Folic acid antagonist linker-drug and antibody-drug conjugates

Abstract

The present invention relates to novel antifolate linker-drugs, conjugates comprising such antifolate linker-drugs, and their use in the treatment of diseases such as cancer, autoimmune diseases, and infectious diseases, optionally in combination with other therapeutic agents.

Description

[Technical Field]

[0001] The present invention relates to novel folate antagonist linker-drugs, conjugates comprising such folate antagonist linker-drugs, and their use in the treatment of diseases such as cancer, autoimmune diseases, and infections, possibly in combination with other therapeutic agents. [Background technology]

[0002] Folic acid antagonists are a type of antimetabolite compound that antagonizes the action of folic acid (vitamin B9).

[0003] [ka]

[0004] Folic acid acts as a cofactor for various methyltransferases involved in the biosynthesis of serine, methionine, thymidine, and purines. As a result, folic acid antagonists inhibit cell division, DNA and RNA synthesis and repair, and protein synthesis. Most folic acid antagonists function by inhibiting dihydrofolate reductase (DHFR).

[0005] The folate antagonists proguanil, pyrimethamine, and trimethoprim selectively inhibit the action of folate in microorganisms such as bacteria, protozoa, and fungi. Other folate antagonists, such as methotrexate, pemetrexed, pralatrexate, and talotrexin, are used to treat certain cancers and / or inflammatory conditions such as rheumatoid arthritis, an autoimmune disease.

[0006] Methotrexate, formerly known as ametopterin, is the oldest and most well-known folic acid analog. Discovered in the 1950s, it is a chemotherapeutic and immunosuppressant. Originally developed for chemotherapy, either alone or in combination with other drugs, it is still used today to treat bladder cancer, breast cancer, head and neck cancer, leukemia, lung cancer, lymphoma, gestational trophoblastic disease, and osteosarcoma. Methotrexate is also used as a disease-modifying agent for several autoimmune diseases, including rheumatoid arthritis, juvenile dermatomyositis, psoriasis, psoriatic arthritis, lupus, sarcoidosis, Crohn's disease, eczema, and vasculitis. Other uses include treating ectopic pregnancies and inducing drug-induced abortions.

[0007] Pemetrexed is used to treat signs of various cancers, including mesothelioma, lung cancer, and head and neck cancer. It works by inhibiting three enzymes chemically similar to folic acid and used in the synthesis of purines and pyrimidines: DHFR, thymidylate synthase (TS), and glycinamide ribonucleotide formyltransferase (GARFT). By inhibiting the formation of their precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA necessary for the growth and survival of both normal and tumor cells.

[0008] Pralatrexate is approved as a treatment for peripheral T-cell lymphoma. It is designed to increase affinity for reduced folate carrier 1 (RFC-1), which increases intracellular uptake, and for folylpolyglutamate synthase, which increases cytotoxic metabolites.

[0009] Talotrexin is a 4-amino derivative of folic acid and an antimetabolite analog of aminopterin, a synthetic derivative of pterin. Talotrexin exhibits antitumor activity, binding to and inhibiting the function of DHFR. Water-soluble talotrexin is actively transported into cells by reduced folate carriers (RFCs), and is therefore unlikely to be associated with multidrug resistance mediated by P-glycoprotein.

[0010] [ka]

[0011] Folic acid antagonists act specifically during DNA and RNA synthesis, primarily exhibiting cytotoxicity during the S phase of the cell cycle. Therefore, they have a significant toxic effect on rapidly dividing cells that frequently replicate DNA (such as malignant cells and bone marrow cells). However, folic acid antagonists not only inhibit the growth and proliferation of tumor cells, but also inhibit the growth and proliferation of non-cancer cells, such as bone marrow cells, gastrointestinal cells, and oral mucosal cells, causing adverse events in the bone marrow, intestines, oral mucosa, skin, and hair.

[0012] Systemic side effects caused by cytotoxic small molecule drugs, such as folic acid antagonists, may be reduced by conjugating such drugs to target molecules, such as antibodies, antigen-binding fragments of antibodies, or fusion proteins (e.g., receptor ligands fused to the Fc of an antibody), via a chemical linker. By combining these two components—the cytotoxic small molecule drug and the target molecule—into a single new molecule, the target molecule can be specifically delivered to the cells or tissues that the cytotoxic small molecule drug targets (the site where cytotoxic activity is exerted), thereby reducing exposure of non-target tissues to the small molecule.

[0013] To improve the targeting of methotrexate and reduce its toxicity, several antibody-drug conjugates (ADCs) of methotrexate were manufactured in the late 1980s. The drug-to-antibody ratio (DAR) of these lysine-binding ADCs ranged from 9.4 to 45. Although the concept of ADCs was successful, the efficacy obtained with these ADCs in vitro (Shen et al, Cancer Res. 1986, 46, 3912-3916; Umemoto et al, Int. J. Cancer 1989, 43, 677-684) and in vivo (Deguchi et al, Cancer Res. 1986, 46, 3751-3755; Rowland et al, Br. J. Cancer 1990, 61, 702-708) was moderate to poor. Even with a high DAR, methotrexate did not appear to be sufficiently potent as a toxin for ADCs.

[0014] A short peptide conjugate targeting tumors with pemetrexed was created and tested (Miklan et al, J. Pept. Sci. 2011, 17, 805-811), but this idea was not explored further. No ADCs of more potent folate antagonists such as pemetrexed, pralatrexate, or talotrexin have been reported.

[0015] Therefore, there is a need for novel and improved target-specific complexes containing folate antagonists, antibodies, antigen-binding fragments, or other target molecules, suitable for use alone or in combination with other therapeutic agents in the treatment of cancer, autoimmune diseases, and infections. [Disclosure of the Invention]

[0016] The present invention relates to novel folate antagonist linker-drugs, conjugates comprising such folate antagonist linker-drugs, and their use in the treatment of diseases such as cancer, autoimmune diseases, and infections, possibly in combination with other therapeutic agents.

[0017] In the first aspect, the present invention relates to formula (I)

[0018] [Chemical formula]

[0019] [In the formula, R 1-4 is O, NH2 or OH; R 2 and R 2’ are independently N, CH or CMe; R 3 is NH, N(C 1-5 alkyl), CH2, CH(C 1-5 alkyl), CH(C 2-4 alkenyl), CH(C 2-4 alkynyl) or CH(C 1-4 alkoxy); ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​2-4 Alkenil, C 2-4 Alkinyl, C 1-4 Alkoxy or C 1-4 Alkylthio, preferably H, F, CH3, CF3, CH2CH3, CH=CH2, CH2CF3 or CF2CF3, more preferably H or F; R 6 H, C 1-4 Alkyl, C 2-4 Alkenil, C 3-6 Cycloalkyl, preferably H; n is 1, 2, 3, or 4, preferably 3; Q is a single bond, -N(R 7 )-(C=O)-, -(C=O)-N(R 7 )-,-CH2N(R 7 )-,-N(R 7 )CH2-, -N(R 7 )SO2- or -SO2N(R 7 )-(Here, R 7 H, C 1-4 Alkyl, C 1-4 Alkenyl or C 1-4 Alkinyl (preferably H) or Q is

[0022] [ka]

[0023] or

[0024] [ka]

[0025] (Here, R b H and C 1-5 Selected from alkyl, T1, T1' and T1'' are independently selected from CH and N, and W1, W1' and W1'' are independently C, CH, S, N, NH, N(C 1-5 An amide bond biological equivalent selected from the group consisting of alkyl and O; V may be one or more R 4 Replaced by, independently,

[0026] [ka]

[0027] A selected aryl, heteroaryl, heterocyclyl or cycloalkane (where U1, U1', U1'', U2, U2', U2'' and U2'''' are independently selected from C, CH, S, N, NH, N(C1-5 alkyl) and O), or V is

[0028] [ka]

[0029] (Here, Z is O, S, NH, or NR) c And R c H and C 1-5 Selected from the group consisting of alkyl groups; s is 0 or 1, preferably 1; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 A linking group selected from alkynylenes; k is 1, 2, 3, or 4, preferably 1; L is the linker;

[0030] [ka]

[0031] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized. This relates to linker-drug compounds represented by [the specified formula].

[0032] In a second aspect, the present invention relates to formula (III) Ab-(LD) y (III) [In the formula, Ab is an antibody or its antigen-binding fragment; LD is the linker-drug compound of the present invention; y represents the average drug-antibody ratio between 1 and 16; The linker-drug compound of the present invention is preferably bound to the antibody or its antigen-binding fragment via a cysteine ​​residue of the antibody or its antigen-binding fragment. This relates to antibody-drug conjugates represented by [the specified format].

[0033] Other aspects of the present invention include pharmaceutical compositions comprising the linker-drug compound or antibody-drug conjugate of the present invention, methods for synthesizing the same, and the use of the same as pharmaceuticals, in particular, for the treatment of cancer, autoimmune diseases, or infectious diseases. [Brief explanation of the drawing]

[0034] [Figure 1] In vitro efficacy of trastuzumab-XT17 folate antagonist ADC and unbound control ADC in HER2-positive SK-BR-3 cells. [Figure 2] In vitro efficacy of trastuzumab-XT17 folate antagonist ADC and unbound control ADC in HER2-negative SW-620 cells. [Figure 3A] In vivo efficacy of folate antagonist ADC1 (5 mg / kg IV) and vehicle control in xenografts of HER2-positive mouse BT-474 cell line. [Figure 3B] In vivo efficacy of folate antagonist ADC1 (5 mg / kg IV or 1.7 mg / kg Q1W × 3) and vehicle control in xenografts of HER2-positive mouse BT-474 cell line. [Figure 4A] Effects of folate antagonist ADC1 (5 mg / kg IV) on body weight in mouse HER2-positive BT-474 cell line xenografts compared to vehicle control. [Figure 4B]Effects of folate antagonist ADC1 (5 mg / kg IV or 1.7 mg / kg Q1W × 3) on body weight in mouse HER2-positive BT-474 cell line xenografts compared to vehicle control. [Figure 5] In vivo efficacy of folate antagonist ADC1 (3 or 10 mg / kg IV) versus vehicle control in xenografts derived from HER2 2+ MAXF574 mouse patients. [Best Mode for Carrying Out the Invention]

[0035] Detailed explanation The inventors have found that the linker-drug compound represented by formula (I) is particularly suitable for binding to other targeting molecules such as antibodies, antigen-binding fragments, or fusion proteins (e.g., receptor ligands fused to the Fc of an antibody). The small molecule drugs released from these linker-drug conjugates exhibit good efficacy as folate antagonists. They show excellent inhibitory activity against DHFR and exhibit potent antiproliferative effects in various cancer cell lines.

[0036] Linker - Drug compound In the first aspect, the present invention relates to formula (I)

[0037] [ka]

[0038] [In the formula, R 1 is O, NH2, or OH; R 2 and R 2’ These are independently N, CH, or CMe; R 3 NH, N(C 1-5 Alkyl), CH2, CH(C 1-5 Alkyl), CH(C 2-4 Alkenyl), CH(C 2-4 Alkinyl) or CH(C 1-4 It is an alkoxy; R 4H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, benzyloxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 (Selected from alkyl) or R 4 is a carboxylic acid biological equivalent; R 5 H, halogen, CF3, C 1-4 Alkyl, C 2-4 Alkenil, C 2-4 Alkinyl, C 1-4 Alkoxy or C 1-4 Alkylthio, preferably H, F, CH3, CF3, CH2CH3, CH=CH2, CH2CF3 or CF2CF3, more preferably H or F; R 6 H, C 1-4 Alkyl, C 2-4 Alkenil, C 3-6 Cycloalkyl, preferably H; n is 1, 2, 3, or 4, preferably 3; Q is a single bond, -N(R 7 )-(C=O)-, -(C=O)-N(R 7 )-,-CH2N(R 7 )-,-N(R 7 )CH2-, -N(R 7 )SO2- or -SO2N(R 7 )-(Here, R 7 H, C 1-4 Alkyl, C 1-4 Alkenyl or C 1-4 Q is either an alkynyl (preferably H) or an amide bond biological equivalent; V may be one or more R 4 Replaced by, independently,

[0039] [ka]

[0040] Selected from aryl, heteroaryl, heterocyclic or cycloalkane (where U1, U1’, U1’’, U2, U2’, U2’’, and U2’’’ are independently selected from C, CH, S, N, NH, N(C1-5 alkyl) and O), or V is

[0041]

Chemical formula

[0042] (where Z is O, S, NH or NR c and R c is selected from the group consisting of H and C 1-5 alkyl); s is 0 or 1, preferably 1; X is a linking group selected from O, NH, S, C 1-5 alkylene, C 1-5 alkenylene and C 1-5 alkynylene; k is 1, 2, 3 or 4, preferably 1; L is a linker;

[0043]

Chemical formula

[0044] means that the bond may be a single bond or, if not cumulative, may be a non-localized double bond in some cases] To provide a linker-drug compound represented by

[0045] Such linker-drug compounds are hereinafter referred to as the linker-drugs or linker-drug compounds of the present invention.

[0046] In the present invention, the halogen is fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Preferred halogens in the linker-drug compounds of the present invention are fluorine, chlorine and bromine, more preferred halogens are fluorine or chlorine, and the most preferred halogen is chlorine.

[0047] In this invention, the number of carbon atoms in alkyl, alkenyl, alkoxy, alkynyl, cyclil, heterocyclil, aryl or heteroaryl is, for example, C 1-6 as shown, and in a non-limiting example, it is shown that 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5 or 6 carbon atoms) are contemplated. Similarly, C 2-4 The carbon atoms of the alkenyl are 2, 3 or 4. The number of carbon atoms can be represented as the total number of carbon atoms without counting additional substituents, the total number of carbon atoms, and the number of carbon atoms in the longest continuous carbon chain. Preferably, the number of carbon atoms is represented as the total number of carbon atoms without counting additional substituents.

[0048] In this invention, the general formula of an unsubstituted alkyl group is C n H 2n+1 and may be straight-chain or branched-chain. The unsubstituted alkyl group may contain a cyclic moiety, and thus the general formula is C n H 2n-1It may also be the case that the alkyl group is substituted with one or more substituents further described herein. Suitable alkyl groups include, but are not limited to, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH(CH3)2, -CH2CH2CH2CH3, and -C(CH3)3. Preferred alkyl groups are linear or branched, most preferably linear. The cyclyl group is a cyclic alkyl group; preferably, the cyclyl group is cyclopropyl, cyclobutyl, and cyclopentyl. The heterocyclyl group is a cyclyl group in which at least one CH2 is substituted with a heteroatom. Preferably, the heteroatom is S, O, and N. Preferably, the heterocyclyl group is piperidinyl, oxylanil, and oxolanil. Preferably, C 1-4 Alkyl groups include -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH(CH3)2, -CH2CH2CH2CH3, -C(CH3)3, cyclopropyl and cyclobutyl, and more specifically, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH(CH3)2, -CH2CH2CH2CH3 and -C(CH3)3.

[0049] In this invention, the general formula for an unsubstituted alkyl group is C n H 2n-1 The alkenyl group may be linear or branched. Suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, and pentenyl. Unsubstituted alkenyl groups may include a cyclic portion, and therefore the general formula is C n H 2n-3 This may also be the case. The preferred alkenyl group is linear or branched, and most preferably linear.

[0050] In this invention, the general formula for an unsubstituted alkynyl group is C n H 2n―3It may be linear or branched. The unsubstituted alkynyl group may include a cyclic portion, and therefore the general formula is C n H 2n-5 This may also be the case. In some cases, the alkynyl group is substituted with one or more substituents further described herein. Suitable examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, n-butane-2-inyl, and n-butane-3-inyl. Preferred alkyl groups are linear or branched, most preferably linear.

[0051] In this invention, the aryl group is aromatic and contains at least six carbon atoms, and may include monocyclic, dicyclic, and polycyclic structures. In some cases, the aryl group may be substituted with one or more substituents further described herein. Examples of aryl groups include, for example, phenyl, naphthyl, and anthranyl groups. The heteroaryl group is aromatic and contains one to four heteroatoms selected from the group consisting of S, O, and N. Due to the heteroatoms, the ring size may be 5 or less.

[0052] In this invention, the alkoxy has a bridging oxygen atom before the alkyl. Suitable examples of alkoxys are -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -OCH(CH3)CH2CH3, -OCH2CH2CH2CH3, and -OC(CH3)3.

[0053] In this invention, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cyclyl and heterocyclyl are preferably halogens, CH3, and other C3. 1-4 C such as alkyl, OH, and OCH3 1-4The alkyl, alkenyl, alkynyl, and alkoxy groups may be substituted with one or more elements selected from alkoxy and =O, and the alkyl, alkoxy, cyclyl, and heterocyclyl groups may be interrupted with heteroatoms such as O or S, and the alkyl, alkoxy, cyclyl, and heterocyclyl groups may be unsaturated. Interruption by a heteroatom means interruption by one or more heteroatoms. In this application, preferably 20 or fewer heteroatoms, more preferably 3, 4, or 5 heteroatoms are interrupted. Preferably, all the interrupting heteroatoms are of the same element. As a non-limiting example, CH2-CH2-CH2-CH2-CH3 may also be CH2-CH2-O-CH2-CH2-O-CH3 when interrupted by heteroatoms.

[0054] The molecules of the present invention may be substituted in some cases. Any suitable substitution is the substitution of -H with a halogen. Preferably, the halogen is F, Cl, Br, and I, and most preferably F. Another suitable substitution is the substitution of one or more -H with -NH2, -OH, =O, alkyl, alkoxy, haloalkyl, haloalkoxy, alkene, haloalkene, alkyne, haloalkyne, and cycloalkyl. The general formula for alkyl groups is C n H 2n+1 It may be linear or branched. The unsubstituted alkyl group may contain a cyclic portion, and therefore the general formula is C n H 2n-1 This may also be the case. In some cases, the alkyl group is substituted with one or more substituents further described herein. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, and the like.

[0055] In this invention, a bioequivalent is a substituent or group of atoms that possesses physical or chemical properties that produce a wide range of biological properties equivalent to those of other compounds. The purpose of replacing one bioequivalent with another is to enhance the desired biological or physical properties of a compound without significantly altering its chemical structure. The general concept of bioequivalents is described, for example, in Meanwell, J. Med. Chem. 2011, 54, 2529-2591 and Patani and LaVoie, Chem. Rev. 1996, 96, 3147-3176.

