A kind of exitecan derivative and preparation method thereof

Abstract

The present invention provides an exotecan derivative and a preparation method thereof, wherein the exotecan derivative is represented by formula (Ia) or formula (Ib) or a stereoisomer thereof and a pharmaceutically acceptable salt thereof. wherein n is an integer from 2 to 6; X1 is selected from a heteroatom; R1 is selected from H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic group; and each of R2 and R3 is independently selected from absence, H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic group.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an exitecan derivative and a preparation method thereof. Background Art

[0002] Ixitecan and its derivatives are potent DNA topoisomerase I inhibitors that can serve as payloads for antibody-drug conjugates (ADCs) targeting HER2. In practical applications, ixitecan derivatives are often required to exhibit high activity and membrane permeability. Therefore, there is a need for ixitecan derivatives with enhanced activity and membrane permeability, as well as methods for their preparation. Summary of the Invention

[0003] One embodiment of this specification provides an exotecan derivative. The exotecan derivative is as shown in formula (Ia):

[0004]

[0005] or its stereoisomers and pharmaceutically acceptable salts thereof, wherein n is an integer of 2-6; X1 is selected from a heteroatom; R1 is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon group; each of R2 and R3 is independently selected from not existing, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon group.

[0006] In some embodiments, the R1 is selected from substituted or unsubstituted C1-C6 alkyl, and the exitecan derivative is as shown in formula (Ia1): or its stereoisomers and pharmaceutically acceptable salts thereof, wherein n is an integer of 2-6; m is an integer of 0-5; and each of X2, X3 and X4 is independently selected from H, halogen, substituted or unsubstituted methyl.

[0007] In some embodiments, X2, X3, and X4 are the same.

[0008] In some embodiments, X2, X3, and X4 are the same halogen.

[0009] In some embodiments, X2, X3, and X4 are F.

[0010] In some embodiments, two of X2, X3, and X4 are methyl groups, and the remaining one is H.

[0011] In some embodiments, X1 is selected from N or O.

[0012] In some embodiments, X1 is selected from N, and each of R2 and R3 is independently selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted aromatic hydrocarbon.

[0013] In some embodiments, one of R2 and R3 is selected from H and the other is selected from substituted C1-C6 alkyl.

[0014] In some embodiments, X1 is selected from O, and the exitecan derivative is as follows: or its stereoisomers and pharmaceutically acceptable salts thereof, wherein R2 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon.

[0015] In some embodiments, the substituents in the alkyl group, the cycloalkyl group, and the aromatic hydrocarbon group are each independently selected from halogen, hydroxyl, amino, methyl, or methylamino.

[0016] One of the embodiments of this specification also provides a method for preparing an exotecan derivative. The exotecan derivative is as follows: or its stereoisomers and pharmaceutically acceptable salts thereof, wherein n is an integer of 2-6; R1 is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon; the method comprises: in the presence of DMA and DIPEA, reacting the isotecan salt with the compound of formula (II) The mixture is mixed and reacted to obtain the exitecan derivative.

[0017] In some embodiments, the substituents in the alkyl group, the cycloalkyl group, and the aromatic hydrocarbon group are each independently selected from halogen, hydroxyl, amino, methyl, or methylamino.

[0018] One of the embodiments of this specification also provides a method for preparing an exitecan derivative. The exitecan derivative is as shown in formula (Ib): or its stereoisomers and pharmaceutically acceptable salts thereof, wherein X1 is selected from a heteroatom; each of R2 and R3 is independently selected from non-existent, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon; the method comprises: in the presence of DMF and DIPEA, reacting an isotecan salt with a carbonyl-containing compound, a compound of formula (III) Mix and react to obtain a compound of formula (IV) The compound of molecular formula (IV) reacts in the presence of TFA and DCM to generate the isotecan derivative, wherein R4 includes tert-butyloxycarbonyl or tert-butyldimethylsilyl. During the reaction of the compound of molecular formula (IV) to generate the isotecan derivative as described in formula (Ib), the tert-butyloxycarbonyl or tert-butyldimethylsilyl in R4 is replaced by H to obtain R3.

[0019] In some embodiments, the carbonyl-containing compound comprises carbonylimidazole.

[0020] In some embodiments, X1 is selected from N, and each of R2 and R3 is independently selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted aromatic hydrocarbon. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The graph shows the dose-response curves of NCI-H292 cells (seeding density 500 cells / well) exposed to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 after culturing at 37°C for 72 hours.

[0022] Figure 2 Figure 2 is a dose-response curve of OVCAR-3 cells (seeding density 1200 cells / well) exposed to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 after incubation at 37°C for 72 h.

[0023] Figure 3 It is a dose-response curve after OV90 (seeding density of 1500 cells / well) was exposed to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 and cultured at 37°C for 72 hours.

[0024] Figure 4 This is a dose-response curve after exposing Raji (seeding density of 1000 cells / well) to different concentrations of compound PB-D21, compound PB-D22, compound PB-D24 and exatecan and culturing at 37°C for 72 hours. DETAILED DESCRIPTION

[0025] The following description is made in conjunction with specific embodiments. It should be noted that the description and embodiments given here are only for illustrating the specific embodiments of the present application, in order to make the features of the embodiments of the present application easier to understand, and are not intended to limit the scope of the claims.

[0026] The terminology used herein is intended only to describe specific example embodiments and is not intended to be limiting. As used herein and in the appended claims, unless the context clearly indicates otherwise, the terms "a," "an," "an," and / or "the" are not intended to refer to the singular but may include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list; a method or apparatus may also include additional steps or elements.

[0027] As used herein, the term "stereoisomers" refers to molecules in which the atoms or groups are identical in the order of attachment but differ in their arrangement in space.

[0028] As described herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the general formula with an acid or base that is suitable for use as a drug. In some embodiments, a pharmaceutically acceptable salt may include an inorganic salt or an organic salt.

[0029] As used herein, the term "substituted" means that any one or more atoms or groups (eg, hydrogen) on the designated atoms or groups are replaced with the designated atoms or groups selected from the designated atoms or groups.

[0030] As described herein, the term "alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group that is straight or branched. In some embodiments, the alkyl group may include 1-6, 1-5, 1-4, 1-3, or 1-2 carbon atoms. In some embodiments, the alkyl group may include, but is not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, and n-hexyl. In some embodiments, the alkyl group may be methyl, ethyl, n-propyl, or isopropyl.

