a sequence of WAS, and LCDR3 with a sequence of SEQ ID NO: 64; or (i) the VH comprises: a HCDR1 of SEQ ID NO: 76, a HCDR2 of SEQ ID NO: 77, and a HCDR3 of SEQ ID NO: 78; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 79, LCDR2 with a sequence of HTS, and LCDR3 with a sequence of SEQ ID NO: 16; (2) VH and / or VL, where defined according to the Kabat numbering system: (a) the VH comprises: a HCDR1 of SEQ ID NO: 3, a HCDR2 of SEQ ID NO: 4, and a HCDR3 of SEQ ID NO: 6; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 7, LCDR2 with a sequence of SEQ ID NO: 9, and LCDR3 with a sequence of SEQ ID NO: 10; (b) the VH comprises: HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 5, and NO: HCDR3 of 6; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 8, LCDR2 with a sequence of SEQ ID NO: 9, and LCDR3 with a sequence of SEQ ID NO: 10; (c) the VH comprises: a HCDR1 of SEQ ID NO: 21, a HCDR2 of SEQ ID NO: 22, and a HCDR3 of SEQ ID NO: 24; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 25, LCDR2 with a sequence of SEQ ID NO: 26, and LCDR3 with a sequence of SEQ ID NO: 27; (d) the VH comprises: a HCDR1 of SEQ ID NO: 21, a HCDR2 of SEQ ID NO: 23, and a HCDR3 of SEQ ID NO: 24; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 25, LCDR2 with a sequence of SEQ ID NO: 26, and LCDR3 with a sequence of SEQ ID NO: 27; (e) the VH comprises: a HCDR1 of SEQ ID NO: 38, a HCDR2 of SEQ ID NO: 39, and a HCDR3 of SEQ ID NO: 41; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 42, LCDR2 with a sequence of SEQ ID NO: 44, and LCDR3 with a sequence of SEQ ID NO: 45; (f) the VH comprises: a HCDR1 of SEQ ID NO: 38, a HCDR2 of SEQ ID NO: 40, and a HCDR3 of SEQ ID NO: 41; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 43, LCDR2 with a sequence of SEQ ID NO: 44, and LCDR3 with a sequence of SEQ ID NO: 45; (g) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 57, and a HCDR3 of SEQ ID NO: 60; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 61, LCDR2 with a sequence of SEQ ID NO: 63, and LCDR3 with a sequence of SEQ ID NO: 64; (h) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 58, and a HCDR3 of SEQ ID NO: 60; and / or, The VL comprises: LCDR1 of SEQ ID NO: 62, LCDR2 of SEQ ID NO: 63, and LCDR3 of SEQ ID NO: 64; or (i) the VH comprises: a HCDR1 of SEQ ID NO: 56, a HCDR2 of SEQ ID NO: 59, and a HCDR3 of SEQ ID NO: 60; and / or, The VL comprises: LCDR1 with a sequence of SEQ ID NO: 62, LCDR2 with a sequence of SEQ ID NO: 63, and LCDR3 with a sequence of SEQ ID NO: 64; Preferably, the anti-Napi2b antibody or antigen-binding fragment thereof binds to human Napi2b, monkey Napi2b and / or rat Napi2b.

24. The anti-Napi2b antibody or antigen-binding fragment thereof according to claim 22 or 23, wherein The anti-Napi2b antibody or antigen-binding fragment thereof comprises: (a) VH shown in SEQ ID NO: 1 or 17, and / or VL shown in SEQ ID NO: 2 or 18; (b) the VH represented by SEQ ID NO: 19 or 34, and / or the VL represented by SEQ ID NO: 20 or 35; (c) the VH represented by SEQ ID NO: 36 or 52, and / or the VL represented by SEQ ID NO: 37 or 53; (d) the VH represented by SEQ ID NO: 54, 71 or 73, and / or the VL represented by SEQ ID NO: 55 or 72; (e) the VH represented by SEQ ID NO: 74 or 33, and / or the VL represented by SEQ ID NO: 75 or 51; (f) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any VH of (a)-(e); and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any VL of (a); or (g) a VH having one or several amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof, compared to any VH in (a) to (e); and / or a VL having one or several amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof, compared to any VL in (a); preferably, the substitutions are conservative substitutions.