[0056] Carboxylic acid bioequivalents are described, for example, in Ballatore et al, ChemMedChem. 2013, 8, 385-395 and Lassalas et al, J. Med. Chem. 2016, 59, 3183-3203.

[0057] A preferred example of a carboxylic acid biological equivalent is:

[0058] [ka]

[0059] (Here, R a ' is H, CH2F, CHF2, CF3 and C 1-6 Selected from alkyl groups, each R a These are independently H, F, CH2F, CHF2, CF3 and C 1-6 Selected from alkyl groups, and possibly two R groups a (They can come together to form a ring.) That is the case.

[0060] Amide bond bioequivalents are described, for example, in Kumari et al, J. Med. Chem. 2020, 63, 12290-12358 and Recnik et al, Molecules 2020, 25, 3576.

[0061] A preferred example of an amide bond biological equivalent is,

[0062] [ka]

[0063] or

[0064] [ka]

[0065] (Here, R b H and C 1-5 Selected from alkyl, T1, T1' and T1'' are independently selected from CH and N, and W1, W1' and W1'' are independently C, CH, S, N, NH, N(C 1-5 It is selected from alkyl and O.

[0066] Tse et al (J. Med. Chem. 2020, 63, 11585-11601), Mykhailiuk (Org. Biomol. Chem., 2019, 17, 2839-2849), Qiao et al (Bioorg. Med. Chem. Lett. 2008, 18, 4118-4123), and Stepan et al (J. Med. Chem. 2012, 55, 3414-3424) have described various non-classical and / or saturated phenyl bioequivalents.

[0067] Examples of such phenyl biological equivalents are,

[0068] [ka] That is the case.

[0069] As mentioned above, V may be one or more R 4 Replaced by, independently,

[0070] [ka]

[0071] An aryl, heteroaryl, heterocyclyl, or cycloalkane selected from the following. In one embodiment, V is

[0072] [ka] That is the case.

[0073] Preferably, V is

[0074] [ka] That is the case.

[0075] More preferably, V is

[0076] [ka] That is the case.

[0077] In a preferred embodiment, the present invention relates to formula (Ia)

[0078] [ka] This provides a linker-drug compound represented by [the specified formula].

[0079] More preferably, the present invention provides a linker-drug compound represented by formula (Ia), wherein, R 1 is O, NH2, or OH, preferably R 1 is NH2; R 2 and R 2’ These are independently N, CH, or CMe, preferably R 2 and R 2’ is N; R 3NH, N(C 1-5 Alkyl), CH2 or CH(C 1-5 Alkyl), preferably R 3 is NH, N(CH3), or CH2; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 Selected from alkyl groups), preferably R 4 It is -COOH or tetrazole; Q is a single bond, -N(R 7 )-(C=O)-, -(C=O)-N(R 7 )-,-CH2N(R 7 )-,-N(R 7 )CH2-, -N(R 7 )SO2- or -SO2N(R 7 )-(Here, R 7 H, C 1-4 Alkyl, C 1-4 Alkenyl or C 1-4 Q is either an alkynyl (preferably H) or an amide bond bioequivalent, preferably Q is -N(R 7 )-(C=O)- or -(C=O)-N(R 7 )-, more preferably-N(R 7 )-(C=O)-; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 A linking group selected from alkynylenes, preferably where X is NH; L is the linker;

[0080] [ka]

[0081] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized.

[0082] In certain aspects, R 1 NH2 is R 2 and R 2’ is N, and R 4 Q is -COOH, and Q is -NH-(C=O)-.

[0083] In another specific embodiment, R 1 NH2 is R 2 and R 2’ is N, and R 4 Q is -COOH, and Q is tetrazole.

[0084] In another specific embodiment, R 1 NH2 is R 2 and R 2’ is N, and R 4 Q is a tetrazole, and Q is -NH-(C=O)-.

[0085] In another specific embodiment, R 1 NH2 is R 2 and R 2’ is N, and R 4 Q is -SO3H, and Q is -NH-(C=O)-.

[0086] In another preferred embodiment, the present invention relates to formula (Ib)

[0087] [ka] This provides a linker-drug compound represented by [the specified formula].

[0088] More preferably, the present invention provides a linker-drug compound represented by formula (Ib), wherein, R 1 is O, NH2, or OH, preferably R 1 is NH2; R 2 and R2’ These are independently N, CH, or CMe, preferably R 2 and R 2’ is N; R 3 NH, N(C 1-5 Alkyl), CH2 or CH(C 1-5 Alkyl), preferably R 3 is NH, N(CH3), or CH2; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 Selected from alkyl groups), preferably R 4 It is -COOH or tetrazole; n is 1, 2, 3, or 4, preferably 3; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 A linking group selected from alkynylenes, preferably X, is NH; L is the linker;

[0089] [ka]

[0090] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized.

[0091] In certain aspects, R 1 And R 2 and R 2’ is N, and R 4 is -COOH, and n is 3.

[0092] In another specific embodiment, R 1 NH2 is R 2 and R2’ is N, and R 4 is tetrazole, and n is 3.

[0093] In another preferred embodiment, the present invention relates to formula (Ic)

[0094] [ka] This provides a linker-drug compound represented by [the specified formula].

[0095] More preferably, the present invention provides a linker-drug compound represented by formula (Ic), wherein, R 2 and R 2’ These are independently N, CH, or CMe, preferably R 2 and R 2’ is N; R 3 NH, N(C 1-5 Alkyl), CH2 or CH(C 1-5 Alkyl), preferably R 3 is NH, N(CH3), or CH2; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 Selected from alkyl groups), preferably R 4 It is -COOH or tetrazole; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 A linking group selected from alkynylenes, preferably X is NH; and L stands for linker.

[0096] In certain aspects, R 2 and R 2’ is N, and R 4 It is -COOH.

[0097] In another specific embodiment, R 2 and R 2’ is N, and R 4 It is tetrazole.

[0098] In yet another specific embodiment, R 2 and R 2’ C is R 4 H is H.

[0099] In yet another specific embodiment, R 2 and R 2’ is N, and R 4 It is.

[0100] In yet another specific embodiment, R 2 and R 2’ is N, and R 4 It is OH.

[0101] In yet another specific embodiment, R 2 and R 2’ is N, and R 4 It is Cl.

[0102] In yet another specific embodiment, R 2 and R 2’ is N, and R 4 It is -SO3H.

[0103] In another preferred embodiment, the present invention relates to formula (Id)

[0104] [ka]

[0105] The present invention provides a linker-drug compound represented by formula (Id), wherein, R 1 is O, NH2, or OH, preferably R 1 is NH2; R 2 and R2’ These are independently N, CH, or CMe, preferably R 2 and R 2’ is N; R 3 NH, N(C 1-5 Alkyl), CH2 or CH(C 1-5 Alkyl), preferably R 3 is NH, N(CH3), or CH2; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 Selected from alkyl groups), preferably R 4 It is -COOH or tetrazole; n is 1, 2, 3, or 4, preferably 4; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 A linking group selected from alkynylenes, preferably X, is NH; L is the linker;

[0106] [ka]

[0107] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized.

[0108] In certain aspects, R 1 NH2 is R 2 and R 2’ is N, and R 4 is COOH, and n is 4.

[0109] In another specific embodiment, R 1 NH2 is R 2and R 2’ is N, and R 4 H is , and n is 3.

[0110] Preferred linker-drug compounds of the present invention are:

[0111] [ka] That is the case.

[0112] A more preferred linker-drug compound of the present invention is:

[0113] [ka] That is the case.

[0114] The linker-drug compound of the present invention comprises a linker, preferably a synthetic linker. The structure of the linker is such that it can chemically bind readily to a low-molecular-weight drug, and the resulting linker-drug compound can readily bind to another substance (e.g., a polypeptide) to form an inhibitor complex. The choice of linker may affect the stability of the final complex in the blood and may also affect how the low-molecular-weight drug is released. Suitable linkers include, for example, Ducry et al, Bioconjugate Chem. 2010, 21, 5-13, King and Wagner, Bioconjugate Chem. 2014, 25, 825-839, Gordon et al, Bioconjugate Chem. 2015, 26, 2198-2215, Tsuchikama and An (DOI: 10.1007 / s13238-016-0323-0), Polakis (DOI: 10.1124 / pr.114.009373), and Bargh et al. (DOI: This is described in International Publication Nos. 10.1039 / c8cs00676h, 2004 / 043493, 2010 / 062171, 2011 / 133039, 2015 / 177360, and 2018 / 069375. The linker may or may not be cleavable. A cleavable linker includes, for example, a portion that is cleaved when exposed to a lysosomal protease or an environment with an acidic pH or a high reduction potential. Suitable cleavable linkers are known in the art and include, for example, di, tri, or tetrapeptides, i.e., peptides consisting of two, three, or four amino acid residues. Furthermore, the cleavable linker may contain a self-destructing moiety such as an ω-aminoaminocarbonyl cyclization spacer (see Saari et al, J. Med. Chem., 1990, 33, 97-101) or an -NH-CH2-O- bond. Cleavage of the linker makes the folate antagonistic moiety in the linker-drug compound of the present invention available to the surrounding environment.Uncleavable linkers, for example, can still more effectively release the folate antagonistic moiety (or its derivatives) from the linker-drug compound of the present invention even if the polypeptide of the complex is degraded in lysosomes. Uncleavable linkers include, for example, succinimidyl-4-N-maleimidomethyl(cyclohexane)-1-carboxylate and maleimidocaproic acid, as well as their analogues.

[0115] To enable a linker or linker-drug to be bound to a polypeptide such as an antibody, its antigen-binding fragment, or another targeted molecule, the linker side that binds (covalently) to the antibody, its antigen-binding fragment, or another targeted molecule typically has a functional group that can react with amino acid residues of the antibody, its antigen-binding fragment, or another targeted molecule under relatively mild conditions. This functional group is referred to in this specification as the reactive moiety (RM). Examples of reactive moieties include, but are not limited to, carbamoyl halides, acyl halides, active esters, anhydrides, α-haloacetyl, α-haloacetamide, maleimide, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, hydrazides, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyls, methylsulfonates, and cyclooctin. The amino acid residues that react with the functional group may be natural or unnatural amino acid residues. In this specification, the term "non-natural amino acid" is intended to refer to an amino acid modified (by synthesis) or a d-type stereoisomer of a naturally occurring amino acid. Preferably, the amino acid residue to which the functional group reacts is a natural amino acid.

[0116] In a preferred embodiment of the present invention, RM is

[0117] [ka]

[0118] (Here, X 1-Cl, -Br, -I, -F, -OH, -ON-succinimide, -O-(4-nitrophenyl), -O-pentafluorophenyl, -O-tetrafluorophenyl, -OC(O)-R 8 and -OC(O)-OR 8 Selected from or C(O)-X 1 It is an active ester; X 2 -Cl, -Br, -I, -O-mesyl, -O-trifuryl, and -O-tosyl are selected from -Cl, -Br, -I, -O-mesyl, -O-trifuryl, and -O-tosyl; R 8 In some cases, it is replaced, branched or unbranched, C1- 10 Alkyl, C1- 10 Heteroalkyl, C3- 10 Cycloalkyl, C1- 10 Heterocycloalkyl, C5- 10 Aryl or C1- 10 Selected from heteroaryls; U is O or NR 9 and; R 9 C1-C is H, branched or unbranched. 12 Alkyl or C4-C 12 (Selected from hetero-aryl groups) That is the case.

[0119] Preferably, RM is

[0120] [ka] That is the case.

[0121] More preferably, RM

[0122] [ka] That is the case.

[0123] Linker,

[0124] [ka]

[0125] It may further include one or more extension spacers.

[0126] The linker may further include one or more desorption spacers as described, for example, Alouane et al, Angew. Chem. Int. Ed. 2015, 54, 7492-7509, Deng et al, Macromol. Rapid Commun. 2020, 41, e1900531, or Bargh et al, Chem. Soc. Rev. 2019, 48, 4361-4374.

[0127] In one embodiment, linker L is

[0128] [ka]

[0129] [Here, m is an integer between 1 and 10, preferably 5; AA is an amino acid, preferably a neutral amino acid; p is 0, 1, 2, 3, or 4; q is an integer between 1 and 12, preferably 2; ES is a single bond or

[0130] [ka]

[0131] An extension spacer selected from; RL is a single bond or

[0132] [ka]

[0133] (Here, t is an integer from 1 to 10, R10 In some cases, C is substituted. 1-4 It is an alkoxy, R 11 H, and possibly substituted C 1-6 Alkyl, and possibly substituted C 6-14 Aryl, or possibly substituted C-bond C 3-8 [A detachment spacer selected from heteroaryls] That is the case.

[0134] In a preferred embodiment, m is 5, p is 0, ES and RL are single bonds, and L is

[0135] [ka] That is the case.

[0136] In another preferred embodiment, AA is an amino acid selected from the group consisting of alanine, glycine, lysine, phenylalanine, valine, and citrulline.

[0137] In the first embodiment, p is 2, and AA2 is phenylalanyl lysine, valylalanine, valylcitrulline, or valyllysine. More preferably, AA2 is valylalanine or valylcitrulline. Most preferably, AA2 is valylalanine or valylcitrulline, and m is 5.

[0138] In the second embodiment, p is 3, and AA3 is alanylphenylalanyllysine.

[0139] In the third embodiment, p is 4, and AA4 is glycylglycylphenylalanylglycine.

[0140] In a preferred embodiment, m is 5, p is 2, and AA2 is valylalanine or valylcitrulline. Preferably, m is 5, p is 2, AA2 is valylalanine, ES and RL are bound, and L is

[0141] [ka] That is the case.

[0142] In another preferred embodiment, m is 5, p is 4, AA4 is glycylglycylphenylalanylglycine, ES and RL are single bonds, and L is

[0143] [ka] That is the case.

[0144] In one embodiment, L is

[0145] [ka]

[0146] (Here, q is an integer between 1 and 12, preferably 2; AA is an amino acid, preferably a neutral amino acid; p is 0, 1, 2, 3, or 4; ES is a single bond or

[0147] [ka]

[0148] An extension spacer selected from; RL is a single bond or

[0149] [ka]

[0150] (Here, t is an integer from 1 to 10, R 10 In some cases, C is substituted. 1-4 It is an alkoxy, R 11 H, and possibly substituted C 1-6 Alkyl, and possibly substituted C 6-14Aryl, or possibly substituted C-bond C 3-8 (A desorption spacer selected from heteroaryls) That is the case.

[0151] In a preferred embodiment, AA is an amino acid selected from the group consisting of alanine, glycine, lysine, phenylalanine, valine, and citrulline.

[0152] In the first embodiment, p is 2, and AA2 is phenylalanyl lysine, valylalanine, valylcitrulline, or valyllysine. More preferably, AA2 is valylalanine or valylcitrulline. Most preferably, AA2 is valylalanine or valylcitrulline, and q is 2.

[0153] In the second embodiment, p is 3 and AA3 is alanylphenylalanyllysine.

[0154] In the third embodiment, p is 4, and AA4 is glycylglycylphenylalanylglycine.

[0155] In a preferred embodiment, q is 2, p is 2, and AA2 is valylalanine or valylcitrulline. Preferably, q is 2, p is 2, AA2 is valylcitrulline, RL is a single bond, and ES is

[0156] [ka] So, L is

[0157] [ka] That is the case.

[0158] In another preferred embodiment, q is 2, p is 2, AA2 is valylcitrulline, and ES is

[0159] [ka] So, RL is

[0160] [ka] So, L is

[0161] [ka] That is the case.

[0162] In one embodiment, Linker L is

[0163] [ka] That is the case.

[0164] The following are preferred linker-drug compounds of the present invention.

[0165] [Table A-1]

[0166] [Table A-2]

[0167] [Table A-3]

[0168] [Table A-4]

[0169] [Table A-5]

[0170] [Table A-6]

[0171] [Table A-7]

[0172] A more preferred linker-drug compound of the present invention is:

[0173] [ka]

[0174] [ka]

[0175] [ka] That is the case.

[0176] A more preferable linker-drug compound of the present invention is,

[0177] [ka]

[0178] [ka] That is the case.

[0179] A more preferable linker-drug compound of the present invention is,

[0180] [ka] That is the case.

[0181] Most preferably, the linker-drug compound of the present invention is of the formula

[0182] [ka] It is a compound represented by [formula].

[0183] Method for producing linker-drug compounds of the present invention The linker-drug compounds of the present invention can be produced by the examples or by the methods described in, for example, Rosowsky et al (J. Med. Chem. 1988, 31 1332-1337; J. Med. Chem. 1998, 41 5310-5319; J. Med. Chem. 2000, 43 1620-1634) or Itoh et al (Chem. Pharm. Bull. 2000, 48 1270-1280), or by similar methods.

[0184] In one aspect, the present invention relates to the formula for the production of the linker-drug compound of the present invention or the production of a complex containing the linker compound of the present invention.

[0185] [ka]

[0186] Regarding the use of the compound represented by the formula, R 1 is O, NH2, or OH; R 2 and R 2’ These are independently N, CH, or CMe; R 3 NH, N(C 1-5 Alkyl), CH2, CH(C 1-5 Alkyl), CH(C 2-4 Alkenyl), CH(C 2-4 Alkinyl) or CH(C 1-4 It is an alkoxy; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4Alkoxy, benzyloxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 (Selected from alkyl) or R 4 is a carboxylic acid biological equivalent; R 5 H, halogen, CF3, C 1-4 Alkyl, C 2-4 Alkenil, C 2-4 Alkinyl, C 1-4 Alkoxy or C 1-4 Alkylthio, preferably H, F, CH3, CF3, CH2CH3, CH=CH2, CH2CF3 or CF2CF3, more preferably H or F; R 6 H, C 1-4 Alkyl, C 2-4 Alkenil, C 3-6 Cycloalkyl, preferably H; n is 1, 2, 3, or 4, preferably 3; Q is a single bond, -N(R 7 )-(C=O)-, -(C=O)-N(R 7 )-,-CH2N(R 7 )-,-N(R 7 )CH2-, -N(R 7 )SO2- or -SO2N(R 7 )-(Here, R 7 H, C 1-4 Alkyl, C 1-4 Alkenyl or C 1-4 Q is either an alkynyl (preferably H) or an amide bond biological equivalent; V may be one or more R 4 Replaced by, independently,

[0187] [ka]

[0188] A selected aryl, heteroaryl, heterocyclyl or cycloalkane (where U1, U1', U1'', U2, U2', U2'' and U2'''' are independently selected from C, CH, S, N, NH, N(C1-5 alkyl) and O), or V is

[0189] [ka]

[0190] (Here, Z is O, S, NH, or NR) c And R c H and C 1-5 Selected from the group consisting of alkyl groups; s is 0 or 1, preferably 1; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 Selected from alkynylenes; and

[0191] [ka]

[0192] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized.