[0031] As described herein, the term "cycloalkyl" used alone or in combination with other terms refers to a non-aromatic hydrocarbon ring system (e.g., monocyclic, bicyclic, or polycyclic). In some embodiments, cycloalkyl can refer to a cyclized alkyl. In some embodiments, cycloalkyl can be a monocyclic C3-C6 alkyl, C3-C5 alkyl, or C3-C4 alkyl. For example, cycloalkyl can include, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0032] As described herein, the term "aryl" used alone or in combination with other terms refers to a monocyclic or polycyclic aromatic hydrocarbon group. In some embodiments, the aromatic hydrocarbon group may include 6-10 carbon atoms. For example, the aromatic hydrocarbon group may include, but is not limited to, a phenyl group.

[0033] As used herein, "halogen" refers to fluorine, chlorine, bromine, and iodine. In some embodiments, halogen can refer to a halogen atom selected from F, Cl, or Br. In some embodiments, halogen can be F.

[0034] Some commonly used abbreviations in this manual have the following meanings:

[0035] DMA: N,N-dimethylacetamide, chemical formula is CH3C(O)N(CH3)2

[0036] DIPEA: N,N-diisopropylethylamine

[0037] TFA: trifluoroacetic acid, chemical formula is CF3COOH

[0038] DCM: dichloromethane, chemical formula is CH2Cl2

[0039] CDI: Carbonyl imidazole

[0040] 2,6-Lutidine: 2,6-dimethylpyridine

[0041] DMF: N,N-dimethylformamide

[0042] PNPC: Bis(p-nitrobenzene) carbonate

[0043] DMAP: 4-dimethylaminopyridine

[0044] Tert-butyloxycarbonyl: The structural formula is Abbreviated as -Boc.

[0045] Tert-butyldimethylsilyl: The structural formula is The abbreviation is -TBS.

[0046] Tert-butyldimethylsilyloxy: The structural formula is Abbreviated as -OTBS.

[0047] The present invention provides an exotecan derivative. One of the structural formulas of exotecan can be represented as:

[0048]

[0049] Ixitecan derivatives may refer to compounds obtained by replacing the H on the amino group (—NH2) in Ixitecan with other atoms or functional groups.

[0050] In some embodiments, the exitecan derivatives can be represented by formula (Ia):

[0051] (Ia)

[0052]

[0053] or its stereoisomers and pharmaceutically acceptable salts thereof. wherein n is an integer of 2-6; and R1 is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted aromatic hydrocarbon.

[0054] In some embodiments, n can be an integer from 2 to 5. In some embodiments, n can also be an integer from 2 to 4. In some embodiments, n can also be an integer from 2 to 3. In some embodiments, n can be 2, 3, 4, 5, or 6.

[0055] In some embodiments, the substituents in the alkyl, cycloalkyl, and aromatic groups are each independently selected from halogen, hydroxyl (—OH), amino (—NH 2 ), methyl (—CH 3 ), or methylamino (—NH—CH 3 ).

[0056] In some embodiments, R1 can be selected from substituted or unsubstituted C1-C6 alkyl groups. For example, R1 can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, a (a is an integer of 1-3) halogen atoms, hydroxyl, amino, methyl or methylamino substituted methyl, b (b is an integer of 1-5) halogen atoms, hydroxyl, amino, methyl or methylamino substituted ethyl, etc. As an example, R1 can be trifluoromethyl (—CF3), trichloromethyl (—CCl3), tribromomethyl (—CBr3), 2,2,2-trifluoroethyl (—CH2—CF3), 2,2,2-trichloroethyl (—CH2—CCl3), 2,2,2-tribromoethyl (—CH2—CBr3), —CH2CH2OH, —CH2CH2NH2, —CH(CH3)2, —C(CH3)3, —CH2NHCH3, —CH2CH2NHCH3, etc.

[0057] In some embodiments, R1 is selected from substituted or unsubstituted C1-C6 alkyl, and the exitecan derivative can be as shown in formula (Ia1):

[0058] (Ⅰa1)

[0059]

[0060] or its stereoisomers and pharmaceutically acceptable salts thereof. wherein n is an integer of 2-6; m is an integer of 0-5; and each of X2, X3 and X4 is independently selected from H, halogen, substituted or unsubstituted methyl.

[0061] In the compound of formula (Ia1), n ​​is as described above for the compound of formula (Ia). In some embodiments, m can be an integer from 1 to 5. In some embodiments, m can also be an integer from 1 to 4. In some embodiments, m can also be an integer from 1 to 3. In some embodiments, m can be an integer from 1 to 2. In some embodiments, m can be 0, 1, 2, 3, 4, or 5.

[0062] In some embodiments, the substituents of the methyl groups in X2, X3, and X4 may include, but are not limited to, halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3), or methylamino (—NH—CH3).

[0063] In some embodiments, X2, X3, and X4 may be identical. For example, X2, X3, and X4 may all be H. For another example, X2, X3, and X4 may be identical halogens. As an example, X2, X3, and X4 may all be F. As another example, X2, X3, and X4 may all be Cl or Br. For another example, X2, X3, and X4 may all be methyl groups. For another example, X2, X3, and X4 may all be methyl groups substituted with the same substituent. The same substituent may mean that the spatial position and number of H groups of the methyl groups substituted with the substituents are equal.

[0064] In some embodiments, X2, X3, and X4 may be different. For example, any two of X2, X3, and X4 are methyl, and the remaining one is H. For another example, any two of X2, X3, and X4 are the same or different halogens, and the remaining one is H. For another example, any two of X2, X3, and X4 are substituted methyl, and the remaining one is H. For another example, any two of X2, X3, and X4 are methyl or substituted methyl, and the remaining one is halogen. For another example, any two of X2, X3, and X4 are the same or different halogens, and the remaining one is methyl or substituted methyl. In some embodiments, X2, X3, and X4 may be different from each other. For example, one of X2, X3, and X4 is H, one is halogen, and one is methyl or substituted methyl. For another example, X2, X3, and X4 may be different halogens. For another example, X2, X3, and X4 may be methyl with different substituents.