25. The anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 24, wherein The anti-Napi2b antibody or antigen-binding fragment thereof comprises: (a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2; (b) VH of SEQ ID NO: 17 and VL of SEQ ID NO: 18; (c) VH of SEQ ID NO: 19 and VL of SEQ ID NO: 20; (d) VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35; (e) VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37; (f) VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53; (g) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55; (h) VH of SEQ ID NO: 71 and VL of SEQ ID NO: 72; (i) VH of SEQ ID NO: 73 and VL of SEQ ID NO: 72; (j) VH of SEQ ID NO: 74 and VL of SEQ ID NO: 75; (k) VH of SEQ ID NO: 33 and VL of SEQ ID NO: 51; (l) a VH and a VL whose VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the VH and VL in any of groups (a) to (k); and / or whose VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (m) VH and VL, compared with the VH and VL in any one of groups (a) to (k), its VH has one or several amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof; and / or, its VL has one or several amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof; preferably, the substitutions are conservative substitutions.

26. The anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 25, wherein The antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody or a fully human antibody; Optionally, the antibody or antigen-binding fragment thereof is selected from scFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody and multispecific antibody; For example, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

27. The anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 26, wherein The antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region CH of a human immunoglobulin or a variant thereof; and / or (b) a light chain constant region CL of a human immunoglobulin or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the wild-type sequence from which it is derived; or, the variant has one or more amino acid substitutions, deletions or additions or any combination thereof, such as up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof, compared to the wild-type sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; and / or, the light chain constant region is a kappa or lambda light chain constant region; More preferably, the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region; and / or the antibody or antigen-binding fragment thereof comprises a human κ light chain constant region.

28. The anti-Napi2b antibody or antigen-binding fragment thereof according to claim 27, wherein The heavy chain constant region comprises a CH as set forth in SEQ ID NO: 70 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 70, such as up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids; or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO: 70; and / or the light chain constant region comprises the CL set forth in SEQ ID NO:80 or a variant thereof having up to 20 conservative amino acid substitutions, such as up to 20, up to 15, up to 10, or up to 5 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, compared to SEQ ID NO:80, or having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:80; Preferably, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region CH shown in SEQ ID NO: 70 and the light chain constant region CL shown in SEQ ID NO:

80.

29. The anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 28, wherein The antibodies include: (a) a heavy chain comprising the VH shown in SEQ ID NO: 71 and the CH shown in SEQ ID NO: 70, and a light chain comprising the VL shown in SEQ ID NO: 72 and the CL shown in SEQ ID NO: 80; (b) a heavy chain comprising the VH shown in SEQ ID NO: 73 and the CH shown in SEQ ID NO: 70, and a light chain comprising the VL shown in SEQ ID NO: 72 and the CL shown in SEQ ID NO: 80; preferably comprising the heavy chain shown in SEQ ID NO: 81 and the light chain shown in SEQ ID NO: 82; (c) a heavy chain comprising VH shown in SEQ ID NO: 17 and CH shown in SEQ ID NO: 70, and a light chain comprising VL shown in SEQ ID NO: 18 and CL shown in SEQ ID NO: 80; preferably comprising a heavy chain shown in SEQ ID NO: 83 and a light chain shown in SEQ ID NO:

84.

30. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 21, wherein: Tb is selected from the anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 29, 31. The antibody-drug conjugate or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1-21 and 30, characterized in that: in, The antibody drug conjugate is selected from: Wherein, Tb1 is lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2 and 16G5B3-hz1 antibodies; q is selected from any value between 0.1-16.0, preferably any value between 2-8, and more preferably, q is 2, 4, 6 or 8.