[0193] In one embodiment, the present invention relates to the formula for the production of the linker-drug compound of the present invention or the production of a complex containing the linker compound of the present invention.

[0194] [ka]

[0195] Regarding the use of the compound represented by the formula, R 1 is O, NH2, or OH; R 2 and R2’ These are independently N, CH, or CMe; R 3 NH, N(C 1-5 Alkyl), CH2 or CH(C 1-5 Alkyl) is; R 4 H, halogen, -COOH, OH, NH2, -CONH2, -CONHR, -CONHR2, C 1-4 Alkyl, C 1-4 Alkoxy, tetrazole, -SO3H, -OSO3H, -PO3H2, -OPO3H2, -CN or azide (where R is H and C) 1-5 (Selected from alkyl groups); n is 1, 2, 3, or 4, preferably 3; X is O, NH, S, C 1-5 Alkilen, C 1-5 Alkenylene and C 1-5 Selected from alkynylenes;

[0196] [ka]

[0197] This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized.

[0198] In a preferred embodiment, the present invention relates to the formula for the production of the linker-drug compound of the present invention or the production of a complex containing the linker-compound of the present invention.

[0199] [ka]

[0200] [ka]

[0201] [ka]

[0202] This relates to the use of a compound represented by the formula. More preferably, the compound is represented by the formula

[0203] [ka]

[0204] [ka] It is a compound represented by [formula].

[0205] More preferably, the compound is formula

[0206] [ka]

[0207] [ka] It is a compound represented by [formula].

[0208] Polypeptide-folate antagonist linker-drug compound inhibitor complex The present invention further provides inhibitor complexes comprising the linker-drug compound of the present invention conjugated with another substance, such as a polypeptide or a polynucleotide (for forming an aptamer). Preferably, the other substance is a polypeptide. More preferably, the polypeptide is an antibody, its antigen-binding fragment, or another targeting molecule. Such inhibitor complexes are hereafter referred to as the inhibitor complexes of the present invention.

[0209] When bound to a polypeptide, the term “inhibitor complex of the present invention” means throughout this specification a polypeptide to which one or more linker-drug compounds of the present invention are bound, i.e., a polypeptide to which one or more linker-drug compounds represented by general formula (I) are bound.

[0210] Typically, the inhibitor complexes of the present invention include polypeptides that bind to, reactively associate with, or form complexes with receptors, receptor complexes, antigens, enzymes, or other moieties associated with abnormal or malignant cells, but preferably not associated with or only slightly associated with healthy cells. The polypeptides in the inhibitor complexes of the present invention function as means for directing the linker-drug compound of the present invention to abnormal or malignant cells. Suitable polypeptides include antibodies, their antigen-binding fragments, enzyme inhibitors, enzyme substrates, receptor ligands, and fusion proteins.

[0211] The linker-drug compound of the present invention may be linked to a suitable polypeptide via a reactive native amino acid residue present in the polypeptide (e.g., lysine or cysteine), or via the N-terminus or C-terminus. Alternatively, a natural or unnatural reactive amino acid residue may be introduced into the suitable polypeptide by genetic engineering or post-translational modification. Furthermore, if the suitable polypeptide is a glycoprotein, the linker-drug compound of the present invention may be linked to the glycoprotein via a glycan.

[0212] It should be understood that the linker-drug compound of the present invention included in the inhibitor complex of the present invention may lack certain atoms or groups of atoms compared to the same compound of the present invention not included in the inhibitor complex, for example, it may lack hydrogen atoms. This may be because, for example, the linker-drug compound of the present invention binds to the polypeptide by esterification at the hydroxyl moiety.

[0213] Preferably, the polypeptide used in this specification is an antibody or an antigen-binding fragment thereof. Therefore, the present invention preferably relates to an antibody-drug conjugate (ADC) comprising the linker-drug compound of the present invention.

[0214] In one embodiment, the present invention relates to formula (III) Ab-(LD) y (III) [In the formula, Ab is an antibody or its antigen-binding fragment; LD is the linker-drug compound of the present invention; [y represents the mean drug-antibody ratio (DAR) of 1 to 16, preferably 1 to 10.] This relates to ADCs represented by [the specified format].

[0215] As is widely known in the art, DAR and drug load distribution can be determined, for example, using hydrophobic interaction chromatography (HIC) or reversed-phase high-performance liquid chromatography (RP-HPLC). HIC is particularly suitable for determining the mean DAR.

[0216] In a preferred embodiment, the present invention relates to an ADC represented by formula (III), wherein the linker-drug compound of the present invention binds to an antibody or its antigen-binding fragment via a cysteine ​​residue of the antibody or its antigen-binding fragment.

[0217] In a more preferred embodiment, the present invention is formulated

[0218] [ka]

[0219] [In the formula, Ab is an antibody or its antigen-binding fragment; [y represents the average DAR of 1 to 16, preferably 1 to 10] This relates to ADCs represented by [the specified format].

[0220] In its most preferred embodiment, the present invention relates to the formula

[0221] [ka]

[0222] [In the formula, Ab is an antibody or its antigen-binding fragment; [y represents the average DAR of 1 to 16, preferably 1 to 10] This relates to ADCs represented by [the specified format].

[0223] In the present invention, Ab in the formula of ADC above may be any antibody or its antigen-binding fragment, preferably a monoclonal antibody (mAb) or its antigen-binding fragment.

[0224] In this specification, the term “antibody” preferably refers to an antibody comprising two heavy chains and two light chains. Generally, an antibody or its antigen-binding fragment is therapeutically active, but as is known in the art of ADCs, such independent efficacy is not necessarily required. The antibody used in the present invention may be any isotype, such as IgA, IgE, IgG, or IgM antibody. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. The antibody may be a chimeric, humanized, or human antibody. Preferably, the antibody is a humanized or human antibody. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG1 mAb. The antibody may have a kappa (κ) or lambda (λ) light chain, preferably a kappa light chain, i.e., a humanized or human IgG1-κ antibody.

[0225] In this specification, the term “antigen-binding fragment” includes Fab, Fab', F(ab')2, Fv, scFv, or reduced IgG (rIgG) fragments, single-chain (sc) antibodies, single-domain (sd) antibodies, diabodies, or minibodies.

[0226] The "humanization" of non-human (e.g., rodent) antibodies is the antibody with the smallest sequence derived from the non-human antibody (e.g., a non-human-human chimeric antibody). Various methods for humanizing non-human antibodies are known in the art. For example, the antigen-binding complementarity-determining regions (CDRs) in the variable regions (VRs) of the heavy chain (HC) and light chain (LC) are derived from antibodies of non-human species, usually mouse, rat, or rabbit. These non-human CDRs can be combined with human framework regions (FRs, i.e., FR1, FR2, FR3, and FR4) of the variable regions of the HC and LC so that antibody functions such as binding affinity and specificity are at least partially preserved. Further improvements to antibody properties can be made, such as improving binding affinity while maintaining low immunogenicity, by substituting selected amino acids in the human FRs with the corresponding amino acids from the original non-human species. The humanized variable regions thus are usually combined with the human constant region. A representative method for humanizing non-human antibodies is that of Winter and his collaborators (Jones et al, Nature 1986, 321, 522-525; Riechmann et al, Nature 1988, 332, 323-327; Verhoeyen et al, Science 1988, 239, 1534-1536). Alternatively, non-human antibodies can be humanized by modifying their amino acid sequence to increase their similarity to naturally produced antibody variants in humans. For example, selected amino acids from the original non-human species FR are replaced with corresponding human amino acids to reduce immunogenicity while maintaining antibody binding affinity. For further details, see Jones et al, Nature 1986, 321, 522-525; Riechmann et al, Nature 1988, 332, 323-327 and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596.See also the following review articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1998, 1, 105-115; Harris, Biochem. Soc. Transactions 1995, 23, 1035-1038 and Hurle and Gross, Curr. Op. Biotech. 1994, 5, 428-433.

[0227] CDR can be determined using the methods of Kabat (in Kabat, EA et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, NIH publication no. 91-3242, pp. 662, 680, 689 (1991)), Chothia (et al, Nature 1989, 342, 877883), or IMGT (Lefranc, The Immunologist 1999, 7, 132-136).

[0228] Typically, an antibody is a monospecific antibody (i.e., specific to one antigen; such antigens may be common across species and may have similar amino acid sequences across species) or a bispecific antibody (i.e., specific to two different antigens of a certain kind) containing at least one HC and LC variable region that binds to a target antigen, preferably an antigen that is either internalized or non-internalized, preferably an internalized membrane-bound target antigen.

[0229] In certain embodiments, the target antigens are Annexin Al, B7H3, B7H4, BCMA, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242 (cancer antigen 242), CAIX, CCR2, CCR5, CD2, CD19, CD20, CD22, CD24, CD30 (tumor necrosis factor 8), CD33, CD37, CD38 (cyclic ADP-ribose hydrolase), CD40, CD44, CD47 (integrin-related proteins), CD56 (neuronal cell adhesion molecules), CD70, CD71, CD73, CD74, CD79, CD115 (colony-stimulating factor 1 receptor), CD123 ( (Centaleukin 3 receptor), CD138 (Syndecane 1), CD203c (ENPP3), CD303, CD333, CDCP1, CEA, CEACAM, Claudin 4, Claudin 7, CLCA-1 (Type C lectin-like molecule 1), CLL1, c-MET (Hepatocyte growth factor receptor), Cripto, DLL3, EGFL, EGFR, EPCAM, EphA2, EPhB3, ETBR (Endothelin type B receptor), FAP, FcRL5 (Fc receptor-like protein 5, CD307), FGFR3, FOLR1 (Folic acid receptor α), FRβ, GCC (Guanylyl cyclase C), GD2, GITR, GLOBO The group is selected from H, GPA33, GPC3, GPNMB, HER2, p95HER2, HER3, HMW-MAA (high molecular weight melanoma-associated antigen), integrin α (e.g., αvβ3 and αvβ5), IGF1R, TM4SF1 (L6), Lewis A-like carbohydrate, Lewis X, Lewis Y (CD174), LGR5, LIV1, mesothelin (MSLN), MN (CA9), MUC1, MUC16, NaPi2b, nectin-4, notch 3, PD-1, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4 (or TPBG, trophoblast glycoprotein), TF (tissue factor, thromboplastin, CD142), TF-Ag, Tag72, TNFα, TNFR, TROP2 (tumor-associated calcium signaling transducer 2), uPAR, VEGFR, and VLA.

[0230] Examples of suitable antibodies include blinatumomab (CD19), epratuzumab (CD22), iratumumab and brentuximab (CD30), vadasutuximab (CD33), tetulumab (CD37), isatuximab (CD38), vivacuzumab (CD44), rorbotuzumab (CD56), borsetuzumab (CD70), milatuzumab (CD74), polatuzumab (CD79), and lovalpituzumab. These include bu (DLL3), futuximab (EGFR), oportuzumab (EPCAM), farletuzumab (FOLR1), glenbatumumab (GPNMB), trastuzumab, pertuzumab and margetuximab (HER2), etalacizumab (integrin), anetumab (mesotelin), pancomab (MUC1), enfortumab (nectin-4), and H8, A1 and A3 (5T4).

[0231] The antibody or its antigen-binding fragment may, where applicable, include (1) a modified constant region, i.e., a constant region into which one or more mutations may have been introduced, for example, to extend the half-life, to provide a linker-drug binding site, and / or to increase or decrease effector function; or (2) a modified variable region, i.e., a variable region into which one or more mutations may have been introduced, for example, to provide a linker-drug binding site. The antibody or its antigen-binding fragment may be manufactured by recombination, synthesis, or by other known appropriate methods.

[0232] The ADC of the present invention may be wild-type, site-specific, or a combination thereof, and can be manufactured by methods known in the art as described below.

[0233] Method for manufacturing ADC of the present invention Wild-type ADCs may be produced, for example, by conjugating a linker-drug containing an amine-reactive group, such as an activated ester, to an antibody or its antigen-binding fragment via the ε-amino group of the antibody's lysine, preferably using such a linker-drug. The ADC is obtained by contacting the activated ester with the antibody or its antigen-binding fragment. Alternatively, wild-type ADCs can be produced by conjugating a linker-drug via a free thiol in the side chain of cysteine ​​generated by reduction of interchain disulfide bonds, using methods and conditions known in the art. See, for example, Doronina et al, Bioconjugate Chem. 2006, 17, 114-124. This production method involves partially reducing interchain disulfides exposed to a solvent and modifying the resulting thiol with a Michael receptor-containing linker-drug, such as a maleimide-containing linker-drug, α-haloacetamide, or ester. In the cysteine-binding strategy, up to two linker-drugs are generated per reduced disulfide. Most human IgG molecules have four solvent-exposed disulfide bonds, and therefore, 0 to 8 linker-drugs are possible per antibody. The exact number of linker-drugs per antibody depends on the degree of disulfide reduction and the molar equivalent of the linker-drug used in the subsequent binding reaction. Complete reduction of all four disulfide bonds yields a homogeneous construct with 8 linker-drugs per antibody, while partial reduction typically yields a heterogeneous mixture with 0, 2, 4, 6, or 8 linker-drugs per antibody.

[0234] Site-directed ADCs are preferably produced by linking a linker-drug to a mutant antibody or its antigen-binding fragment via a side chain of a modified cysteine ​​residue at an appropriate position on the mutant antibody or its antigen-binding fragment. The modified cysteine ​​is usually capped with cysteine ​​or another thiol such as glutathione to form a disulfide. These capped residues need to be uncapped before linker-drug binding can occur. Linker-drug binding to the modified residue is achieved by (1) reducing both the natural interchain disulfide and the mutant disulfide, then re-oxidizing the natural interchain cysteine ​​with a mild oxidizing agent such as CuSO4 or dehydroascorbic acid, and then by standard linkage formation between the uncapped modified cysteine ​​and the linker-drug, or (2) using a mild reducing agent that reduces the mutant disulfide faster than the interchain disulfide bond, and by standard linkage formation between the uncapped modified cysteine ​​and the linker-drug. Under optimal conditions, two linker-drugs (i.e., DAR = 2) bind to one antibody or its antigen-binding fragment (when modifying one cysteine ​​of the HC or LC of the antibody or fragment). Suitable methods for site-specific linker-drug binding can be found, for example, in International Publication 2015 / 177360 describing methods of reduction and reoxidation, International Publication 2017 / 137628 describing the use of mild reducing agents, and International Publication 2018 / 215427 describing methods for binding reduced interchain cysteine ​​to uncapped modified cysteine.

[0235] Pharmaceutical composition In another aspect, the present invention provides compositions containing the linker-drug compound or inhibitor complex of the present invention, preferably pharmaceutical compositions, and more preferably further comprising pharmaceutically acceptable additives. Such compositions are hereafter referred to as compositions of the present invention. The compositions may be, for example, liquid formulations, lyophilized formulations, or in the form of capsules or tablets. Typically, pharmaceutical formulations in the form of capsules or tablets containing low molecular weights such as the linker-drug compound of the present invention include excipients. Suitable water-soluble excipients include sugars, sugar alcohols, polysaccharides, and cyclodextrins. Suitable non-water-soluble excipients include calcium phosphate, calcium sulfate, starch, modified starch, and crystalline cellulose. Furthermore, pharmaceutical formulations containing low molecular weights such as the linker-drug compound of the present invention may also include binders. Suitable binders include gelatin, cellulose derivatives, polymers such as cross-linked polyvinylpyrrolidone (crospovidone) or copolividone, and polyethylene glycol. Pharmaceutical formulations containing low molecular weights such as the linker-drug compound of the present invention may further include disintegrants. Suitable disintegrants include cross-linked polymers such as crospovidone, cross-linked carboxymethylcellulose sodium (croscarmellose sodium), and sodium starch glycolate. Furthermore, pharmaceutical formulations containing low molecular weights such as the linker-drug compounds of the present invention may also contain flow enhancers such as fumed silica, talc, and magnesium carbonate; lubricants such as talc or silica, vegetable stearin, magnesium stearate, or stearic acid; antioxidants or preservatives such as parabens; colorants; sweeteners and / or fragrances.

[0236] Typically, pharmaceutical compositions comprising the inhibitor complex, ADC, or linker-drug compound of the present invention take the form of a lyophilized cake (lyophilized powder) that requires dissolution (i.e., reconstitution) (in water) before intravenous infusion, or a frozen (water) solution that requires thawing before use. Therefore, in a preferred embodiment, the present invention provides a lyophilized composition containing the linker-drug compound or inhibitor complex of the present invention, preferably a pharmaceutical composition, and more preferably further comprising pharmaceutically acceptable additives. In a further preferred embodiment, the present invention provides a frozen composition containing water and the linker-drug compound or inhibitor complex of the present invention, preferably a pharmaceutical composition, and more preferably further comprising pharmaceutically acceptable additives. In this specification, the frozen solution is preferably at atmospheric pressure, and the frozen solution is preferably obtained by freezing the liquid composition of the present invention at a temperature below 0°C. Suitable pharmaceutically acceptable additives for incorporation into the pharmaceutical composition (before freeze-drying) of the present invention include buffer solutions (e.g., succinates containing amino acids such as citrate and histidine, or salts in water), cryoprotectants (e.g., sucrose, trehalose), isotonic agents (e.g., chlorides such as sodium chloride), surfactants (e.g., polysorbates), and bulking agents (e.g., mannitol, glycine). Additives used in freeze-dried protein formulations are selected based on their ability to prevent protein denaturation during freeze-drying and storage.