[0065] In some embodiments, R1 can be selected from substituted or unsubstituted cycloalkyl. For example, R1 can be selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl substituted with halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3) or methylamino (—NH—CH3). As an example, R1 can be In some embodiments, R1 can also be selected from substituted or unsubstituted heterocyclic groups. Wherein, the heteroatom can be N, O or S. The substituent of the heterocyclic group can be selected from halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3) or methylamino (—NH—CH3). For example, R1 can be

[0066] In some embodiments, R1 can be selected from substituted or unsubstituted aromatic hydrocarbon groups. For example, R1 can be substituted or unsubstituted phenyl. The substituents of the phenyl group can include but are not limited to halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3) or methylamino (—NH—CH3). For example, R1 can be a phenyl group substituted with d (d is an integer from 1 to 5) of the above substituents. As an example, R1 can be

[0067] In some embodiments, the exitecan derivatives may also be methylene (—(CH2) n —) Compounds connected to R1 to form epoxy ethers. For example, the ixitecan derivative can be represented by formula (Ia2):

[0068] (Ia2)

[0069]

[0070] or its stereoisomers and pharmaceutically acceptable salts thereof. wherein p is an integer from 0 to 4, and q is an integer from 1 to 4; or p is an integer from 1 to 4, and q is an integer from 0 to 4. As an example, p may be 1, and q may be 1. As another example, p may be 2, and q may be 1. As yet another example, p may be 2, and q may be 2.

[0071] The present invention also provides another exotecan derivative. The exotecan derivative can be as shown in formula (Ib):

[0072] (Ib)

[0073]

[0074] or its stereoisomers and pharmaceutically acceptable salts thereof. wherein X1 is selected from a heteroatom; and each of R2 and R3 is independently selected from absence, H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic group.

[0075] In some embodiments, the heteroatom may include N, O, or S atoms. In some embodiments, X 1 may be selected from N or O atoms.

[0076] In the compound of formula (Ib), the substituents in the alkyl, cycloalkyl and aromatic groups are independently selected from halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3) or methylamino (—NH—CH3).

[0077] In some embodiments, when X1 is selected from N, each of R2 and R3 is independently selected from H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic group. In some embodiments, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from C1-C6 alkyl group. In some embodiments, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from a substituted C1-C6 alkyl group. For example, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from a hydroxy-substituted C1-C6 alkyl group. As an example, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from —CH2OH. As another example, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from —CH2CH2OH. In some embodiments, when X1 is selected from N, one of R2 and R3 can be selected from C1-C6 alkyl group, and the other can be selected from a substituted C1-C6 alkyl group. For example, when X1 is selected from N, one of R2 and R3 can be selected from a methyl group, and the other can be selected from a methylamino-substituted ethyl group (—CH2CH2NHCH3). For another example, when X1 is selected from N, one of R2 and R3 can be selected from an ethyl group, and the other can be selected from an amino-substituted methyl group. In some embodiments, when X1 is selected from N, one of R2 and R3 can be selected from H, and the other can be selected from a substituted or unsubstituted cycloalkyl group. In some embodiments, when X1 is selected from N, one of R2 and R3 can be selected from a C1-C6 alkyl group, and the other can be selected from a substituted or unsubstituted aromatic hydrocarbon group.

[0078] In some embodiments, when X1 is selected from N, N, R2 and R3 can be linked to form a heterocyclic group. For example, the Xitecan derivative can be represented by formula (Ib2):

[0079] (Ⅰb2)

[0080]

[0081] or its stereoisomers and pharmaceutically acceptable salts thereof. Wherein, x is an integer of 1-4, and y is an integer of 1-4. As an example, x can be 1 and y can be 1. As another example, x can be 2 and y can be 2.

[0082] In some embodiments, X1 is selected from O, and the exitecan derivative can be represented by formula (Ib1):

[0083] (Ⅰb1)

[0084]

[0085] or its stereoisomers and pharmaceutically acceptable salts thereof. Wherein, R2 is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon. In the compound of molecular formula (Ib1), the substituents in the alkyl, cycloalkyl and aromatic hydrocarbon groups are each independently selected from halogen, hydroxyl (—OH), amino (—NH2), methyl (—CH3) or methylamino (—NH—CH3). For example, R2 can be a C1-C6 alkyl substituted with the above substituents. For example, R2 can be a C3-C6 cycloalkyl substituted with the above substituents. As an example, R2 can be selected from For another example, R2 can be a phenyl group substituted by the above substituents. As an example, R2 can be selected from

[0086] The present invention also provides a method for preparing an isotecan derivative. The isotecan derivative is represented by the above formula (Ia), (Ia1), (Ia2) or its stereoisomers and pharmaceutically acceptable salts thereof. The preparation method may include: in the presence of DMA and DIPEA, reacting an isotecan salt with a compound of formula (II')

[0087] (II')

[0088]

[0089] Mix and react to obtain the ixetine derivatives shown in formula (Ia), (Ia1), and (Ia2). In formula (Ia1) In formula (Ia2) wherein m, n, p, q, R1, X2, X3, and X4 are as defined above.

[0090] In some embodiments, the exitecan salt may include exitecan hydrochloride or exitecan mesylate, etc.

[0091] In some embodiments, the reaction of an exitecan salt with a compound of formula (II') to produce an exitecan derivative as shown in formula (Ia), (Ia1), or (Ia2) can be carried out at a temperature between 20°C and 50°C. In some embodiments, the reaction temperature can be between 20°C and 45°C. In some embodiments, the reaction temperature can be between 20°C and 40°C. In some embodiments, the reaction temperature can be room temperature. In the embodiments of this specification, room temperature can be 25°C ± 5°C, or room temperature can refer to a temperature between 20°C and 30°C.

[0092] In some embodiments, the reaction time of mixing the isotecan salt with the compound of formula (II') to form the isotecan derivatives represented by formula (Ia), (Ia1), and (Ia2) can be within the range of 15 h to 20 h. In some embodiments, the reaction time can be within the range of 18 h to 20 h.

[0093] During the reaction, one H in -NH2 of the isotecan salt is replaced by R5 in the compound of molecular formula (II') to obtain isotecan derivatives shown in formulas (Ia), (Ia1) and (Ia2).

[0094] In some embodiments, the isotecan derivative is as shown in the above formula (Ia) or its stereoisomers and pharmaceutically acceptable salts thereof, and the preparation method may include: in the presence of DMA and DIPEA, reacting isotecan salt with a compound of formula (II)

[0095] (II)

[0096]

[0097] The mixture is reacted to obtain an isotecan derivative as shown in formula (Ia). Wherein, n is an integer of 2-6, and R1 is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic hydrocarbon. Specifically, n and R1 can be as defined above. In some embodiments, the substituents in the alkyl, cycloalkyl, and aromatic hydrocarbon groups can be independently selected from halogen, hydroxyl, amino, methyl, or methylamino. During the reaction, one H in the -NH2 of the isotecan salt is replaced by the H in the compound of molecular formula (II). Substitution can give the exitecan derivative shown in formula (Ia).