32. A method for preparing the antibody drug conjugate of any one of claims 1-21, 30, and 31, comprising: Conjugate Tb with the drug linker shown in formula III Carry out the coupling reaction in a suitable solvent and conditions; in: L1, L2, L3, L4, Tb are as defined in any one of claims 1 to 21; R1, R2, R3, X, and W are as defined in claims 1 to 21; Lg is a leaving group, and Lg is selected from halogen, sulfone group, tertiary amine salt group (Me3N+, Et3N+), diazonium salt group, -OMs, MeSO2-, CF3SO3-.

33. The method of claim 32, wherein The method comprises conjugating Tb with a drug linker shown in formula III The step of performing a coupling reaction to form a C-S bond in a suitable solvent and conditions; Preferably, the molar ratio of the Tb to the drug-linker conjugate is 1:(1-20); Preferably, the coupling reaction is carried out in water and / or an organic solvent; preferably, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (such as methanol, ethanol) or any combination thereof; the nitrile may be acetonitrile, and the alcohol may be methanol or ethanol; Preferably, the method further comprises a step of purifying the coupled product; preferably, the coupled product is purified by a chromatography method; preferably, the chromatography method comprises one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography.

34. An isolated nucleic acid molecule encoding the anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 29.

35. A vector comprising the isolated nucleic acid molecule of claim 34; preferably, the vector is a cloning vector or an expression vector.

36. A host cell comprising the isolated nucleic acid molecule of claim 34 or the vector of claim 35.

37. A method for preparing the anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 29, comprising culturing the host cell according to claim 36 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

38. An antibody-drug conjugate, wherein the antibody is the anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22 to 29, which is connected to a coupling portion via a linker, and the coupling portion is selected from: a detectable label, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide with binding activity, a protein, a receptor, a ligand, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

39. A population of antibody drug conjugates comprising the antibody drug conjugate of any one of claims 1-21, 30, 31, and 38, wherein the antibody drug conjugate has one, two, or more q values; 40. A pharmaceutical composition comprising a stereoisomer of the antibody-drug conjugate of any one of claims 1-21, 30, 31, 38, a prodrug thereof, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, or an anti-Napi2b antibody or antigen-binding fragment thereof according to any one of claims 22-29, or a nucleic acid molecule according to claim 34, or a vector according to claim 35, or a host cell according to claim 36, or a group of antibody-drug conjugates according to claim 39; preferably further comprising a pharmaceutically acceptable carrier and / or excipient.

41. The group according to claim 39 or the pharmaceutical composition according to claim 40, characterized in that The drug to antibody ratio (DAR) is an integer or decimal selected from 1-10; Preferably, the drug to antibody ratio (DAR) of the population is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5 and 7.5-8.5; Preferably, the drug to antibody ratio (DAR) of the population is selected from the group consisting of about 2.0, 4.0, 6.0, and 8.0; Preferably, the drug to antibody ratio (DAR) of the population is selected from the group consisting of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.

42. Use of a stereoisomer of the antibody-drug conjugate of any one of claims 1-21, 30, 31, 38, a prodrug thereof, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, or the anti-Napi2b antibody or antigen-binding fragment thereof of any one of claims 22-2, 9, or the nucleic acid molecule of claim 34, or the vector of claim 35, or the host cell of claim 36, or the population of antibody-drug conjugates of claim 39, or the pharmaceutical composition of claim 40 in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity; Optionally, the disease associated with abnormal cell activity may be cancer; Preferably, the cancer disease is selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumor, prostate cancer or thyroid cancer; the esophageal cancer is such as esophageal adenocarcinoma or esophageal squamous cell carcinoma; the lung cancer is such as small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma; the central nervous system tumor is such as glioma, glioblastoma multiforme, glioma or sarcoma; the colon cancer is such as human colon adenocarcinoma; Preferably, the cancer disease is selected from colon cancer, colorectal cancer, colon adenocarcinoma, small cell lung cancer, ovarian cancer; More preferably, the cancer disease is a cancer disease associated with Napi2b.

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