[0237] Pharmaceutical use In another aspect, the present invention provides linker-drug compounds, inhibitor conjugates (preferably antibody-drug conjugates), or compositions for use as pharmaceuticals, preferably for use in the treatment of cancer, autoimmune diseases, or infectious diseases. These linker-drug compounds, conjugates, and compositions will hereafter be referred to collectively as products used in the applications of the present invention.

[0238] In the first aspect, the product used in the applications of the present invention is for use in the treatment of solid tumors or hematological cancers.

[0239] In a second aspect, the product used in the applications of the present invention is for use in the treatment of autoimmune diseases.

[0240] In a third aspect, the product used in the applications of the present invention is for use in treating infections caused by bacteria, viruses, parasites, or other organisms.

[0241] In the present invention, cancer is preferably a tumor that expresses the antigen targeted by the product used in the applications of the present invention. Such cancers may be solid tumors or hematological cancers. Examples of solid tumors or hematological cancers that can be treated with the product used in the applications of the present invention as described above include: breast cancer; brain cancer (e.g., glioblastoma); head and neck cancer; thyroid cancer; parotid gland cancer; adrenal cancer (e.g., neuroblastoma, paraganglioma, or pheochromocytoma); bone cancer (e.g., osteosarcoma); soft tissue sarcoma (STS); eye cancer (e.g., uveal melanoma); esophageal cancer; stomach cancer; small intestine cancer; colorectal cancer; urothelial cell carcinoma (e.g., bladder cancer, penile cancer, ureteral cancer, or kidney cancer); ovarian cancer; uterine cancer; vaginal, vulvar, and cervical cancer; lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)); melanoma; mesothelioma (e.g., malignant pleural and abdominal mesothelioma); liver cancer (e.g., hepatitis Cellular cancer; pancreatic cancer; skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, or dermatofibrosarcoma protuberans); testicular cancer; prostate cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); myelodysplastic syndrome (MDS); blastic plasmacytoid dendritic cell neoplasm (BPDCN); Hodgkin lymphoma; non-Hodgkin lymphoma (NHL) (including follicular lymphoma (FL), CNS lymphoma, and diffuse large B-cell lymphoma (DLBCL)); light chain amyloidosis; plasma cell leukemia; and multiple myeloma (MM) may be included, but are not limited to these.

[0242] The autoimmune diseases in this invention are preferably autoimmune diseases related to the antigen targeted by the product used in the applications of this invention. Autoimmune diseases describe conditions resulting from an abnormal immune response against normal somatic cells and tissues. There are at least 80 types of autoimmune diseases. Some diseases are organ-specific and limited to specific tissues, while others are similar to systemic inflammatory diseases that affect many tissues throughout the body. The appearance and severity of these signs and symptoms may vary over time, depending on the site and type of inflammatory response that occurs. Examples of autoimmune diseases that can be treated with the products used in the applications of the present invention described above include: rheumatoid arthritis; juvenile dermatomyositis; psoriasis; psoriatic arthritis; lupus; sarcoidosis; Crohn's disease; eczema; nephritis; uveitis; polymyositis; neuritis including Guillain-Barré syndrome; encephalitis; arachnoiditis; systemic sclerosis; autoimmune diseases of the muscles, skeleton and connective tissue; Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and Kawasaki disease of large, medium, and small vessels. Neuromuscular degenerative diseases, including Henoch-Schönlein vasculitis; cold and warm agglutinin diseases; autoimmune hemolytic anemia; type 1 diabetes mellitus; Hashimoto's thyroiditis; Graves' disease; Graves' ophthalmopathy; adrenal nephritis; hypophysitis; pemphigus vulgaris; Addison's disease; ankylosing spondylitis; Behçet's syndrome; celiac disease; Goodpasture syndrome; myasthenia gravis; sarcoidosis; scleroderma; primary sclerosing cholangitis; acquired epidermolysis bullosa; and bullous pemphigoid may be included, but are not limited to, these conditions.

[0243] Preferably, the autoimmune disease treated by the present invention is rheumatoid arthritis.

[0244] The infectious diseases in the present invention are preferably those related to the antigens targeted by the products used in the applications of the present invention. Such infectious diseases may be caused by bacteria, viruses, parasites, or other organisms. Examples of infectious diseases that can be treated with the products used in the applications of the present invention as described above include, but are not limited to, malaria; toxoplasmosis; meridianus caused by Pneumocystis jirovecii; bacterial dysentery; listeriosis; cyclosporiasis; leprosy; tuberculosis; and infection prevention in immunocompromised individuals, such as those who are HIV-positive, those receiving immunosuppressive therapy, and those with congenital abnormalities such as cystic fibrosis or benign proliferative disorders (e.g., hydatidiform mole, endometriosis).

[0245] The products used in the applications of the present invention as described in this specification can be used in the manufacture of pharmaceuticals as described herein. The products used in the applications of the present invention as described in this specification are preferably for therapeutic methods, and the products used in the applications are administered in a therapeutically effective dose to a subject, preferably a subject that needs it. Accordingly, in some embodiments, or in combination with other embodiments, the present invention relates to the use of the products used in the applications of the present invention in the manufacture of pharmaceuticals for the treatment of cancer, autoimmune diseases or infectious diseases, in particular cancer. For exemplary and non-limiting cancers or other diseases treated by the present invention, please refer to the preceding description.

[0246] In some embodiments, or in combination with other embodiments, the present invention relates to a method for treating cancer, autoimmune diseases, or infectious diseases, particularly cancer, the method comprising administering a therapeutically effective amount of the product used in the present invention to a subject requiring such treatment. For exemplary and non-limiting cancers or other diseases treated by the present invention, please refer to the preceding description.

[0247] The products used in the applications of the present invention are for administration to subjects. The products used in the applications of the present invention can be used in the therapeutic methods described herein by administering an effective amount of the composition to a subject in need. In this specification, the term "subject" refers to all animals classified as mammals, including but not limited to primates and humans. The subject is preferably human. The expression "therapeutic effective amount" means an amount sufficient to produce a desired response or to improve a symptom or sign. The therapeutic effective amount in a particular subject may vary depending on factors such as the symptoms, the subject's overall condition, the method, route and dose of administration, and the severity of side effects.

[0248] In another aspect, the present invention provides a product for use in the applications of the present invention, which is used in combination with one or more other therapeutic agents. The product for use in the applications of the present invention can be used simultaneously with or in succession with one or more other therapeutic agents.

[0249] Appropriate chemotherapy agents include alkylating agents such as nitrogen mustards, hydroxyureas, nitrosourias, tetrazines (e.g., temozolomide), and aziridines (e.g., mitomycin); agents that inhibit the DNA damage response, such as PARP inhibitors, ATR and ATM inhibitors, CHK1 and CHK2 inhibitors, DNA-PK inhibitors, and WEE1 inhibitors; antimetabolites such as folic acid antagonists (e.g., pemetrexed), fluoropyrimidines (e.g., gemcitabine), deoxynucleoside analogs, and thiopurines; antimicrotubule agents such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics such as anthracyclines and bleomycin; methylation inhibitors such as decitabine and azacitidine; histone deacetylase inhibitors; all-trans retinoic acids; and arsenic trioxide. Appropriate radiotherapy includes 131 I-Metaiodobenzylguanidine (MIBG), as sodium phosphate 32 P, chloride 223 Radium, chloride 89 Strontium and 153Radioisotopes such as Sm-diaminetetramethylenephosphonate (EDTMP) are included. Appropriate drugs used as hormonal therapies include hormone synthesis inhibitors such as aromatase inhibitors and GnRH analogs; hormone receptor antagonists such as selective estrogen receptor modulators (e.g., tamoxifen and fulvestrant) and antiandrogens such as bicalutamide, enzalutamide, and flutamide; CYP17A1 inhibitors such as abiraterone; and somatostatin analogs.

[0250] Targeted therapies are therapeutic agents that interfere with specific proteins involved in tumorigenesis and proliferation, and may be small molecule drugs; proteins such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids such as ADCs. Examples of small molecule targeted drugs include mTor inhibitors such as everolimus, temsirolimus, and rapamycin; kinase inhibitors such as imatinib, dasatinib, and nilotinib; VEGF inhibitors such as sorafenib and regorafenib; EGFR / HER2 inhibitors such as gefitinib, lapatinib, and erlotinib; and CDK4 / 6 inhibitors such as palbociclib, ribociclib, and abemaciclib. Examples of peptide or peptide derivative targeted therapies include proteasome inhibitors such as bortezomib and carfilzomib.

[0251] Appropriate anti-inflammatory drugs include D-penicillamine, azathioprine and 6-mercaptopurine, cyclosporine, anti-TNF biological agents (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab or certolizumab pegol), lenflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acetretin, and JAK inhibitors (e.g., tofacitinib, baricitinib or upadacitinib).

[0252] Immunotherapy agents include cytokines (IL-2 and IFNα); immunomodulatory imides (e.g., thalidomide, lenalidomide, pomalidomide, or imiquimod); cancer vaccines (e.g., tarimodine laherparepbec); cell-based immunotherapies (e.g., dendritic cell vaccines, adoptive T cells, or chimeric antigen receptor-modified T cells); and therapeutic antibodies that can bind to cell membrane ligands and cause antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) via their Fc domain, thereby inducing, enhancing, or suppressing immune responses.

[0253] In the present invention, treatment preferably prevents, reverses, cures, improves, and / or delays cancer, autoimmune disease, or infection. This may mean that the severity of at least one symptom of cancer, autoimmune disease, or infection is reduced, and / or that at least one parameter related to cancer, autoimmune disease, or infection is improved. Preferably, such parameter is related to folate activity.

[0254] In this invention, the subject may survive and / or be considered disease-free. Alternatively, the disease or condition may cease or be delayed. In this invention, improvement in quality of life and observed pain relief may mean that the amount of analgesic required by the subject may be less than at the start of treatment. In this specification, “less” may mean less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, or less than 90%. The subject no longer requires any analgesic. This improvement in quality of life and observed pain relief may be observed, detected, or evaluated at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or more after treatment in the subject, and may be compared to the quality of life and observed pain relief at the start of treatment in the subject.

[0255] General definition The linker-drug compounds of the present invention may contain one or more chiral centers and / or double bonds, and therefore may exist as stereoisomers such as double bond isomers (i.e., geometric isomers), positional isomers, enantiomers, or diastereomers. Accordingly, the chemical structures shown herein encompass all possible enantiomers and stereoisomers of the illustrated or specified compounds, including sterically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and mixtures of stereoisomers. Mixtures of enantiomers and mixtures of stereoisomers can be separated into their constituent enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. The compounds may exist in several tautomers, including enol forms, keto forms, and mixtures thereof. Accordingly, the chemical structures shown herein encompass all possible tautomers of the illustrated or specified compounds. It is also understood that some isomers, such as diastereomers, enantiomers, and geometric isomers, can be separated by physical and / or chemical methods by those skilled in the art. Where a chemical formula or name indicates the presence of a chiral center, each chiral center is referred to individually for all three: racemic mixtures, pure R enantiomers, and pure S enantiomers, even if chirality is not explicitly stated. Where the structure of a compound is presented as a specific enantiomer, it should be understood that the present invention is not limited to that specific enantiomer. When two parts combine to form a bond, this means that these parts do not exist as atoms, and that the valence rules are satisfied by electronic bond substitution. All of this is known in the art.

[0256] The compounds disclosed in this specification and claims may also exist as exo and endo-positional isomers. Unless otherwise stated, the descriptions of compounds in this specification and claims include both the individual exo-positional isomers and individual endo-positional isomers of the compound, as well as mixtures thereof. Furthermore, the compounds disclosed in this specification and claims may also exist as cis and trans isomers. Unless otherwise stated, the descriptions of compounds in this specification and claims include both the individual cis isomers and individual trans isomers of the compound, as well as mixtures thereof. As an example, if the structure of a compound is shown as a cis isomer, it should be understood that the corresponding trans isomer or mixtures of cis and trans isomers are not excluded from the present invention.

[0257] In this specification and claims, the verb “comprise” and its conjugations are used in a non-restrictive sense, meaning that the item following it is included, but not that items not specifically mentioned are excluded. In addition, references to elements with the indefinite article “a” or “an” do not rule out the possibility that there are two or more elements unless the context explicitly indicates that there is only one element. Thus, the indefinite article “a” or “an” usually means “at least one.”

[0258] When the words "about" or "approximately" are used with numbers (e.g., about 10), it is preferable to mean that the value may be greater than or less than 1% of that value.

[0259] Wherever parameters of a substance are described in this specification, unless otherwise specified, it is assumed that the parameters are determined, measured, or expressed under physiological conditions. Physiological conditions are known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, pH 6–8, room temperature to about 37°C (about 20°C to about 40°C), and appropriate concentrations of buffer salts or other components. It is understood that charge is often associated with equilibrium. A portion referred to as having or bearing a charge is a portion that is more frequently found in such a charged or bearing state than in such an uncharged or unbearing state. Thus, as understood by those skilled in the art, atoms indicated in this disclosure as being charged may be uncharged under certain conditions, and neutral portions may be charged under certain conditions.

[0260] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0261] The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Examples]

[0262] general All solvents used were reagent grade or HPLC grade from various vendors. NMR spectra were obtained using a Bruker AVANCE400. 1 H is 400MHz; 13The 100 MHz (C) reading was recorded. Chemical shifts are expressed in ppm relative to tetramethylsilane or residual non-deuterated solvent as an internal standard. Mass spectra were recorded using a Waters UPLC-MS (ESI with SQ detector 3100) equipped with a reversed-phase C18 cross-linked ethylsiloxane-silica hybrid column (Waters ACQUITY UPLC® BEH C18, particle size 1.7 μm, 2.1 × 50 mm) at a flow rate of 0.4 mL / min (acetonitrile (MeCN) / water × 0.1% formic acid (FA)). Purification by preparative HPLC was performed using a Shimadzu Prominence 20AP system fitted with a Waters SunFire Prep C18 OBD 5 μm column (19 × 150 mm) at a flow rate of 17 mL / min (MeCN / water × 0.1% trifluoroacetic acid (TFA)). Microwave reactions were performed using a Biotage Initiator+ instrument.

[0263] Hydrophobic interaction chromatography (HIC) for evaluating the properties of ADCs For HIC analysis, 5–10 μL of sample (1 mg / mL) was injected into a TSKgel butyl-NPR column (4.6 mm ID × 3.5 cm L, Tosoh Bioscience, Cat. no. 14947). Elution was performed with a linear gradient of 0.4 mL / min for 20 minutes from 100% buffer A (25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 6.95) to 100% buffer B (25 mM sodium phosphate, pH 6.95, 20% isopropanol). A Waters Acquity H-Class UPLC system with a PDA detector and Empower software was used. Absorbance was measured at 214 nm to determine the retention time in the ADC.

[0264] Size exclusion chromatography (SEC) for evaluating the properties of ADCs. For SEC analysis, 5 μL of sample (1 mg / mL) was injected into a TSKgel G3000SWXL column (5 μm, 7.8 mm ID × 30 cm L, Tosoh Bioscience, Cat. no. 08541) equipped with a TSKgel SWXL Guard column (7 μm, 6.0 mm ID × 4.0 cm L, Tosoh Bioscience, Cat. no. 08543). Elution was performed using 100% 50 mM sodium phosphate, 300 mM NaCl, pH 7.5 at a rate of 0.6 mL / min for 30 minutes. The column temperature was maintained at 25°C. A Waters Acquity H-Class UPLC system with a PDA detector and Empower software was used. HMW species were quantified by measuring absorbance at 214 nm.

[0265] General Procedure XXA: HATU-Assisted Amide Coupling A carboxylic acid (1.0 equivalent) and an amine (1.0-1.5 equivalents) were dissolved in dimethylformamide (DMF; 0.175 M). 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (hexafluorophosphate azabenzotriazole tetramethyl uronium, HATU; 1.2 equivalents) and N,N-diisopropylethylamine (DIPEA; 6.0 equivalents) were added at room temperature, and the mixture was stirred for 30 minutes. After concentration, the product was recrystallized from methanol (MeOH) or purified by flash chromatography, as shown in each example.

[0266] General Procedure XXB: Reduction of Phenylazide with Pd / C and Hydrogen To a solution of azide (1.0 equivalent) in DMF (0.074 M), 10 wt% of palladium supported on activated carbon was added under an N2 atmosphere. The mixture was purged with hydrogen gas and vigorously stirred until the UPLC indicated completion of the reaction (approximately 45 minutes). The reaction product was purged with N2 and filtered through Celite®. The filtrate was concentrated under reduced pressure and purified as shown in each example.

[0267] General Procedure XXC: Deprotection and / or Hydrolysis of Formamide Ester (1.0 equivalent) was dissolved / suspended in methanol / dimethyl sulfoxide (MeOH / DMSO; 5:1, 0.36 M) and cooled to 0°C. NaOH aqueous solution (2.0 M, 3-24 equivalents) was added dropwise, and the resulting solution was stirred at room temperature until ULC analysis was complete (1-6 hours). The reaction mixture was diluted with water (approx. 4×), cooled to 0°C, and the pH was adjusted to approximately 8.5 with hydrochloric acid (1.0 M). If no precipitate formed, the solution was washed with ethyl acetate (HCl; 3×). This step was omitted for suspensions. The aqueous phase was further diluted with water (approx. 3×), and the pH was adjusted to approximately 4.7 with acetic acid (AcOH) aqueous solution (1.0 M). The resulting suspension was vigorously stirred for 30 minutes and filtered. Occasionally, a gelatinous mixture was obtained, which was then gently heated with a heat gun to obtain a suspension. The solid was washed with MeOH and ether and dried under vacuum.