[0098] In some embodiments, the isotecan derivative is as shown in the above formula (Ib) or its stereoisomers and pharmaceutically acceptable salts thereof, and the preparation method may include: in the presence of DMF and DIPEA, reacting an isotecan salt with a carbonyl-containing compound and a compound of formula (III)

[0099] (III)

[0100]

[0101] Mix the reaction to obtain a compound of formula (IV)

[0102] (IV)

[0103] The compound of formula (IV) is reacted in the presence of TFA and DCM to produce the exitecan derivative shown in formula (Ib). wherein X1 is selected from N, and R2 and R3 are as defined above. Each of R2 and R3 is independently selected from H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted aromatic group. In some embodiments, R4 is selected from an alkyl group substituted with tert-butyloxycarbonyl or tert-butyldimethylsilyl in R3, a cycloalkyl group substituted with tert-butyloxycarbonyl or tert-butyldimethylsilyl in R3, or an aromatic group substituted with tert-butyloxycarbonyl or tert-butyldimethylsilyl in R3. In some embodiments, R4 comprises tert-butyloxycarbonyl or tert-butyldimethylsilyl. In some embodiments, the tert-butyloxycarbonyl or tert-butyldimethylsilyl group in R4 is attached to the nitrogen atom. During the reaction of the compound of formula (IV) to produce the exitecan derivative shown in formula (Ib), the tert-butyloxycarbonyl or tert-butyldimethylsilyl group in R4 is replaced by H to produce R3. For example, R4 is R3 is For another example, R4 is R3 is For another example, R4 is R3 is

[0104] A carbonyl-containing compound may refer to a compound containing a carbonyl group In some embodiments, the carbonyl-containing compound may include, but is not limited to, carbonylimidazole.

[0105] In some embodiments, the reaction of the isocyanate salt with the carbonyl-containing compound and the compound of formula (III) to produce the compound of formula (IV) in the presence of DMF and DIPEA can be carried out at room temperature. In some embodiments, the reaction time of the isocyanate salt with the carbonyl-containing compound and the compound of formula (III) to produce the compound of formula (IV) can be in the range of 3 hours to 16 hours. In some embodiments, the reaction time can be in the range of 4.5 hours to 14 hours. In some embodiments, the reaction time can be in the range of 4.5 hours to 10 hours. In some embodiments, the reaction time can be in the range of 6 hours to 8 hours.

[0106] In some embodiments, the reaction of the compound of formula (IV) in the presence of TFA and DCM to produce the isotecan derivative represented by formula (Ib) can be carried out at room temperature. In some embodiments, the reaction time for the compound of formula (IV) in the presence of TFA and DCM to produce the isotecan derivative represented by formula (Ib) can be in the range of 0.5 h to 3 h. In some embodiments, the reaction time can be in the range of 1 h to 3 h. In some embodiments, the reaction time can be in the range of 1 h to 2 h. During this reaction, the tert-butyloxycarbonyl or tert-butyldimethylsilyl group on R4 in the compound of formula (IV) is replaced by H, such that R4 in the compound of formula (IV) becomes R3, to produce the compound represented by formula (Ib).

[0107] Example 1 Preparation of Compound PB-D5

[0108] To 32 mg (approximately 0.069 mmol) of isotecan hydrochloride, add 1.0 mL of DMA. Then, add 90.30 mg (approximately 0.344 mmol) of 2-(trifluoromethoxy)ethyl trifluoromethanesulfonate and 44.52 mg (approximately 0.344 mmol) of DIPEA dropwise at room temperature. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0109]

[0110] The reaction solution was separated and purified by preparative HPLC to obtain 13.0 mg of compound PB-D5 as a white solid with a yield of 34.4%.

[0111] The compound PB-D5 prepared in Example 1 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 548.5 [M+H] + , HPLC retention time 6.624min.

[0112] The compound PB-D5 obtained in Example 1 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400MHz, DMSO-d6) δ7.73(d,J=11.2Hz,1H),7.30(s,1H),6.52(s,1H),5.43(s,2H),5.40-5.30(m,2H),4.32-4.27(m,1H),4.21 -4.11(m,2H),3.28-2.98(m,5H),2.37(s,3H),2.27-2.16(m,1H),2.13-2.03(m,1H),1.93-1.79(m,2H),0.87(t,J=7.6Hz,3H)ppm.

[0113] Example 2 Preparation of Compound PB-D6

[0114] To 54 mg (approximately 0.115 mmol) of isotecan hydrochloride, add 3.5 mL of DMA. Then, add 118.35 mg (approximately 0.574 mmol) of 3-oxetane trifluoromethanesulfonate and 44.52 mg (approximately 0.344 mmol) of DIPEA dropwise at room temperature. After the addition is complete, stir and react at 45°C for 20 hours to obtain a reaction solution. The synthesis process is as follows:

[0115]

[0116] The reaction solution was separated and purified by preparative high performance liquid chromatography to obtain 13.0 mg of compound PB-D6 as a white solid with a yield of 21.85%.

[0117] The compound PB-D6 prepared in Example 2 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 492.4 [M+H] + , HPLC retention time 5.886min.

[0118] The compound PB-D6 prepared in Example 2 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR(400MHz,DMSO-d6)δ7.73(d,J=11.2Hz,1H),7.30(s,1H),6.52(s,1H),5.42(s,2 H),5.40-5.29(m,2H),4.73(t,J=6.4Hz,1H),4.66(t,J=6.4Hz,1H),4.42(t,J=6.4H z,1H),4.36(t,J=6.4Hz,1H),4.29-4.23(m,1H),4.14(t,J=6.4Hz,1H),3.05-2.93( m,2H),2.37(s,3H),2.07-1.93(m,2H),1.92-1.81(m,2H),0.87(t,J=7.2Hz,3H)ppm.