[0268] Preparation of (S)-5-(4-azidobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX5) and (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX7) [ka]

[0269] (S)-2-amino-5-(4-azidobenzamide)methyl pentanoate (XX3) [ka]

[0270] Thionyl chloride (3.76 mL, 51.5 mmol) and DMF (5 drops) were added to a suspension of 4-azidobenzoic acid (7.00 g, 42.9 mmol) in dichloromethane (DCM; 150 mL) under a nitrogen atmosphere. The reaction mixture was heated and gently refluxed for 2 hours, cooled to room temperature, concentrated, and the solid was gently ground into a powder. In a separate flask, at room temperature, an aqueous solution of L-ornithine hydrochloride (L-Orn-OH·HCl) (6.70 g, 38.9 mmol) and NaOH (3.11 g, 77.8 mmol) (67 mL) was treated with an aqueous solution of CuSO4·5H2O (4.86 g, 19.5 mmol) (67 mL) to obtain a deep blue solution. NaHCO3 (3.93 g, 46.7 mmol) was added to this solution. After dissolution, the dark blue solution was poured directly onto unpurified solid 4-azidobenzoic acid chloride (7.78 g, 42.8 mmol) at room temperature, and the mixture was vigorously stirred at room temperature for 16 hours. The suspension was filtered, and the solid was washed with water (2 × 20 mL), ethanol (2 × 20 mL), and ether (3 × 40 mL) to obtain 4.97 g of solid. The material was dissolved in MeOH (140 mL), and thionyl chloride (13.0 mL) was added over 45 minutes at 0°C under a nitrogen atmosphere. The reaction mixture was then stirred at room temperature for 18 hours and then concentrated. Subsequently, a portion of the material was purified by solid-phase extraction (SPE; Biotage, SCX-2) to obtain XX3 as a colorless waxy solid (1.20 g). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.49 (t, J = 5.5 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 3.61 (s, 3H), 3.37-3.31 (m, 1H), 3.24 (q, J = 6.1 Hz, 2H), 2.13 (br s, 2H), 1.64-1.43 (m, 4H). MS (ESI + ) C 13 H 18 N5O3 + [M+H] + Calculated value: 292.14, measured value: 292.20.

[0271] 4-(2-(2,4-diaminopteridine-6-yl)ethyl)methyl benzoate (XX1) A mixture of pyrimidine-2,4,5,6-tetraamine sulfate (6.00 g, 25.2 mmol), barium chloride dihydrate (6.16 g, 25.2 mmol), and water (145 mL) was stirred at room temperature for 90 minutes. The mixture was warmed to 70°C, filtered, and the filtrate was cooled to room temperature, and the pH was adjusted to 3.5 with 10% NaOH (aqueous solution). A portion of this solution (116 mL) was mixed with unpurified methyl 4-(3-bromo-4-oxobutyl)benzoate (6.06 g, 21.2 mmol, prepared as described in Chen et al, J. Heterocycl. Chem. 2015, 52, 1565-1569) in MeCN (43.8 mL), and then AcOH (34.7 mL) was added while stirring on a water bath at room temperature. After 5 minutes, MnO2 (11.6 g) was added, and the mixture was stirred at room temperature for 1 hour and 45 minutes. The reaction product was filtered through Celite®, and the residue was washed with MeCN / water (7:3). The filtrate was concentrated and washed with water (125 mL). The remaining water was removed azeotropically with MeCN. The residue was then triturated twice with hot MeCN (125 mL), and the solid was washed with ether and dried in vacuum to obtain XX1 as a brown solid (1.31 g, 20%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.65 (s, 1H), 8.38 (br s, 1H), 8.31 (br s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.27 (br s, 2H), 3.82 (s, 3H), 3.19 (s, 4H). MS (ESI + ) C 16 H 17 N6O2 + [M+H] + Calculated value: 325.14, measured value: 325.12.

[0272] 4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzoic acid (XX2) Ester (XX1) (245 mg, 0.755 mmol) was suspended in 2-methoxyethanol (6 mL), and 0.5 N NaOH (aqueous solution, 6 mL) was added at room temperature. The suspension was stirred for 24 hours and then filtered. The residue was washed with 2-methoxyethanol / water (1:1, 1 mL), and then glacial acetic acid (0.325 mL) was added to the filtrate to adjust the pH to 4.5. The mixture was allowed to stand at 4°C for 16 hours, filtered, washed with water (2 × 3 mL), and dried in vacuum to obtain XX2 as a brown solid (140 mg, 60%). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.78 (br s, 1H), 8.56 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.56 (br s, 2H), 7.37 (d, J = 8.1 Hz, 2H), 6.54 (br s, 2H), 3.19-3.11 (s, 4H). MS (ESI + ) C 15 H 15 N6O2 + [M+H] + Calculated value: 311.13, measured value: 311.34.

[0273] (S)-5-(4-azidobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XX4) Following general procedure XXA, acid XX2 (235 mg, 0.757 mmol) and amine XX3 (243 mg, 0.833 mmol) were reacted and purified by recrystallization from MeOH to obtain ester XX4 as a brown solid (321 mg, 73%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.66 (d, J = 7.4 Hz, 1H), 8.55 (s, 1H), 8.47 (t, J = 5.6 Hz, 1H), 7.90-7.86 (m, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.59 (br s, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.21-7.17 (m, 2H), 6.54 (br s, 2H), 4.47-4.42 (m, 1H), 3.63 (s, 3H), 3.30-3.25 (m, 2H), 3.17-3.12 (m, 4H), 1.91-1.75 (m, 2H), 1.70-1.52 (m, 2H). MS (ESI + ) C 28 H 30 N 11 O4 + [M+H] + Calculated value: 584.25, measured value: 584.49.

[0274] (S)-5-(4-azidobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX5) Hydrolysis of ester XX4 (36 mg, 0.062 mmol) with NaOH (3 equivalents) was carried out according to the general procedure XXC, and acid XX5 was obtained as a yellow solid (27 mg, 75%). 1H NMR (400 MHz, DMSO-d6) ppm = 12.60 (br s, 1H), 8.56 (s, 1H), 8.52 (d, J = 7.7 Hz, 1H), 8.48 (t, J = 5.6 Hz, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.65 (br s, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 6.60 (s, 2H), 4.42-4.33 (m, 1H), 3.27 (q, J = 6.4 Hz, 2H), 3.15 (s, 4H), 1.92-1.73 (m, 2H), 1.70-1.54 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 173.8, 166.4, 165.2, 162.8, 162.3, 154.2, 150.4, 148.4, 144.9, 142.1, 131.7, 131.2, 129.1, 128.3, 127.5, 121.4, 118.8, 52.4, 38.9, 35.3, 33.9, 28.2, 26.2. MS (ESI + ) C 27 H 28 N 11 O4 + [M+H] + Calculated value: 570.23, measured value: 570.54.

[0275] (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XX6) Azide XX4 (250 mg, 0.482 mmol) was reduced according to the general procedure XXB. Aniline XX6 was obtained as a yellow solid (151 mg, 63%) by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3). 1H NMR (400 MHz, DMSO-d6) ppm = 8.68 (d, J = 7.5 Hz, 1H), 8.55 (s, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.63 (br s, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 6.58 (br s, 2H), 6.52 (d, J = 8.6 Hz, 2H), 5.59 (br s, 2H), 4.45-4.40 (m, 1H), 3.63 (s, 3H), 3.22 (q, J = 6.2 Hz, 2H), 3.15 (s, 4H), 1.88-1.74 (m, 2H), 1.67-1.49 (m, 2H). MS (ESI + ) C 28 H 32 N9O4 + [M+H] + Calculated value: 558.26, measured value: 558.51.

[0276] (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX7) Hydrolysis of ester XX6 (151 mg, 0.271 mmol) with NaOH (3 equivalents) was carried out according to the general procedure XXC, yielding acid XX7 as a yellow solid (112 mg, 75%). 1H NMR (400 MHz, DMSO-d6) ppm = 12.52 (br s, 1H), 8.56 (s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.65 (br s, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.62 (br s, 2H), 6.52 (d, J = 8.5 Hz, 2H), 5.58 (br s, 2H), 4.41-4.33 (m, 1H), 3.22 (td, J = 6.6, 6.0 Hz, 2H), 3.15 (s, 4H), 1.91-1.71 (m, 2H), 1.67-1.50 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 173.8, 166.4, 166.2, 162.8, 162.3, 154.2, 151.5, 150.4, 148.4, 144.9, 131.8, 128.6, 128.3, 127.6, 121.4, 112.5, 52.6, 38.6, 35.3, 33.9, 28.2, 26.5. MS (ESI + C 27 H 30 N9O4 + [M+H] + The calculated value is 544.24 and the measured value is 544.52.

[0277] Preparation of (S)-5-(4-aminobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)-benzamide)pentanoic acid (XX12)

change

[0278] 4-(((2,4-ジアミノプテリジン-6-イル)メチル)(メチル)アミノ)benzoic acid (XX9) (2,4-diaminopteridine-6-yl)methanol hydrochloride (8.80 g, 38.5 mmol) was dissolved in hot water (300 mL). After cooling to room temperature, aqueous NaOH solution (40 mL, 1.0 M) was added until the pH exceeded 7. The solution was filtered, washed with water (2 × 25 mL), and dried in vacuum at room temperature for 3 hours. The remaining water was removed azeotropically with ethanol (EtOH). The residue was triturated with hot EtOH, cooled to room temperature, filtered, washed with EtOH (50 mL) and ether (2 × 50 mL), and dried in vacuum to obtain (2,4-diaminopteridine-6-yl)methanol as a free base (6.41 g). In a separate flask, bromine (1.60 mL, 31.2 mmol) was added dropwise over 50 minutes to a suspension of PPh3 (8.19 g, 31.2 mmol) in cooled (0°C) dimethylacetamide (DMA; 13.5 mL). The addition rate was controlled so that the temperature of the solution did not exceed 8°C. The concentrated slurry was stirred at room temperature for 75 minutes to obtain an orange slurry. The solid free base (2,4-diaminopteridine-6-yl)methanol (2.00 g, 10.4 mmol) was added, and the temperature was raised to 38°C. The mixture was stirred at room temperature for 24 hours, then 4-(methylamino)benzoic acid (2.36 g, 15.6 mmol), followed by DIPEA (3.81 mL, 21.9 mmol). After stirring for 5 days, the mixture was added to aqueous NaOH solution (136 mL, 0.33 M), and the reaction was completed using DMA (4 mL). Water (40 mL) was added, and the precipitate was filtered off. The filtrate was diluted to pH 4.5 with 10% aqueous acetic acid solution (approximately 25.0 mL). The precipitate was filtered off, washed with water, triturated with hot MeOH (16 mL), filtered, and the residue was suspended in dioxane. Freeze-dried to obtain acid XX9 as a yellow solid (3.55 g, quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.15 (br s, 1H), 8.65 (s, 1H), 8.35 (br s, 1H), 8.12 (br s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.20 (br s, 2H), 6.83 (d, J = 8.8 Hz, 2H), 4.83 (s, 2H), 3.24 (s, 3H). MS (ESI + ) C 15 H 16 N7O2 + [M+H] + Calculated value: 326.14, measured value: 326.38.

[0279] (S)-5-(4-azidobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)-methylpentanoate(XX10) Acid XX9 (250 mg, 0.768 mmol) and amine XX3 (323 mg, 1.11 mmol) were reacted according to general procedure XXA. Ester XX10 was obtained as a yellow solid by purification using flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) (434 mg, 94%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.66 (d, J = 7.5 Hz, 1H), 8.55 (s, 1H), 8.47 (t, J = 5.6 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.59 (br s, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 6.54 (br s, 2H), 4.47-4.42 (m, 1H), 3.63 (s, 3H), 3.30-3.25 (m, 2H), 3.12 (s, 4H), 1.91-1.75 (m, 2H), 1.69-1.53 ​​(m, 2H). MS (ESI + ) C 28 H 30 N 11 O4 + [M+H] + Calculated value: 584.25, measured value: 584.49.

[0280] (S)-5-(4-aminobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)-methylpentanoate(XX11) Azide XX10 (195 mg, 0.326 mmol) was reduced according to the general procedure XXB. Aniline XX11 was obtained as a yellow solid (111 mg, 60%) by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.56 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H), 7.96 (t, J = 5.4 Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.67 (br s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.45 (br s, 1H), 6.81 (d, J = 8.9 Hz, 2H), 6.62 (br s, 2H), 6.51 (d, J = 8.6 Hz, 2H), 5.56 (br s, 2H), 4.78 (s, 2H), 4.41-4.35 (m, 1H), 3.60 (s, 3H), 3.24-3.18 (m, 2H), 3.21 (s, 3H), 1.85-1.70 (m, 2H), 1.64-1.46 (m, 2H). MS (ESI + ) C 28 H 33 N 10 O4 + [M+H] + Calculated value: 573.27, measured value: 573.52.

[0281] (S)-5-(4-aminobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)pentanoic acid (XX12) Ester XX11 (111 mg, 0.194 mmol) was hydrolyzed according to the general procedure XXC, yielding acid XX12 as a yellow solid (72 mg, 66%). 1H NMR (400 MHz, DMSO-d6) ppm =12.43 (br s, 1H), 8.61 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.16 (br s, 1H), 7.97 (t, J = 5.3 Hz, 1H), 7.94 (br s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.08 (br s, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 8.5 Hz, 2H), 6.04 (br s, 2H), 4.81 (s, 2H), 4.36-4.31 (m, 1H), 3.22 (s, 3H), 3.21-3.18 (m, 2H), 1.86-1.69 (m, 2H), 1.64-1.47 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 174.1, 166.2, 166.2, 162.7, 160.7, 152.2, 151.4, 150.8, 149.0, 147.6, 129.0, 128.6, 121.6, 121.4, 121.3, 112.5, 111.1, 54.9, 52.4, 39.2, 38.6, 28.3, 26.5. MS (ESI + C 27 H 31 N 10 O4 + [M+H] + The calculated value is 559.25 and the measured value is 559.53.

[0282] Preparation of (S)-2-amino-5-(5-amino-1,3-dioxoisoindolin-2-yl)methyl pentanoate (XT6)

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[0283] 5-アミノイソインドリン-1,3-ジオン(XT2) NH4Cl (11.1 g, 208 mmol) in water (25 mL) was added to a solution of compound XT1 (10.0 g, 52.0 mmol) in tetrahydrofuran (THF; 75 mL). Then, zinc powder (13.6 g, 208 mmol) was carefully added little by little (caution: exothermic reaction!), and the resulting suspension was stirred for 1 hour. The yellow reaction mixture was filtered with Celite®, then rinsed with methanol (250 mL), concentrated under reduced pressure to obtain Crop 1. The Celite® cake was suspended in DMF (40 mL), stirred overnight, then filtered, concentrated under reduced pressure to obtain Crop 2. Crop 1 in water (50 mL) was stirred for 15 minutes, filtered, the cake was washed with ether, and air-dried overnight. Crop 2 in ether (150 mL) was stirred overnight, filtered, and the solid was stirred in water (100 mL) for 15 minutes. After filtering, the cake was washed with ether, and the solid was air-dried. The two batches were combined to obtain XT2 as a yellow solid (12g, quantified). MS (ESI + ) C8H7N2O2 + [M+H] + Calculated value: 163.05, measured value: 163.06.

[0284] N-(1,3-dioxoisoindolin-5-yl)formamide (XT3) To a 250 mL round-bottom flask containing FA (22.2 mL, 578 mmol), Ac2O (10.9 mL, 116 mmol) was slowly added, and the mixture was stirred for 10 minutes. Subsequently, crushed aniline XT2 (3.75 g, 23.1 mmol) was added, and the resulting mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure, and water (25 mL) was added to the unpurified product. The resulting suspension was stirred for 15 minutes. After filtration, the solid was air-dried overnight to obtain formamide XT3 as a yellow solid (2.70 g, 14.2 mmol, 61%). 1H NMR (400 MHz, DMSO-d6) ppm = 11.24 (br s, 1H), 10.80 (s, 1H), 8.42 (d, J = 1.1 Hz, 1H), 8.12 (d, J = 1.1 Hz, 1H), 7.87 (dd, J = 8.2, 1.1 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H). MS (ESI + ) C9H7N2O3 + [M+H] + Calculated value: 191.05, measured value: 191.13.

[0285] 5-Formamide-1,3-Dioxoisoindoline-2-carboxylate ethyl(XT4) To a solution of formamide XT3 (2.50 g, 13.2 mmol) in cooled (0°C) DMF (25 mL), Et3N (1.83 mL, 13.2 mmol) was added, and ethyl chloroformate (1.25 mL, 13.2 mmol) in DMF (12.5 mL) was added dropwise. The resulting mixture was stirred at 0°C for 1 hour. Additional Et3N (1.83 mL, 13.2 mmol) and ethyl chloroformate (1.25 mL, 13.2 mmol) in DMF (12.5 mL) were added (the latter dropwise), and the mixture was stirred at 0°C for 30 minutes. Finally, Et3N (0.92 mL, 6.6 mmol) and ethyl chloroformate (0.625 mL, 6.6 mmol) in DMF (6 mL) were added, and the mixture was stirred at 0°C for a final 30 minutes. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, dimethyl:DCM 0:1 to 1:0) to obtain formamide XT4 as a yellow solid (1.4 g, 41%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.90 (br s, 1H), 8.45 (br s, 1H), 8.24 (br s, 1H), 7.95-7.90 (m, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1Hz, 3H). MS (ESI + ) C 12 H 11N2O5 + [M+H] + Calculated value: 263.07, measured value: 263.14.

[0286] (S)-2-amino-5-(5-amino-1,3-dioxoisoindolin-2-yl)methyl pentanoate (XT6) To an aqueous solution (9 mL) of L-Orn-OH hydrochloride (0.835 g, 4.85 mmol) and NaOH (0.388 g, 9.71 mmol), an aqueous solution (9 mL) of CuSO4·5H2O (0.606 g, 2.43 mmol) was added at room temperature to obtain a deep blue solution. To the solution, NaHCO3 (0.489 g, 5.82 mmol) and formamide XT4 (1.40 g, 5.34 mmol) were added, and the pale blue suspension was stirred at room temperature for 4 hours. The mixture was filtered, and the solid was washed with water (2 × 2.5 mL), ethanol (2 × 2.5 mL), and ether (2 × 2.5 mL), and air-dried overnight to obtain a yellow / gray copper salt (1.6 g). The material was suspended in methanol (45 mL) and cooled to -20°C. While maintaining the temperature below 0°C, thionyl chloride (4.21 mL, 57.7 mmol) was added over 45 minutes. After warming to room temperature, the mixture was stirred for 18 hours. The reaction mixture was concentrated under reduced pressure, and the solvent was evaporated along with toluene (12 mL). The unpurified residue was triturated with a mixture of MeOH (4 mL) / siRNA (3.6 mL) / acetone (3.6 mL), the solid was filtered, washed with ether (20 mL), and dried under vacuum to obtain succinimide XT6 as a yellow solid (1.50 g, 87%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.57 (br s, 3H), 7.50 (d, J =8.1 Hz, 1H), 7.32 (br s, 3H), 6.98 (s, 1H), 6.85 (d, J = 8.0 Hz, 1H), 4.05-3.97 (m, 1H), 3.71 (s, H), 3.55-3.47 (m, 2H), 1.83-1.54 (m, 4H). MS (ESI + ) C 14 H 18 N3O4 + [M+H]+ Calculated value: 292.13, measured value: 292.17.

[0287] Production of (S)-4-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT9) and (S)-5-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT10) [ka]

[0288] (S)-5-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)-formamide)benzamide)methyl pentanoate (XT8) To a suspension of acid XT7 (470 mg, 1.39 mmol; synthesized as described in U.S. Patent Application Publication No. 2004 / 0072837) and Et3N (2.32 mL, 16.6 mmol) in DMF (10 mL), isobutyl chloroformate (0.182 mL, 1.385 mmol) was added at room temperature. The resulting mixture was stirred for 1 hour, then succinimide XT6 (499 mg, 1.52 mmol) was added and stirred for 1 hour. Additional isobutyl chloroformate (0.091 mL, 0.692 mmol) was added, and after 20 minutes, succinimide XT6 (250 mg, 0.762 mmol) was added and the mixture was stirred for 1 hour. Finally, an additional isobutyl chloroformate (0.045 mL, 0.347 mmol) was added, and after 20 minutes, succinimide XT6 (125 mg, 0.381 mmol) was added, and the mixture was stirred for 1 hour. After concentration, the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 20:80). The product was suspended in water (6 mL), filtered, and washed with water (2 mL) and Et2O (4 mL) to obtain formamide XT8 as a yellow solid (552 mg, 65%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.81 (s, 1H), 8.74 (br s, 1H), 8.69 (d, J = 7.3 Hz, 1H), 8.01 (br s, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.72 (br s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.2 Hz, 1H), 6.97 (br s, 2H), 6.91 (d, J = 1.8 Hz, 1H), 6.78 (dd, J = 8.2, 1.8 Hz, 1H), 6.45 (br s, 2H), 5.25 (s, 2H), 4.46-4.37 (m, 1H), 3.61 (s, 3H), 3.51 (t, J = 6.4 Hz, 2H), 1.83-1.46 (m, 4H). MS (ESI + ) C 29 H 29 N 10 O6 + [M+H] + Calculated value: 613.23, measured value: 613.30.

[0289] (S)-4-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT9) and (S)-5-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT10) Formamide XT8 (120 mg, 0.196 mmol) was hydrolyzed with NaOH (6 equivalents) according to the general procedure XXC. The solid was dissolved in a 10% MeCN aqueous solution containing 0.1% TFA and purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain acid XT9 (25 mg, 22%) and acid XT10 (5 mg, 4%) as yellow solids.

[0290] XT9: 1 1H NMR (400 MHz, DMSO-d6) ppm = 12.44 (broad singlet, 1H), 9.31 (broad singlet, 1H), 9.26 (broad singlet, 1H), 8.57 (broad singlet, 1H), 8.14 (doublet, J = 7.7 Hz, 1H), 7.99 (triplet, J = 5.6 Hz, 1H), 7.80 - 7.63 (multiplet, 2H) 7.74 (doublet, J = 8.6 Hz, 1H), 7.56 (doublet, J = 8.5 Hz, 1H), 6.90 (broad singlet, 1H), 6.75 (doublet, J = 2.2 Hz, 1H), 6.52 (doublet of doublets, J = 8.5, 2.2 Hz, 1H), 6.41 (doublet, J = 2.2 Hz, 1H), 5.91 (broad singlet, 2H), 4.61 (singlet, 2H), 4.40 - 4.29 (multiplet, 1H), 3.20 - 3.10 (multiplet, 2H), 2.84 - 2.76 (multiplet, 2H), 1.94 - 1.70 (multiplet, 2H), 1.67 - 1.47 (multiplet, 2H). MS (ESI + ) C 27 H 29 N 10 O6 + [M+H] + Calculated value 589.23, measured value 589.37. XT10: 1H NMR (400 MHz, DMSO-d6) ppm = 12.53 (br s, 2H), 9.34 (br s, 1H), 9.30 (br s, 1H), 8.84 (s, 1H), 8.62 (br s, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.07 (t, J = 5.5 Hz, 1H), 7.79 (br s, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.2 Hz, 1H), 7.07-6.70 (m, 1H) 6.82 (d, J = 2.2 Hz, 1H), 6.74 (d, J = 8.6 Hz, 2H), 6.61 (dd, J = 8.3, 2.3 Hz, 1H), 4.62 (s, 2H), 4.37-4.29 (m, 1H), 3.16 (q, J = 5.9 Hz 2H) 1.91-1.69 (m, 2H), 1.67-1.47 (m, 2H). MS (ESI + ) C 27 H 29 N 10 O6 + [M+H] + Calculated value: 589.23, measured value: 589.47.

[0291] Preparation of 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-butyl)carbamoyl)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT16) and 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT17) [ka]

[0292] (S)-5-(5-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-1,3-Dioxoisoindoline-2-yl)-2-(4-(N-((2,4-Diaminopteridine-6-yl)methyl)formamide)benzamide)Methyl pentanoate (XT11) To a suspension of succinimide XT8 (170 mg, 0.278 mmol) in pyridine (5 mL) at 0°C, (S)-(1-chloro-1-oxopropan-2-yl)carbamate (9H-fluoren-9-yl)methyl (Fmoc-Ala-Cl, 320 mg, 0.971 mmol) (prepared according to Unsworth et al, Angew. Chem. Int. Ed. 2015, 52, 15794-15798) was added in six separate additions. The reaction mixture was quenched with MeOH (6 mL), and after UPLC indicated completion of the reaction, it was concentrated under reduced pressure. The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 20:80) to obtain amide XT11 as a yellow solid (298 mg, quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.71 (s, 1H), 9.07 (br s, 1H), 8.81 (s, 1H), 8.79 (br s, 1H), 8.71 (d, J = 7.7 Hz, 1H), 8.21 (s, 1H), 7.93-7.84 (m, 4H), 7.83-7.78 (m, 2H), 7.74 (t, J = 6.7 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.45-7.38 (m, 2H), 7.37-7.30 (m, 2H), 5.30 (s, 2H), 4.47-4.38 (m, 1H), 4.29 (d, J = 6.9 Hz, 2H), 4.22 (t, J = 6.6 Hz, 2H), 4.09 (br s, 4H), 3.64-3.52 (m, 5H), 1.86-1.60 (m, 4H), 1.34 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) ppm = 173.2, 173.0, 168.2, 168.1, 166.2, 163.1, 156.4, 145.0, 144.3, 144.2, 144.0, 141.2, 133.6, 131.2, 129.2, 128.1, 127.5, 126.0, 125.8, 124.7, 123.8, 122.2, 120.6, 113.3, 89.8, 66.1, 52.8, 52.4, 51.1, 47.1, 37.5, 28.3, 25.4, 18.2. MS (ESI + ) C 47 H 44 N 11 O9 + [M+H] + Calculated value: 906.33, measured value: 906.40.

[0293] (S)-5-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-1,3-dioxoisoindorin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XT12) In a 25 mL round-bottom flask, amide XT11 (250 mg, 0.276 mmol) and tetrabutylammonium fluoride trihydrate (TBAF·3H2O) (178 mg, 0.552 mmol) were placed, and the flask was thoroughly purged with N2 (at least 3 vacuum cycles per N2 cycle). DMF (6 mL) was added, and after all solids had dissolved, decanethiol (0.608 mL, 2.76 mmol) was added directly into the solution at room temperature using a syringe, and the mixture was stirred for 90 minutes. Next, Boc-Val-OSu (130 mg, 0.414 mmol) and DIPEA (0.096 mL, 0.552 mmol) were added, and the resulting mixture was stirred for 1.5 hours. Another 130 mg of Boc-Val-OSu (0.414 mmol) was added, and the mixture was stirred for a further 30 minutes. The reaction mixture was then concentrated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 20:80) to obtain dipeptide XT12 containing tetrabutylammonium salt. MS (ESI + ) C 42 H 51 N 12 O 10 + [M+H] + Calculated value: 883.38, measured value: 883.71.

[0294] (S)-5-(5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-1,3-dioxoisoindorin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XT13) To a solution of dipeptide XT12 (244 mg, 0.276 mmol) containing impurities in DCM (3 mL), TFA (3 mL) was added at 0°C. The resulting mixture was stirred for 15 minutes while being warmed to room temperature. The reaction mixture was concentrated under reduced pressure, and the solvent was evaporated along with the DCM to obtain unpurified, deprotected amine XT13 as an orange TFA salt, which was used directly without further analysis. MS (ESI + ) C 37 H 43 N 12O8 + [M+H] + Calculated value: 783.33, measured value: 783.32.

[0295] 4-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT14) and 5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT15) Hydrolysis of formamide XT13 (190 mg, 0.243 mmol) was performed using the general procedure XXC with the following modifications: Formamide XT13 was first reacted with NaOH (12 equivalents) at 0°C for 1 hour, then 2 parts NaOH (12 equivalents) were added, and the mixture was reacted at room temperature for 6 hours. Washing of the solid with MeOH was changed to washing with water (3 mL). A mixture of dioxides XT14 and XT15 (approximately 1:2) was obtained. If necessary, the mixture was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%) to obtain dioxides XT14 (15 mg, 8%, 3 steps) and XT15 (30 mg, 16%, 3 steps) as yellow solids.

[0296] X T14: 11H NMR (400 MHz, DMSO-d6) ppm = 13.37 - 12.23 (m, 2H), 10.46 (s, 1H), 8.96 (br s, 1H), 8.81 (s, 1H), 8.75 (d, J = 6.7 Hz, 1H), 8.22 (t, J = 5.4 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.08 (br s, 2H), 7.80 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.67 (dd, J = 8.5, 1.7 Hz, 1H), 7.58 (d, J = 1.7 Hz, 1H), 6.91 - 6.86 (m, 1H), 6.75 (d, J = 8.6 Hz, 2H), 6.54 (s, 2H), 4.59 (s, 2H), 4.49 (p, J = 6.9 Hz, 1H), 4.38 - 4.30 (m, 1H), 3.61 (s, 1H), 3.17 (q, J = 5.6 Hz, 2H), 2.13 - 2.04 (m, 1H), 1.93 - 1.83 (m, 1H), 1.83 - 1.71 (m, 1H), 1.69 - 1.49 (m, 2H), 1.36 (d, J = 7.0 Hz, 3H), 0.95 (t, J = 6.2 Hz, 6H). MS (ESI + ) C 35 H 43 N 12 O8 + [M+H] + Calculated value: 759.33, Measured value: 759.50. XT15: 11H NMR (400 MHz, DMSO-d6) ppm = 13.20 - 12.32 (m, 2H), 10.38 (s, 1H), 9.01 (br s, 1H), 8.81 (s, 1H), 8.74 (d, J = 6.8 Hz, 1H), 8.24 (t, J = 5.4 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.08 (br s, 2H), 7.97 (d, J = 2.0 Hz, 1H), 7.77 - 7.69 (m, 3H), 7.39 (d, J = 8.3 Hz, 1H), 6.91 - 6.86 (m, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.54 (br s, 2H), 4.60 (d, J = 3.4 Hz, 2H), 4.48 (p, J = 7.0 Hz, 1H), 4.39 - 4.31 (m, 1H), 3.62 (s, 1H), 3.19 (q, J = 5.9 Hz, 2H), 2.15 - 2.04 (m, 1H), 1.93 - 1.83 (m, 1H), 1.83 - 1.72 (m, 1H), 1.68 - 1.50 (m, 2H), 1.37 (d, J = 7.1 Hz, 3H), 0.96 (t, J = 6.1 Hz, 6H). MS (ESI + ) C 35 H 43 N 12 O8 + [M+H] + Calculated value 759.33, measured value 759.68.

[0297] 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT16) and 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT17) A mixture of XT14 and XT15 diacitates (0.209 g, 0.276 mmol) (approximately 1:2) in cooled (0°C) DMF (10 mL) was mixed with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.085 g, 0.276 mmol), followed by DIPEA (0.289 mL, 1.66 mmol). The resulting mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain a mixture of XT16 and XT17 (1:2) as a yellow solid (65 mg, 25%, 4 steps). By reacting the isomers with pure XT14 or XT15, it became possible to isolate pure XT16 or XT17, respectively.

[0298] XT16: 1H NMR (400 MHz, DMSO-d6) ppm = 13.27-12.28 (m, 3H), 10.15 (s, 1H), 9.31 (br s, 1H), 9.27 (br s, 1H), 8.84 (s, 1H), 8.57 (br s, 1H), 8.26-8.18 (m, 2H), 8.16 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 2.0 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.76-7.71 (m, 3H), 7.60 (br s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.00 (s, 2H), 6.89 (br s, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.53 (br s, 1H), 4.62 (s, 2H), 4.41-4.30 (m, 2H), 4.17 (dd, J = 8.2, 7.2 Hz, 1H), 3.19 (q, J = 6.2 Hz, 2H), 2.23-2.06 (m, 2H), 2.01-1.72 (m, 3H), 1.68-1.42 (m, 6H), 1.31 (d, J = 7.0 Hz, 3H), 1.25-1.13 (m, 2H), 0.84 (dd, J = 16.7, 6.8 Hz, 6H). MS (ESI + C 45 H 54 N 13 O 11 + [M+H] + The calculated value is 952.41 and the measured value is 952.75. XT17: (31 mg, 82%) yellow solid color. 11H NMR (400 MHz, DMSO-d6) ppm = 13.31 - 12.29 (m, 2H), 10.25 (s, 1H), 9.29 (br s, 1H), 9.25 (br s, 1H), 8.83 (s, 1H), 8.56 (br s, 1H), 8.26 - 8.18 (m, 2H), 8.15 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.69 (dd, J = 8.7, 1.9 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.00 (s, 2H), 6.89 (br s, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.54 (br s, 1H), 4.62 (s, 2H), 4.40 - 4.30 (m, 2H), 4.17 (dd, J = 8.3, 7.3 Hz, 1H), 3.38 (q, J = 5.9 Hz, 2H), 2.23 - 2.05 (m, 2H), 1.98 - 1.71 (m, 3H), 1.68 - 1.41 (m, 6H), 1.31 (d, J = 7.2 Hz, 3H), 1.25 - 1.13 (m, 2H), 0.84 (dd, J = 17.7, 6.7 Hz, 6H). MS (ESI + ) C 45 H 54 N 13 O 11 + [M + H] + Calculated value 952.41, measured value 952.82.

[0299] Manufacturing of (S)-4-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)-benzamide)butyl)-carbamoyl)benzoic acid (XT20) and (S)-5-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT21)

Chemical Structure

[0300] (S)-5-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)-amino)benzamide)methyl pentanoate (XT19) To a suspension of acid XX9 (150 mg, 0.461 mmol) and triethylamine (1.16 mL, 8.30 mmol) in DMF (5 mL), isobutyl chloroformate (0.061 mL, 0.461 mmol) was added at room temperature, and the reaction mixture was stirred for 1 hour. Amine XT6 (166 mg, 0.507 mmol) was added, and the reaction mixture was stirred for 1 hour. Additional isobutyl chloroformate (0.030 mL, 0.230 mmol) was added, and after 20 minutes, additional amine XT6 (83 mg, 0.254 mmol) was added. A further isobutyl chloroformate (0.015 mL, 0.115 mmol) was added, and after 20 minutes, additional XT6 (42 mg, 0.127 mmol) was added. Despite the multiple additions of chloroformic acid, the reaction stopped at a conversion rate of 30%. HATU (175 mg, 0.461 mmol) was added at room temperature for 30 minutes, followed by the addition of amine XT6 (166 mg, 0.507 mmol). After 30 minutes, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 25:75) to obtain aniline XT19 as a yellow solid (294 mg, quantified). MS (ESI + ) C 29 H 31 N 10 O5 + [M+H] + Calculated value: 599.25, measured value: 599.29.

[0301] (S)-4-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT20) and (S)-5-amino-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)(methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT21) Hydrolysis of ester XT19 (276 mg, 0.461 mmol) with NaOH (12 equivalents) was carried out according to the general procedure XXC, except that only ether was used in the washing step during solid recovery by filtration. The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain diacitates XT20 (10 mg, 0.017 mmol, 4%) and XT21 (76 mg, 0.126 mmol, 27%) as yellow solids.

[0302] XT20: 1 H NMR (400 MHz, DMSO-d6) ppm = 12.35 (br s, 2H), 9.26 (s, 1H), 9.05 (s, 1H), 8.72 (s, 1H), 8.58 (br s, 1H), 8.19 (dd, J = 7.7 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.75-7.67 (m, 1H), 7.56 (d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.8 Hz, 2H), 6.63-6.53 (m, 1H), 6.52 (dd, J = 8.5, 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.87 (s, 2H), 4.40-4.27 (m, 1H), 3.25 (s, 3H), 3.14 (q, J = 5.8 Hz, 2H), 1.93-1.70 (m, 2H), 1.67-1.46 (m, 2H). MS (ESI + ) C 28 H 31 N 10 O6 + [M+H] + Calculated value: 603.24, measured value: 603.40. XT21: 1H NMR (400 MHz, DMSO-d6) ppm = 9.27 (s, 1H), 9.07 (s, 1H), 8.72 (s, 1H), 8.61 (br s, 1H), 8.21 (d, J = 7.7 Hz, 1H), 8.07 (t, J = 5.6 Hz, 1H), 7.86 (br s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.3 Hz, 1H), 6.85-6.79 (m, 3H), 6.62 (dd, J = 8.3, 2.2 Hz, 1H), 4.87 (s, 2H), 4.39-4.28 (m, 1H), 3.25 (s, 3H), 3.16 (q, J = 6.1 Hz, 2H), 1.93-1.70 (m, 2H), 1.66-1.46 (m, 2H). MS (ESI + ) C 28 H 31 N 10 O6 + [M+H] + Calculated value: 603.24, measured value: 603.27.