[0119] Example 3 Preparation of Compound PB-D7

[0120] To 5.0 g (48.0 mmol) of isopropoxyethanol was added 60 mL of DCM and 9.31 g (72.0 mmol) of DIPEA. At -20°C, 9.56 mL (57.6 mmol) of trifluoromethanesulfonic anhydride was added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 18 hours to obtain a reaction solution. The reaction solution was washed with water, and the aqueous layer was extracted with DCM. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 14.17 g of compound N7. Compound N7 was a brown liquid with a yield of 100%.

[0121] To 27 mg (approximately 0.0575 mmol) of isotecan hydrochloride, add 0.5 mL of DMA. Then, add 40 mg (approximately 0.1723 mmol) of compound N7 and 37 mg (approximately 0.287 mmol) of DIPEA dropwise at room temperature. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0122]

[0123]

[0124] The reaction solution was separated and purified by preparative HPLC to obtain 10.5 mg of compound PB-D7 as a brown solid with a yield of 34.9%.

[0125] The compound PB-D7 prepared in Example 3 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 522.3 [M+H] + , HPLC retention time 5.891min.

[0126] The compound PB-D7 prepared in Example 3 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR(400MHz,DMSO-d6)δ7.74(d,J=11.2Hz,1H),7.30(s,1H),6.51(s,1H),5.4 7-5.32(m,4H),4.30(t,J=4.0Hz,1H),3.63-3.43(m,5H),3.06-2.95(m,1H),3 .94-2.87(m,1H),2.85-2.75(m,1H),2.37(s,3H),2.28-2.15(m,1H),2.13-2. 00(m,1H),1.93-1.81(m,1H),1.11(t,J=6.4Hz,6H),0.88(t,J=7.2Hz,3H)ppm.

[0127] Example 4 Preparation of Compound PB-D19

[0128] To 65 mg (approximately 0.115 mmol) of exitecan mesylate, add 2 mL of DMA. Then, add 119.5 mg (approximately 0.574 mmol) of 2-methoxyethyl trifluoromethanesulfonate and 74.2 mg (approximately 0.574 mmol) of DIPEA dropwise at room temperature. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0129]

[0130] The reaction solution was separated and purified by preparative HPLC to obtain 26.0 mg of compound PB-D19 as a white solid with a yield of 44.96%.

[0131] The compound PB-D19 prepared in Example 4 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 494.3 [M+H] + , HPLC retention time 5.385min.

[0132] The compound PB-D19 prepared in Example 4 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400MHz, DMSO-d6): δ9.04(s,1H),8.74(s,1H),7.90(d,J=10.8Hz,1H),7.35(s,1H),6.59(s,1H),5.64-5.27(m,4H),5.07(s,1H),3.64-3 .59(m,2H),3.35(s,3H),3.25-3.07(m,4H),2.81-2.78(m,1H),2.42(s ,3H),2.15-2.20(m,1H),1.93-1.84(m,2H),0.87(t,J=7.6Hz,3H)ppm.

[0133] Example 5 Preparation of Compound PB-D21

[0134] To 76 mg (approximately 0.161 mmol) of isotecan hydrochloride, add 1 mL of DMA. Then, add 178.44 mg (approximately 0.804 mmol) of 2-ethoxyethyl trifluoromethanesulfonate and 103.89 mg (approximately 0.804 mmol) of DIPEA dropwise at room temperature. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0135]

[0136] The reaction solution was separated and purified by preparative HPLC to obtain 25 mg of compound PB-D21 as a white solid with a yield of 30.64%.

[0137] The compound PB-D21 prepared in Example 5 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 508.3 [M+H] + , HPLC retention time 5.510min.

[0138] The compound PB-D21 prepared in Example 5 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR(400MHz,DMSO-d6)δ9.01(s,1H),8.72(s,1H),7.91(d,J=12.8Hz,1H),7.36(s,1H),6 .50(s,1H),5.53(d,J=19.2Hz,1H),5.47(s,2H),5.40(d,J=19.2Hz,1H),5.09-5.08(m,1H ),3.72-3.64(m,2H),3.61-3.53(m,2H),3.23-3.11(m,4H),2.81-2.76(m,1H),2.42(s,3H ), 2.23-2.11(m,1H),1.95-1.82(m,2H),1.22(t,J=7.2Hz,3H),0.88(t,J=7.6Hz,3H)ppm.

[0139] Example 6 Preparation of Compound PB-D22

[0140] To 300.0 mg (1.561 mmol) of 2-(2,3,6-trifluorophenoxy)ethanol and 0.218 mL (approximately 1.874 mmol) of 2,6-lutidine was added 10 mL of DCM, followed by 0.311 mL (approximately 1.874 mmol) of trifluoromethanesulfonic anhydride at 0°C. After the addition was complete, the mixture was stirred at 0°C for 1 hour, then warmed to room temperature and stirred for another hour to obtain a reaction solution. The reaction solution was extracted with DCM, washed with water, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 420 mg of compound N22. Compound N22 was a brown oily liquid with a yield of 66.40%.

[0141] Compound N22 was analyzed by nuclear magnetic resonance, and the results are as follows: 1 H NMR (400MHz, CDCl3) δ6.98-6.79 (m, 2H), 4.79 (dd, J = 9.2, 5.2Hz, 2H), 4.48 (dd, J = 5.2, 3.6Hz, 2H) ppm.

[0142] To 65 mg (approximately 0.138 mmol) of isoproterenol hydrochloride, add 1 mL of DMA. Then, at room temperature, add 420 mg (approximately 1.296 mmol) of compound N7 and 89.05 mg (approximately 0.689 mmol) of DIPEA dropwise. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0143]

[0144] The reaction solution was separated and purified by preparative HPLC to obtain 16 mg of compound PB-D22 as a yellow solid with a yield of 17.72%.

[0145] The compound PB-D22 prepared in Example 6 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 610.4 [M+H] + , HPLC retention time 7.148min.

[0146] The compound PB-D22 prepared in Example 6 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR(400MHz,DMSO)δ9.38(s,1H),9.10(s,1H),7.98-7.83(m,1H),7.36-7.25(m,3H),6.56(s,1H),5.67-5.45(m,4H),5.25-5.20(m,1H),4.5 0-4.31(m,2H),3.68-3.54(m,2H),3.30-3.16(m,2H),2.80-2.70(m,1H ), 2.42 (s, 3H), 2.23 (s, 1H), 2.00-1.74 (m, 2H), 0.88 (t, J = 7.2Hz, 3H).