[0303] Manufacturing of (S)-4-amino-2-((4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-butyl)carbamoyl)benzoic acid (XT24) and (S)-5-amino-2-((4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)carbamoyl)benzoic acid (XT25) [ka]

[0304] (S)-5-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-methylpentanoate (XT23) Acid XX2 (140 mg, 0.451 mmol) and amine XT6 (222 mg, 0.677 mmol) were reacted according to general procedure XXA. Ester XT23 was obtained as a yellow solid by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) (263 mg, quantified). MS (ESI + ) C 29 H 30 N9O5 + [M+H] +Calculated value: 584.24, Measured value: 584.24.

[0305] (S)-4-amino-2-((4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)carbamoyl)benzoic acid (XT24) and (S)-5-amino-2-((4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)carbamoyl)benzoic acid (XT25) Hydrolysis of ester XT23 (263 mg, 0.451 mmol) with NaOH (3 equivalents) was carried out according to the general procedure XXC, except that only ether was used in the washing step during solid recovery by filtration. The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain diacitates XT24 (7 mg, 0.012 mmol, 3%) and XT25 (93 mg, 0.158 mmol, 35%) as colorless solids.

[0306] XT24: 1 H NMR (400 MHz, DMSO-d6) ppm = 13.40-11.80 (m, 2H), 9.22 (br s, 1H), 8.75 (s, 1H), 8.61-8.47 (m, 1H), 8.50 (d, J =7.7 Hz, 1H), 8.01 (t, J =5.6 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.77-7.65 (m, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 8.3 Hz, 2H), 6.67-6.43 (m, 4H), 6.52 (dd, J = 8.5, 2.2 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 4.43-4.32 (m, 1H), 3.29-3.12 (m, 6H), 1.95-1.72 (m, 2H), 1.69-1.49 (m, 2H). MS (ESI + ) C 28 H30 N9O6 + [M+H] + Calculated value: 588.23, measured value: 588.46. XT25: 1 H NMR (400 MHz, DMSO-d6) ppm = 9.25 (br s, 1H), 9.16 (br s, 1H), 8.75 (s, 1H), 8.64-8.50 (m, 1H), 8.55 (d, J = 7.7 Hz, 1H), 8.09 (t, J = 5.5 Hz, 1H), 7.93 (br s, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 2.3 Hz, 1H), 6.64 (dd, J = 8.3, 2.3 Hz, 1H), 4.41-4.33 (m, 1H), 3.30-3.12 (m, 6H), 1.95-1.72 (m, 2H), 1.67-1.49 (m, 2H). MS (ESI + ) C 28 H 30 N9O6 + [M+H] + Calculated value: 588.23, measured value: 588.48.

[0307] (S)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)benzoic acid (XT31) and (S)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)benzoic acid (XT32) Manufacturing [ka]

[0308] (S)-5-(5-(6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexaneamide)-1,3-dioxoisoindoline-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XT27) To a suspension of aniline XT8 (100 mg, 0.163 mmol) in pyridine (3 mL), acid chloride XT26 (182 mg, 0.490 mmol) (prepared according to Unsworth et al, Angew. Chem. Int. Ed. 2015, 52, 15794-15798) was gradually added (30 mg at a time) at 0°C, and the reaction was immediately confirmed by UPLC-MS. After completion, the reaction product was quenched with MeOH (2 mL) and concentrated under reduced pressure. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded amide XT27 (160 mg, quantified) together with a trace amount of pyridine. 1 H NMR (400 MHz, DMSO-d6) ppm = 10.52 (s, 1H), 8.81 (s, 1H), 8.69 (d, J = 7.4 Hz, 1H), 8.19 (s, 1H), 7.98 (br s, 1H), 7.90-7.81 (m, 5H), 7.80-7.73 (m, 1H), 7.67 (d, J =, 7.4 Hz, 2H), 7.59 (d, J =8.3 Hz, 2H), 7.43-7.36 (m, 2H), 7.34-7.25 (m, 3H), 6.94 (br s, 2H), 5.25 (s, 2H), 4.46-4.35 (m, 1H), 4.30-4.25 (m, 2H), 4.21-4.15 (m, 1H), 4.10 (q, J = 4.5 Hz, 2H), 3.60 (s, 3H), 3.60-3.52 (m, 2H), 2.99 (q, J = 6.1 Hz, 2H), 2.42-2.34 (m, 2H), 1.85-1.56 (m, 6H), 1.48-1.38 (m, 2H), 1.35-1.25 (m, 2H). 13C NMR (100 MHz, DMSO-d6) ppm = 173.0, 172.7, 168.2, 168.1, 166.3, 156.6, 145.2, 144.4, 144.1, 141.2, 133.6, 131.1, 129.2, 128.0, 127.7, 127.5, 125.6, 125.6, 124.6, 123.5, 122.1, 120.6, 113.1, 65.6, 52.8, 52.3, 47.2, 46.9, 40.4, 37.4, 36.9, 29.6, 28.3, 26.3, 25.4, 25.1. MS (ESI + ) C 50 H 50 N 11 O9 + [M+H] + Calculated value: 948.38, measured value: 948.45.

[0309] (S)-4-(6-aminohexaneamide)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-butyl)carbamoyl)benzoic acid (XT28) and (S)-5-(6-aminohexaneamide)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT29) Hydrolysis of ester XT27 (145 mg, 0.153 mmol) with NaOH (12 equivalents) was carried out according to the general procedure XXC, except that only ether was used in the washing step during solid recovery by filtration. An unpurified mixture of diacitates XT28 and XT29 (80 mg, 75%, ratio 1:2) was obtained and used in the next step without further purification. MS (ESI + ) C 33 H 40 N 11 O7 + [M+H] + Calculated value: 702.31, measured value: 702.56.

[0310] (S)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)benzoic acid (XT31) and (S)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanamide)benzoic acid (XT32) To a suspension of diacitates XT28 and XT29 (60 mg, 0.086 mmol, ratio 1:2) in DMF (3 mL), 260 μL, 1 M aqueous solution of Na2CO3 was added at room temperature, followed by the addition of N-methoxycarbonylmaleimide XT30 (13.3 mg, 0.086 mmol). Additional aqueous solution of Na2CO3 (0.170 mL, 1 M) was added three times at 20-minute intervals, followed by the final aqueous solution of Na2CO3 (0.085 mL, 1 M). After 2 hours, the reaction mixture was cooled to 0°C and quenched with aqueous solution of AcOH (2.06 mL, 1 M). The resulting suspension was concentrated under reduced pressure. The crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain a mixture of amides XT31 and XT32 in a 1:2 ratio as a yellow solid (15 mg, 22%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.41 (br s, 2H), 10.19 (s, 1H), 10,12 (s, 1H), 9.16 (d, J = 21.3 Hz, 2H), 8.83 (s, 1H), 8.27-8.12 (m, 2H), 7.94 (d, J = 2.0 Hz, 1H), 7.80-7.62 (m, 4H), 7.57 (d, J = 1.9 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.00 (s, 2H), 6.89 (br s, 1H), 6.75 (d, J = 8.8 Hz, 2H), 6.55 (br s, 1H), 4.61 (s, 2H), 4.39-4.30 (m, 1H), 3.17 (q, J = 5.9 Hz, 2H), 2.35-2.26 (m, 2H), 1.92-1.71 (m, 2H), 1.69-1.45 (m, 6H), 1.31-1.19 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 174.6, 172.2, 172.0, 171.6, 169.2, 168.5, 168.4, 167.3, 166.8, 163.3, 150.9, 150.7, 149.8, 142.6, 141.2, 140.3, 134.9, 133.1, 132.2,131.3, 129.6, 128.8, 123.8, 122.2, 121.9, 121.0, 119.6, 118.6, 117.9, 111.9, 52.9, 46.1, 37.4, 36.7, 36.6, 28.7, 28.2, 26.6, 26.2, 24.9. MS (ESI + C 37 H 40 N 11 O9 + [M+H] + The calculated value is 782.30 and the measured value is 782.52.

[0311] Preparation of (S)-5-(4-aminobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XT35)

change

[0312] (S)-5-(4-azidobenzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-methylpentanoate (XT33) Acid XT7 (150 mg, 0.442 mmol) and amine XX3 (142 mg, 0.486 mmol) were reacted according to general procedure XXA. Ester XT33 was obtained as a yellow solid by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) (270 mg, quantified). MS (ESI + ) C 28 H 29 N 12 O5 + [M+H] + Calculated value: 613.24, measured value: 613.50.

[0313] (S)-5-(4-aminobenzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-methylpentanoate (XT34) Azide XX33 (270 mg, 0.441 mmol) was reduced according to the general procedure XXB. Aniline XT34 was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain a yellowish-gray solid (190 mg, 0.324 mmol, 74%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.75 (d, J = 7.3 Hz, 1H), 8.68 (s, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.87-7.80 (m, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.57-7.48 (m, 1H), 6.81 (br s, 2H), 6.52 (d, J = 8.6 Hz, 2H), 6.40-4.80 (m, 2H), 5.23 (s, 2H), 4.47-4.38 (m, 1H), 3.62 (s, 3H), 3.22 (q, J = 6.0 Hz, 2H), 1.89-1.71 (m, 2H), 1.66-1.48 (m, 2H). MS (ESI + ) C 28 H 31 N 10 O5 + [M+H] + Calculated value: 587.25, measured value: 587.55.

[0314] (S)-5-(4-aminobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XT35) Hydrolysis of ester XT34 (190 mg, 0.324 mmol) with NaOH (6 equivalents) was carried out using the general procedure XXC with the following modifications. After filtration and washing of the solid, the material was dissolved in a 2% aqueous ammonia solution and freeze-dried to obtain XT35 as a yellow-orange solid (143 mg, 0.26 mmol, 81%). 13 C NMR (100 MHz, DMSO-d6) ppm = 174.5, 166.6, 166.4, 163.4, 163.3, 155.7, 151.9, 151.1, 150.1, 146.0, 129.3, 129.1, 122.2, 121.9, 121.3, 113.0, 111.9, 53.3, 46.1, 40.9, 29.4, 26.7. MS (ESI + ) C 26 H 29 N 10 O4 + [M+H] + Calculated value: 545.24, measured value: 545.23.

[0315] 2-(((S)-4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT41) Manufacturing [ka]

[0316] (S)-5-(5-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-1,3-Dioxoisoindoline-2-yl)-2-(4-(2-(2,4-Diaminopteridine-6-yl)ethyl)benzamide)Methyl pentanoate (XT36) Aniline XT23 (280 mg, 0.480 mmol) and Fmoc-Ala-Cl (396 mg, 1.20 mmol) were reacted in the same manner as with XT11. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded amide XT36 as a yellow solid (350 mg, 83%). MS (ESI + ) C 47 H 45 N 10 O8 + [M+H] + Calculated value: 877.34, measured value: 877.44.

[0317] (S)-5-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-1,3-dioxoisoindorin-2-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT37) Amine XT36 (350 mg, 0.399 mmol) protected with Fmoc was deprotected using TBAF·3H2O and decanethiol, and subsequently reacted with Boc-Val-OSu and DIPEA, as in the case of XT12. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded dipeptide XT37 contaminated with tetrabutylamine salt (quantified). MS (ESI + ) C 42 H 52 N 11 O9 + [M+H] + Calculated value: 854.39, measured value: 854.49.

[0318] (S)-5-(5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-1,3-dioxoisoindorin-2-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT38) Dipeptide XT37 (340 mg, 0.400 mmol) was deprotected in the same manner as for XT13. After concentration, the TFA salt XT38 was used directly in the next step. MS (ESI + ) C 37 H 44 N 11 O7 + [M+H] + Calculated value: 754.34, measured value: 754.47.

[0319] 5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-(((S)-4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)carbamoyl)benzoic acid (XT40) The hydrolysis of formamide XT38 (300 mg, 0.400 mmol) was performed using the general procedure XXC with the following modifications: Formamide XT38 was first reacted with NaOH (12 equivalents) at 0°C for 1 hour, then another 12 equivalents of NaOH were added and the reaction was carried out at room temperature for 6 hours. The washing of the solid with MeOH was changed to washing with water (3 mL). The ring-opening of phthalimide proceeded with moderate selectivity, and the desired positional isomer XT40 was dominant (approximately 1:2). The material was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 25%) to obtain XT40 as a white solid (50 mg, 16%, 3 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 13-83-11.98 (m, 2H), 10.40 (s, 1H), 9.06 (br s, 1H), 8.96 (br s, 1H), 8.76 (d, J = 6.7 Hz, 1H), 8.72 (s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 8.26 (t, J = 5.6 Hz, 1H), 8.11 (br s, 3H), 7.98 (d, J = 2.1 Hz, 1H), 7.83 (d, J = 8.2 Hz, 2H), 7.72 (dd, J = 8.4, 2.1 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 6.58 (br s, 1H), 4.49 (p, J = 7.0 Hz, 1H), 4.43-4.28 (m, 1H), 3.63 (d, J = 5.1 Hz, 1H), 3.27-3.14 (m, 6H), 2.09 (sextet, J = 6.6 Hz, 1H), 1.96-1.86 (m, 1H), 1.86-1.73 (m, 1H), 1.70-1.51 (m, 2H), 1.37 (d, J = 7.1 Hz, 3H), 0.96 (dd, J = 6.7, 4.8 Hz, 6H). MS (ESI + ) C 36 H 44 N 11 O8 +[M+H] + Calculated value: 758.34, measured value: 758.61.

[0320] 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methyl-butanamide)propanamide)benzoic acid (XT41) Amine XT40 (45 mg, 0.059 mmol) was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (28 mg, 0.089 mmol) and DIPEA (0.062 mL, 0.36 mmol) in the same manner as with XT17. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%) yielded XT41 as a white solid (30 mg, 53%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.25-12.33 (m, 2H), 10.15 (s, 1H), 9.25 (s, 1H), 9.16 (s, 1H), 8.75 (s, 1H), 8.57 (br s, 1H), 8.52 (d, J = 7.7 Hz, 1H), 8.24 (t, J = 5.6 Hz, 1H), 8.21 (d, J = 6.7 Hz, 1H), 7.97 (d, J = 1.9 Hz, 1H), 7.85-7.78 (m, 3H), 7.74 (dd, J = 8.4, 1.9 Hz, 1H), 7.72-7.54 (m, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.00 (s, 2H), 4.42-4.31 (m,2H), 4.17 (dd, J = 8.0, 7.4 Hz, 1H), 3.32-3.14 (m, 8H), 2.22-2.07 (m, 2H), 2.01-1.73 (m, 3H), 1.69-1.41 (m, 6H), 1.31 (d, J = 7.1 Hz, 3H), 1.24-1.13 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + C 45 H 55 N 13 O 11 + [M+H] + The calculated value is 951.41 and the measured value is 951.91.

[0321] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzamide)production of pentanoic acid (XT46)

change

[0322] (S)-5-(4-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT42) Aniline XX6 (150 mg, 0.269 mmol) and Fmoc-Ala-Cl (310 mg, 0.942 mmol) were reacted in the same manner as for XT11. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded amide XT42 as an off-white solid (180 mg, 79%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.25 (s, 1H), 9.07 (br s, 1H), 8.98 (br s, 1H), 8.72 (s, 1H), 8.70 (d, J = 7.5 Hz, 1H), 8.58 (d, J = 4.0 Hz, 1H), 8.38 (t, J = 5.1 Hz, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.86-7.77 (m, 4H), 7.76-7.70 (m, 3H), 7.67 (d, J = 8.6 Hz, 2H), 7.59 (br s, 1H), 7.45-7.28 (m, 7H), 7.10 (br s, 1H), 4.49-4.41 (m, 1H), 4.31-4.25 (m, 2H), 4.25-4.16 (m, 2H), 3.63 (s, 3H), 3.28-3.16 (m, 4H), 1.92-1.76 (m, 2H), 1.72-1.52 (m, 2H), 1.32 (d, J = 7.1 Hz, 3H). MS (ESI + ) C 46 H 47 N 10 O7 + [M+H] + Calculated value: 851.36, measured value: 851.82.

[0323] (S)-5-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT43) To a solution of XT42 (180 mg, 0.212 mmol) in DMF (4 mL), piperidine (0.419 mL, 4.23 mmol) was added, and the resulting mixture was stirred at room temperature for 15 minutes. The reaction mixture was concentrated, and the solvent was evaporated with toluene. Ether (50 mL) was added, and the resulting suspension was stirred at room temperature for 10 minutes. After filtration, the solid was collected, and the unpurified amine was dissolved in DMF (4 mL). Next, Boc-Val-OSu (100 mg, 0.317 mmol) and DIPEA (0.074 mL, 0.42 mmol) were added, and the resulting mixture was stirred for 4 hours. Subsequently, the reaction mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain dipeptide XT43 (170 mg, 97%). 1H NMR (400 MHz, DMSO-d6) ppm = 10.45 (s, 1H), 10.21 (s, 1H), 8.85 (br s, 1H), 8.68 (d, J = 7.4 Hz, 1H), 8.56 (s, 1H), 8.36 (t, J = 5.5 Hz, 1H), 8.12 (d, J = 6.8 Hz, 1H), 7.79 (d, J = 8.5 Hz, 4H), 7.67 (br s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 6.71 (d, J = 8.8 Hz, 1H), 6.60 (br s, 2H), 4.49-4.38 (m, 2H), 3.83 (dd, J = 8.0, 7.5 Hz, 1H), 3.63 (s, 3H), 3.29-3.23 (m, 2H), 3.19-3.07 (m, 4H), 2.02-1.90 (m, 1H), 1.90-1.74 (m, 2H), 1.70-1.50 (m, 2H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 7.0 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H). MS (ESI + ) C 41 H 54 N 11 O8 + [M+H] + Calculated value: 828.42, measured value: 828.81.

[0324] (S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT44) Dipeptide XT43 (340 mg, 0.400 mmol) was deprotected in the same manner as for XT13. After concentration, the TFA salt XT44 was used directly in the next step. MS (ESI + ) C 36 H 46 N 11 O6+ [M+H] + Calculated value: 728.36, measured value: 728.66.