[0147] Example 7 Preparation of Compound PB-D23

[0148] To 1 g (6.940 mmol) of 2-(2,2,2-trifluoroethoxy)ethanol was added 20 mL of DCM, followed by 1.728 mL (approximately 10.410 mmol) of trifluoromethanesulfonic anhydride at -20°C. After the addition was complete, 1.79 g (approximately 13.880 mmol) of DIPEA was slowly added, and the mixture was stirred at -20°C for 3 h to yield a reaction solution. The reaction solution was washed with water and extracted with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to yield 2 g of compound N23. Compound N23 was a brown oily liquid.

[0149] To 65 mg (approximately 0.138 mmol) of isoproterenol hydrochloride, add 2 mL of DMA. Then, at room temperature, add 114.15 mg (approximately 0.413 mmol) of compound N23 and 89.05 mg (approximately 0.689 mmol) of DIPEA dropwise. After the addition is complete, stir and react at room temperature for 18 hours to obtain a reaction solution. The synthesis process is as follows:

[0150]

[0151] The reaction solution was separated and purified by preparative HPLC to obtain 12 mg of compound PB-D23. Compound PB-D22 was a white solid with a yield of 14.89%.

[0152] The compound PB-D23 prepared in Example 7 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 562.3 [M+H] + , HPLC retention time 7.205min.

[0153] The compound PB-D23 prepared in Example 7 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400MHz, DMSO) δ9.14 (s, 1H), 8.90 (s, 1H), 7.89 (d, J = 10.8Hz, 1H), 7.36 (s, 1H), 6. 59(s,1H),5.52(d,J=19.6Hz,1H),5.46(s,2H),5.40(d,J=19.2Hz,1H),5.06(brs,1H) ,4.16(q,J=9.2Hz,2H),3.86(s,2H),3.59-3.55(m,2H),3.124-3.10(m,2H),2.84-2.7 0(m,1H),2.42(s,3H),2.24-2.11(m,1H),1.94-1.82(m,2H),0.88(t,J=7.2Hz,3H)ppm.

[0154] Example 8 Preparation of Compound PB-D11

[0155] 2 mL of DMF was added to 50 mg (about 0.094 mmol) of isotecan mesylate, and 12.16 mg (about 0.094 mmol) of DIPEA and 18.3 mg (about 0.113 mmol) of CDI were added at room temperature. After the addition was complete, the reaction was stirred at room temperature for 3 hours, and then 21.25 mg (about 0.113 mmol) of methyl (2- (methylamino) ethyl) carbamate was added and the reaction was continued for 1.5 hours to obtain a reaction solution. The reaction solution was separated and purified by reverse phase chromatography to obtain 34 mg of compound N11. Compound N11 was a yellow solid with a yield of 51.73%. Compound N11 was analyzed by liquid chromatography-mass spectrometry, and the results were as follows: ESI m / z: 650.3 [M+H] + .

[0156] To 27 mg (approximately 0.00574 mmol) of compound N11, add 2 mL of DCM, cool to 0°C, and add 1 mL (approximately 13.463 mmol) of TFA dropwise. After the addition is complete, stir at room temperature for 3 hours to obtain a reaction solution. The synthesis process is as follows:

[0157]

[0158] The reaction solution was concentrated under reduced pressure and separated and purified by preparative HPLC to obtain 15 mg of compound PB-D11 as a white solid with a yield of 48.13%.

[0159] Compound PB-D11 prepared in Example 8 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 550.3 (M+H) + , HPLC retention time: 7.492 min.

[0160] The compound PB-D11 prepared in Example 8 was analyzed by nuclear magnetic resonance, and the results were as follows: 1H NMR (400MHz, DMSO-d6) δ8.41(brs,2H),7.78(d,J=10.9Hz,1H),7.32(s,1H),7.00(d,J=8.5Hz,1 H),6.56(s,1H),5.50-5.44(m,1H),5.43(s,2H),5.24(d,J=18.8Hz,1H),5.15(dd,J=19.2Hz,1H ),3.71-3.62(m,1H),3.57-3.49(m,1H),3.31-3.20(m,1H),3.13-3.10(m,3H),2.90(s,3H),2.6 5(t,J=5.3Hz,3H),2.40(s,3H),2.21-2.17(m,2H),1.94-1.78(m,2H),0.87(t,J=7.3Hz,3H)ppm.

[0161] Example 9 Preparation of Compound PB-D12

[0162] 1 mL of DMF was added to 30 mg (approximately 0.064 mmol) of isotecan hydrochloride, and 8.22 mg (approximately 0.064 mmol) of DIPEA and 12.37 mg (approximately 0.076 mmol) of CDI were added at room temperature. After the addition was complete, the reaction was stirred at room temperature for 3 hours, and then 14.21 mg (approximately 0.076 mmol) of tert-butyl piperazine-1-carboxylate was added and the reaction was continued for 1.5 hours to obtain a reaction solution. The reaction solution was separated and purified by reverse phase chromatography to obtain 32 mg of compound N12. Compound N12 was a white solid with a yield of 77.71%. Compound N12 was analyzed by liquid chromatography-mass spectrometry, and the results were as follows: ESI m / z: 648.3 [M+H] + .

[0163] To 32 mg (approximately 0.049 mmol) of compound N12, add 2 mL of DCM, cool to 0°C, and add 1 mL (approximately 13.463 mmol) of TFA dropwise. After the addition is complete, stir at room temperature for 3 hours to obtain a reaction solution. The synthesis process is as follows:

[0164]

[0165] The reaction solution was concentrated under reduced pressure and separated and purified by preparative HPLC to obtain 13 mg of compound PB-D12 as a white solid with a yield of 46.62%.

[0166] Compound PB-D12 prepared in Example 9 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 548.3 (M+H) + , HPLC retention time 5.922 min.

[0167] The compound PB-D12 prepared in Example 9 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 6.55 (s, 1H), 5.42 (m, 3H), 5.23 (d, J = 18.8 Hz, 1H), 5.15 (d, J = 18.8 Hz, 1H), 3.74-3.54 (m, 4H), 3.26-3.07 (m, 6H), 2.39 (s, 3H), 2.23-2.13 (m, 2H), 1.86 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H) ppm. Example 10 Preparation of Compound PB-D17

[0168] 5 mL of DMF was added to 300.0 mg (about 1.393 mmol) of N-BOC-cyclohexylamino alcohol, and 540.28 mg (about 4.180 mmol) of DIPEA and 1271.72 mg (about 4.180 mmol) of PNPC were added at room temperature. After the addition was complete, the reaction was stirred at room temperature for 1 hour to obtain a reaction solution. The reaction solution was separated and purified by reverse phase chromatography to obtain 400 mg of compound N17-1. Compound N17-1 was a yellow solid with a yield of 75.4%. Compound N17-1 was analyzed by liquid chromatography-mass spectrometry, and the results were as follows: ESI m / z: 403.1 (M+Na) + .

[0169] 4 mL of DMA was added to 32 mg (approximately 0.069 mmol) of isotecan hydrochloride, and 26.21 mg (approximately 0.069 mmol) of compound N17-1, 13.36 mg (approximately 0.103 mmol) of DIPEA, and 4.21 mg (approximately 0.034 mmol) of DMAP were added at room temperature. After the addition was complete, the reaction was stirred at room temperature for 16 hours to obtain a reaction solution. The reaction solution was separated and purified by reverse phase chromatography to obtain 40 mg of compound N17-2. Compound N17-2 was a yellow solid with a yield of 85.8%. Compound N17-2 was analyzed by liquid chromatography-mass spectrometry, and the results were as follows: ESI m / z: 621.3 [M-55] + .

[0170] To 40 mg (approximately 0.059 mmol) of compound N17-2, 3 mL of DCM was added, and 0.5 mL (approximately 0.074 mmol) of TFA was added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 0.5 h to obtain a reaction solution. The synthesis process is as follows:

[0171]

[0172] The reaction solution was concentrated under reduced pressure and separated and purified by preparative HPLC to obtain 2.2 mg of compound PB-D17-P1 and 2.8 mg of compound PB-D17-P2 as white solids, with a yield of 14.38%.

[0173] The compound PB-D17-P1 prepared in Example 10 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 577.3 (M+H) + , HPLC retention time 5.925 min.

[0174] The compound PB-D17-P1 prepared in Example 10 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400MHz, DMSO-d6) δ8.29(s,1H),7.99(d,J=6.8Hz,1H),7.76(d,J=10.8Hz,1H),7 .30(s,1H),6.54(brs,1H),5.42(s,2H),5.28-5.10(m,3H),4.43(t,J=5.2Hz,1H),3. 45-3.33(m,1H),3.16-3.08(m,1H),2.37(s,3H),2.28-2.20(m,1H),2.15-1.99(m,2H ),1.92-1.81(m,4H),1.73-1.62(m,2H),1.36-1.23(m,6H),0.87(t,J=7.2Hz,3H)ppm.

[0175] The compound PB-D17-P2 prepared in Example 10 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 577.3 (M+H) + , HPLC retention time 6.203 min.

[0176] The compound PB-D17-P2 prepared in Example 10 was analyzed by nuclear magnetic resonance, and the results were as follows: 1H NMR(400MHz,DMSO)δ8.27(s,1H),7.98(d,J=9.2Hz,1H),7.75(d,J=10.8Hz,1H), 7.31(s,1H),5.41(s,2H),5.36-5.12(m,3H),4.423(t,J=5.2Hz,1H),3.41-3.22( m,1H),3.14-3.05(m,1H),2.82-2.70(m,1H),2.36(s,3H),2.27-1.96(m,3H),1. 94-1.78(m,3H),1.73-1.60(m,2H),1.37-1.17(m,6H),0.87(t,J=7.2Hz,3H)ppm.

[0177] Example 11 Preparation of Compound PB-D24

[0178] 1 mL of DMF was added to 60 mg (about 0.113 mmol) of isotecan mesylate, and 14.59 mg (about 0.113 mmol) of DIPEA and 25.62 mg (about 0.158 mmol) of CDI were added at room temperature. After the addition was complete, the reaction was stirred at room temperature for 3 hours, and then 23.75 mg (about 0.135 mmol) of 2-(tert-butyldimethylsilyloxy)ethylamine was added and the reaction was continued for 1.5 hours to obtain a reaction solution. The reaction solution was separated and purified by reverse phase chromatography to obtain 30 mg of compound N24. Compound N24 was a light brown solid with a yield of 39.65%. Compound N24 was analyzed by liquid chromatography-mass spectrometry, and the results were as follows: ESI m / z: 637.3 [M+H] + .

[0179] To 30 mg (approximately 0.047 mmol) of compound N24, 1 mL of DCM was added. 0.5 mL (approximately 6.731 mmol) of TFA was added dropwise at room temperature. After the addition was complete, the mixture was stirred at room temperature for 2 hours to obtain a reaction solution. The synthesis process is as follows:

[0180]

[0181] The reaction solution was concentrated under reduced pressure and separated and purified by preparative HPLC to obtain 8 mg of compound PB-D24 and 8 mg of compound PB-D12 as a light yellow solid with a yield of 32.17%.

[0182] Compound PB-D24 prepared in Example 11 was analyzed by liquid chromatography-mass spectrometry. The results were as follows: ESI m / z: 523.1 (M+H) + , HPLC retention time: 6.124 min.

[0183] The compound PB-D24 prepared in Example 11 was analyzed by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400MHz, DMSO-d6) δ7.77(d,J=10.8Hz,1H),7.31(s,1H),6.72(d,J=8.8Hz,1 H),6.51-6.49(m,1H),5.98(t,J=5.6Hz,1H),5.43(s,2H),5.38-5.33(m,2H),5. 23(d,J=18.8Hz,1H),3.44(t,J=5.6Hz,2H),3.18-3.11(m,4H),2.57-2.51(m,1H ), 2.39 (s, 3H), 2.21-2.10 (m, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.2Hz, 3H) ppm.

[0184] The liquid chromatography separation conditions in the above embodiment are: Waters SunFire 10μm C18 chromatographic column ( 250 x 19 mm). Solvent A was 0.01% trifluoroacetic acid (TFA) in water, and solvent B was acetonitrile. The elution condition was a linear gradient increase of solvent B from 5% to 100% at a flow rate of 30 mL / min over 20 minutes.

[0185] In the examples of this specification, the ionized group (amino group—NH2) in exitecan is converted into a non-ionized group by substitution. Specifically, the group that replaces the H atom in the amino group is a lipophilic group, which can improve its membrane permeability.

[0186] Cell culture and killing experiments

[0187] In the examples herein, NCI-H292 (Kebai, catalog number CBP60187) and OVCAR-3 (Kebai, catalog number CBP60294) were purchased from Kebai Biotechnology. Raji (ATCC, catalog number CCL-86) was purchased from ATCC. OV90 was purchased from Meisen.

[0188] Cell lines were cultured in a CO2 incubator at 37°C with 5% CO2. NCI-H292 and Raji cells were cultured in RPMI1640 (Gibco, catalog number 11875093) medium containing 10% FBS (Cellmax, catalog number SA211.02). OVCAR-3 cells were cultured in RPMI1640 (Gibco, catalog number 11875093) medium containing 20% ​​FBS (Cellmax, catalog number SA211.02). OV90 cells were cultured in MCDB105 medium containing 15% FBS (Cellmax, catalog number SA211.02), 42.5% (1.5 g / L) sodium bicarbonate, and 42.5% (2.2 g / L) sodium bicarbonate in 199 medium. Cells were subcultured every 1-2 days using 0.25% trypsin to maintain a reasonable cell density. When the cells are in the logarithmic growth phase and the number meets the experimental requirements, the cells are collected and counted for subsequent cell experiments.

[0189] The day before drug addition, cells were harvested and seeded into 96-well, flat-bottomed culture plates. The next day, cells were exposed to different concentrations of drug and cultured at 37°C for 72 hours. Then, 40 μL of Cell-titer Glo (CTG) was added to each well of the culture plate, incubated for 5 minutes, and luciferase analysis was performed using a microplate reader. All readings were normalized according to the percentage of viable cells in the untreated control wells, and the IC was calculated using Prism software. 50 value.

[0190] Figure 1 The graph shows the dose-response curves of NCI-H292 cells (seeding density 500 cells / well) exposed to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 after culturing at 37°C for 72 hours.

[0191] The half-inhibitory concentration IC of compounds PB-D5, PB-D7, exatecan and SN38 was calculated 50 The apoptosis-inducing ability of the compound PB-D5 synthesized in this specification is stronger than that of exatecan and SN38 in NCI-H292. The apoptosis-inducing ability of the compound PB-D7 synthesized in this specification is stronger than that of SN38 in NCI-H292.

[0192] Figure 2The graph shows the dose-response curves after exposure of OVCAR-3 (seeding density 1200 cells / well) to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 and culture at 37°C for 72 hours.

[0193] The half-inhibitory concentration IC of compounds PB-D5, PB-D7, exatecan and SN38 was calculated 50 The apoptosis-inducing ability of the compound PB-D5 synthesized in this specification is stronger than that of exatecan and SN38 in inducing apoptosis of OVCAR-3.

[0194] Figure 3 It is a dose-response curve after OV90 (seeding density of 1500 cells / well) was exposed to different concentrations of compound PB-D5, compound PB-D7, exatecan and SN38 and cultured at 37°C for 72 hours.

[0195] The half-inhibitory concentration IC of compounds PB-D5, PB-D7, exatecan and SN38 was calculated 50 The results are as follows: 0.0004623 μM, 0.002162 μM, 0.0009749 μM and 0.002835 μM, respectively. Therefore, compared with exatecan and SN38, the compound PB-D5 synthesized in this specification has a stronger ability to induce apoptosis of OV90. Compared with SN38, the compound PB-D7 synthesized in this specification has a stronger ability to induce apoptosis of OV90.

[0196] Figure 4 This is a dose-response curve after exposing Raji (seeding density of 1000 cells / well) to different concentrations of compound PB-D21, compound PB-D22, compound PB-D24 and exatecan and culturing at 37°C for 72 hours.

[0197] The half-inhibitory concentration IC of compound PB-D21, compound PB-D22, compound PB-D24 and exatecan was calculated 50 The concentrations of PB-D21 and PB-D22 in the samples were 1.831*10^(-5)μM, 3.419*10^(-5)μM, 0.0002015μM, and 8.989*10^(-5)μM, respectively. Therefore, compared with exatecan, the compounds PB-D21 and PB-D22 synthesized in this specification have stronger apoptosis-inducing abilities on Raji.

[0198] It should be noted that the above embodiments are only used to explain this specification and do not constitute a limitation to this application.

[0199] The beneficial effects that may be brought about by the embodiments of this application include, but are not limited to: (1) Compared with isotecan, the isotecan derivatives provided in the embodiments of this specification have higher lipophilicity, which can improve their membrane permeability; (2) Compared with isotecan, the isotecan derivatives provided in the embodiments of this specification have higher activity, and their application in antibody-drug conjugates can produce a better bystander effect. It should be noted that different embodiments may produce different beneficial effects. In different embodiments, the beneficial effects that may be produced may be any one or a combination of the above, or any other possible beneficial effects.

[0200] While the basic concepts have been described above, it will be apparent to those skilled in the art that the detailed disclosure is merely illustrative and does not limit this specification. Although not explicitly stated herein, various modifications, improvements, and revisions to this specification may be made by those skilled in the art. Such modifications, improvements, and revisions are suggested in this specification and remain within the spirit and scope of the exemplary embodiments of this specification.

[0201] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[0202] Similarly, it should be noted that, in order to simplify the presentation of this specification and thus facilitate understanding of one or more embodiments of the invention, the foregoing descriptions of the embodiments of this specification sometimes combine multiple features into a single embodiment, figure, or description thereof. However, this disclosure method does not imply that the subject matter of this specification requires more features than those recited in the claims. In fact, an embodiment may have fewer features than all of the features of a single disclosed embodiment.

[0203] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values ​​are as accurate as possible within the feasible range.

[0204] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, and documents, cited in this specification is hereby incorporated by reference in its entirety. This includes application history documents that are inconsistent with or conflict with the content of this specification, as well as documents (currently or subsequently attached to this specification) that limit the broadest scope of the claims of this specification. It should be noted that if the descriptions, definitions, and / or terminology used in the accompanying materials are inconsistent or conflicting with the content of this specification, the descriptions, definitions, and / or terminology used in this specification will control.

[0205] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. An exitecan derivative, characterized in that: The exitecan derivatives are as follows: or its stereoisomers and pharmaceutically acceptable salts thereof.

2. A method for preparing an exitecan derivative, characterized in that: The exitecan derivatives are as follows: or its stereoisomers and pharmaceutically acceptable salts thereof; The method comprises: In the presence of DMA and DIPEA, the isotecan salt and the compound of the formula The mixture is mixed and reacted to obtain the exitecan derivative.

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