[0325] (S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XT45) The hydrolysis of ester XT44 (145 mg, 0.153 mmol) with NaOH (12 equivalents) was carried out with a modification to the general procedure XXC, which involved freeze-drying the solution obtained after evaporation of MeOH, because the product did not solidify after treatment with aqueous AcOH solution (1 M). The resulting cake was stirred with DMF (5 mL), filtered, and the resulting DMF solution containing XT45 was used directly in the next step. MS (ESI + ) C 35 H 44 N 11 O6 + [M+H] + Calculated value: 714.35, measured value: 714.71.

[0326] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzamide)pentanoic acid (XT46) Amine XT45 was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (49 mg, 0.16 mmol) and DIPEA (0.168 mL, 0.960 mmol) in the same manner as with XT17. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%) yielded XT46 as a white solid (67 mg, 46%, 3 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 13.13 (br s, 1H), 10.11 (s, 1H), 9.25 (s, 1H), 9.16 (s, 1H), 8.75 (s, 1H), 8.57 (br s, 1H), 8.55 (d, J = 7.7 Hz, 1H), 8.36 (t, J = 5.6 Hz, 1H), 8.20 (d, J = 6.7 Hz, 1H), 7.92-7.75 (m, 6H), 7.66 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 6.99 (s, 2H), 4.44-4.33 (m, 2H), 4.17 (dd, J = 7.9, 7.5 Hz, 1H), 3.37 (t, J = 6.9 Hz, 2H), 3.31-3.16 (m, 6H), 2.23-2.06 (m, 2H), 2.01-1.73 (m, 3H), 1.71-1.54 (m, 2H), 1.53-1.41 (m, 4H), 1.32 (d, J = 7.1 Hz, 3H), 1.23-1.13 (m, 2H), 0.86 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 45 H 55 N 12 O9 + [M+H] + Calculated value: 907.42, measured value: 907.84.

[0327] Manufacturing of (S)-5-amino-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-benzamide)methyl pentanoate hydrochloride (XT48) [ka]

[0328] (S)-5-((tert-butoxycarbonyl)amino)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-benzamide)methyl pentanoate (XT47) Acid XT7 (1.30 g, 3.83 mmol) and H-Orn(Boc)-OMe (1.30 g, 4.60 mmol) were reacted according to general procedure XXA. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded ester XT47 as a firm, orange / red foam (1.80 g, 83%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.81 (s, 1H), 8.72-8.67 (m, 1H), 8.70 (s, 1H), 7.99 (br s, 1H), 7.90 (d, J = 8.5 Hz, 2H), 7.68 (br s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 6.96 (br s, 2H), 6.79 (t, J = 5.2 Hz, 1H), 5.25 (s, 2H), 4.44-4.35 (m, 1H), 3.63 (s, 3H), 2.99-2.88 (m, 2H), 1.86-1.76 (m, 2H), 1.55-1.38 (m, 2H), 1.36 (s, 9H). MS (ESI + ) C 26 H 34 N9O6 + [M+H] + Calculated value: 568.26, measured value: 568.56.

[0329] (S)-5-amino-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate hydrochloride (XT48) Ester XT47 (1.60 g, 2.82 mmol) was heated in dioxane (16 mL) until dissolved. After cooling to room temperature, the solution was added dropwise to HCl in dioxane (4 M, 30 mL) which was vigorously stirred. The resulting yellow suspension was stirred for 2 hours. The reaction mixture was then filtered, the residue was washed with ether (50 mL), dissolved in water (30 mL), and freeze-dried to obtain XT48 as a yellow solid (1.42 g, 100%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.21 (br s, 1H), 9.29 (s, 1H), 8.98 (s, 1H), 8.87 (d, J = 7.4 Hz, 1H), 8.83 (d, J = 5.2 Hz, 1H), 8.68 (br s, 1H), 8.01 (br s, 4H), 7.95 (d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 5.33 (s, 2H), 4.46-4.38 (m, 1H), 3.63 (s, 3H), 2.85-2.71 (m, 2H), 1.95-1.79 (m, 2H), 1.75-1.57 (m, 2H). MS (ESI + ) C 21 H 26 N9O4 + [M+H] + Calculated value: 468.21, measured value: 468.52.

[0330] Preparation of 5-((S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecanamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT54) [ka]

[0331] (2,2,2-trifluoroacetyl)glycinoyl chloride (XT49) To a solution of glycine (2.50 g, 33.3 mmol) in MeOH (33 mL) and Et3N (6.96 mL, 50.0 mmol), trifluoroethyl acetate (5.17 mL, 43.3 mmol) was added. The resulting mixture was stirred at room temperature for 18 hours. The solution was concentrated, the residue was dissolved in Depositphotos (75 mL), the organic solution was washed with hydrochloric acid (1 M, 150 mL), and the aqueous phase was back-extracted with Depositphotos (2 × 75 mL). The combined organic phases were washed with brine (50 mL), dried over MgSO4, filtered, and the filtrate was concentrated to obtain (2,2,2-trifluoroacetyl)glycine (5.3 g, 93%) as a white solid. A portion of the product (1.00 g, 5.85 mmol) was suspended in DCM (30 mL), cooled to 0°C, and oxalyl chloride (1.54 mL, 17.54 mmol), followed by 2 drops of DMF, was added dropwise. The resulting mixture was stirred at room temperature for 1 hour, then concentrated, and the solvent was evaporated with toluene (2×) to obtain XT49 (quantified). The product was quenched with MeOH to obtain the corresponding methyl ester. MS (ESI + ) C5H7F3NO3 + [M+H] + Calculated value: 186.04, measured value: 186.36.

[0332] (S)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-(1,3-dioxo-5-(2-(2,2,2-trifluoroacetamide)acetamide)isoindorin-2-yl)methyl pentanoate (XT50) A suspension of XT8 (90.0 mg, 0.147 mmol) in pyridine (2.5 mL) was cooled to 0°C, and XT49 (167 mg, 0.88 mmol) was added in six fractions over 1 hour. After UPLC-MS indicated completion of the reaction, the reaction product was quenched with MeOH (3 mL) and concentrated under reduced pressure. The crude residue was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain XT50 as a yellow solid (118 mg, quantified). MS (ESI + ) C 33 H 31 F3N11 O8 + [M+H] + Calculated value: 766.23, measured value: 766.61.

[0333] (S)-5-(2-aminoacetamide)-2-((4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT52) The hydrolysis of formamide XT50 (107 mg, 0.14 mmol) was performed using the general procedure XXC with the following modifications: Formamide XT50 was first reacted with NaOH (12 equivalents) at 0°C for 1 hour, then another 12 equivalents of NaOH were added and the reaction was carried out at room temperature for 3 hours. The washing of the solid with MeOH was changed to washing with water (3 mL). The ring-opening of phthalimide proceeded with moderate selectivity, and the desired positional isomer XT52 was dominant (approximately 1:2). The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%) to obtain diacid XT52 as a yellow solid (36 mg, 40%, 2 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.79-12.05 (m, 2H), 10.67 (s, 1H), 9.17 (br s, 1H), 9.13 (br s, 1H), 8.83 (s, 1H), 8.37 (br s, 1H), 8.28 (t, J = 5.6 Hz, 1H), 8.19-8.05 (m, 3H), 7.96 (d, J = 1.9 Hz, 1H), 7.77-7.69 (m, 4H), 7.42 (d, J = 8.4 Hz, 1H), 6.63 (br s, 1H), 6.75 (d, J = 8.7, 2H), 6.54 (br s, 1H), 4.61 (s, 2H), 4.39-4.30 (m, 1H), 3.81 (s, 2H), 3.19 (q, J = 6.2 Hz, 2H), 1.94-1.83 (m, 1H), 1.83-1.71 (m, 1H), 1.69-1.49 (m, 2H). MS (ESI + ) C29 H 32 N 11 O7 + [M+H] + Calculated value: 646.25, measured value: 646.67.

[0334] 5-((S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecaneamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzoic acid (XT54) To a suspension of tripeptide XT53 (synthesized as described in European Patent Application Publication No. 2907824) (105 mg, 0.22 mmol) in THF (4 mL), N,N′-dicyclohexylcarbodiimide (DCC; 46 mg, 0.22 mmol) and N-hydroxysuccinimide (26 mg, 0.22 mmol) were added, and the resulting suspension was stirred for 18 hours. The resulting suspension was filtered, and a portion of the filtrate (1.0 mL) was added to a solution of diacitate XT52 (32 mg, 0.05 mmol) in DMF (0.5 mL). DIPEA (0.052 mL, 0.30 mmol) was added, and the resulting solution was stirred at room temperature for 30 minutes. After concentration, the crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain XT54 as a yellow solid (22 mg, 40%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.28-12.29 (m, 2H), 10.12 (s, 1H), 9.20 (br s, 1H), 9.15 (br s, 1H), 8.82 (s, 1H), 8.40 (t, J = 5.7 Hz, 1H), 8.25 (t, J = 5.5 Hz, 1H), 8.18-8.12 (m, 2H), 8.07 (t, J = 5.7 Hz, 1H), 8.02 (t, J = 5.6 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.77-7.71 (m, 3H), 7.39 (d, J = 8.4 Hz, 1H), 7.28-7.15 (m, 6H), 6.99 (s, 2H), 6.89 (br s, 1H), 6.74 (d, J = 8.7 Hz, 2H), 6.53 (br s, 1H), 4.61 (d, J = 3.2 Hz, 2H), 4.46-4.48 (m, 1H), 4.39-4.29 (m, 1H), 3.90 (t, J = 4.6 Hz, 2H), 3.80-3.55 (m, 6H), 3.19 (q, J = 5.9 Hz, 2H), 3.08 (dd, J = 13.9, 4.5 Hz, 1H), 2.83 (dd, J = 13.8, 9.8 Hz, 1H), 2.10 (t, J = 7.5 Hz, 2H), 1.94-1.70 (m, 2H), 1.69-1.39 (m, 6H), 1.26-1.14 (m, 2H). MS (ESI + C 52 H 58 N 15 O 13 + [M+H] + The calculated value is 1100.43 and the measured value is 1100.85.

[0335] 2-(((S)-5-carboxy-5-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-pentyl)amino)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT63) manufacturing

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[0336] (S)-2-((5-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexyl)amino)-5-methyl nitrobenzoate (XT57) A suspension of XT55 (1.42 g, 7.13 mmol), XT56 (2.0 g, 7.13 mmol), and K2CO3 (1.48 g, 10.7 mmol) in MeCN (15 mL) was placed in a sealed tube and heated at 90°C for 2 hours. Concentrated hydrochloric acid (0.89 mL, 11 mmol) was added at room temperature, and the unpurified reaction mixture was concentrated. Next, the carboxylic acid intermediate was dissolved in MeOH (100 mL), cooled to 0°C, and thionyl chloride (5.8 mL, 79 mmol) was added. The resulting solution was stirred at 0°C for 1 hour, and then gradually warmed to room temperature over 1 hour. After completion, the reaction mixture was filtered through Celite®, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:9) to obtain aniline XT57 as a yellow foam (2.8 g, 83%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.64 (d, J = 2.8 Hz, 1H), 8.54 (t, J = 5.4 Hz, 1H), 8.19 (dd, J = 9.5, 1.7 Hz, 1H), 7.75 (d, J = 7.8 hz, 1H), 7.42-7.25 (m, 5H), 6.93 (d, J = 9.6 Hz, 1H), 5.03 (s, 2H), 4.08-4.00 (m, 1H), 3.86 (s, 3H), 3.63 (s, 3H), 3.34 (q, J = 6.3 Hz, 2H), 1.79-1.52 (m, 4H), 1.50-1.34 (m, 2H). MS (ESI + ) C 23 H 28 N3O8 + [M+H] + Calculated value: 474.19, measured value: 474.31.

[0337] (S)-2-((5-amino-6-methoxy-6-oxohexyl)amino)-5-methyl hydrobromide nitrobenzoate (XT58) A solution of XT57 (750 mg, 1.58 mmol) in DCM (8 mL) was cooled to 0°C. HBr in ice-cold AcOH (33%, 10 mL) was added, and the resulting solution was stirred at 0°C for 2 hours. Subsequently, the reaction mixture was concentrated under reduced pressure, and the solvent was evaporated with toluene to obtain amine XT58 as a yellow solid (660 mg, 99%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.66 (d, J = 2.8 Hz, 1H), 8.55 (t, J = 5.4 Hz, 1H), 8.34 (br s, 2H), 8.21 (dd, J = 9.5, 2.7 Hz, 1H), 6.96 (d, J = 9.6 Hz, 1H), 4.13-4.05 (m, 1H), 3.88 (s, 3H), 3.76 (s, 3H), 3.37 (q, J = 6.2 Hz, 2H), 1.89-1.75 (m, 2H), 1.69-1.58 (m, 2H), 1.56-1.32 (m, 2H). MS (ESI + ) C 15 H 22 N3O6 + [M+H] + Calculated value: 340.15, measured value: 340.44.

[0338] (S)-2-((5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)-5-nitrobenzoate methyl (XT59) Amine XT58 (310 mg, 0.74 mmol) and acid XT7 (150 mg, 0.74 mmol) were reacted according to general procedure XXA. The product was recrystallized from MeOH (8 mL) to obtain XT59 as an orange solid (390 mg, 80%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.71-8.67 (m, 1H), 8.66 (s, 1H), 8.60 (d, J = 2.8 Hz, 1H), 8.54 (t, J = 5.3 Hz, 1H), 8.15 (dd, J = 9.5, 2.8 Hz, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.65 (br s, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.33 (br s, 1H), 6.92 (d, J = 9.5 Hz, 1H), 6.63 (br s, 2H), 5.22 (s, 2H), 4.46-4.38 (m, 1H), 3.82 (s, 3H), 3.62 (s, 3H), 3.40-3.27 (m, 2H), 1.90-1.78 (m, 2H), 1.71-1.55 (m, 2H), 1.54-1.37 (m, 2H). MS (ESI + ) C 30 H 33 N 10 O8 + [M+H] + Calculated value: 661.25, measured value: 661.65.

[0339] (S)-5-amino-2-((5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)methyl benzoate (XT60) To a solution of XT59 (150 mg, 0.23 mmol) in DMF (1.5 mL), saturated NH4Cl aqueous solution (0.375 mL) and zinc powder (445 mg, 6.81 mmol) were added. The resulting suspension was stirred at room temperature for 2 hours. Next, the reaction mixture was diluted with DMF (4 mL) and filtered through Celite®. The filtrate was stirred in air at room temperature for 18 hours. After stirring for 16 hours, the reaction mixture was concentrated, the crude product was suspended in MeOH (6 mL), filtered, and the residue was washed with ether (4 mL) to obtain aniline XT60 as a gray solid (145 mg, quantified). 1H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.70 (d, J = 7.6 Hz, 1H), 8.66 (s, 1H), 7.88(d, J = 8.5 Hz, 2H), 7.73 (br s, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.54 (t, J = 8.7 Hz, 1H), 7.10 (d, J = 2.6 Hz, 1H), 6.92 (t, J = 4.8 Hz, 1H), 6.80 (dd, J = 8.7, 2.5 Hz, 1H), 6.72 (bs ​​s, 2H), 6.56 (d, J = 8.8 Hz, 1H), 5.22 (s, 2H), 4.58 (s, 2H), 4.40 (q, J = 7.1 Hz, 1H), 3.70 (s, 3H), 3.63 (s, 3H), 3.07 (q, J = 5.7 Hz, 2H), 1.88-1.72 (m, 2H), 1.66-1.35 (m, 4H). MS (ESI + ) C 30 H 35 N 10 O6 + [M+H] + Calculated value: 631.27, measured value: 631.15.

[0340] 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-(((S)-5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)methyl benzoate (XT61) Aniline XT60 (140 mg, 0.22 mmol) and Fmoc-Ala-OH (70 mg, 0.22 mmol) were reacted according to the general procedure XXA. The product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain the amide (60 mg, 82%). A portion of the product (40 mg, 0.04 mmol) was deprotected with piperidine and reacted with Boc-Val-OSu as in the case of XT43. The product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain XT61 as a yellow solid (30 mg, 77%). MS (ESI + ) C 43 H 57 N 12 O 10 + [M+H] + Calculated value: 901.43, measured value: 901.54.

[0341] 5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-(((S)-5-carboxy-5-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentyl)amino)benzoic acid (XT62) Carbamate XT61 (15 mg, 0.02 mmol) was suspended in DCM (1.5 mL), cooled to 0°C, and TFA (1.5 mL) was added. The resulting mixture was stirred at 0°C for 15 minutes, then concentrated, and the solvent was evaporated along with the DCM. Subsequently, hydrolysis of the crude product with NaOH (24 equivalents) was carried out using the general procedure XXC with the following modifications. The product did not readily precipitate upon treatment with 1 M AcOH. Methanol was removed by vacuum evaporation, and the aqueous solution was freeze-dried. The resulting cake was stirred with DMF (5 mL), filtered, and the resulting DMF solution containing XT62 was used directly in the next step. MS (ESI + ) C 35 H 45 N 12 O7 + [M+H] + Calculated value: 745.35, measured value: 745.52.

[0342] 2-(((S)-5-carboxy-5-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentyl)amino)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT63) Amine XT62 (25 mg, 0.034 mmol) was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (10 mg, 0.034 mmol) and DIPEA (0.06 mL, 0.34 mmol) in the same manner as with XT17. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) yielded XT63 as a yellow solid (8 mg, 25%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.25 (br s, 1H), 12.43 (br s, 1H), 9.67 (s, 1H), 9.38-9.13 (m, 3H), 8.84 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 7.1 Hz, 1H), 8.03 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.57 (dd, J = 9.1, 2.4 Hz, 1H), 6.99 (s, 2H), 6.95 (br s, 1H), 6.76 (d, J = 8.7 Hz, 2H), 6.68 (d, J = 9.3 Hz, 1H), 6.53 (br s, 2H), 4.61 (s, 2H), 4.38-4.28 (m, 2H), 4.18-4.12 (m, 1H), 3.17-3.07 (m, 4H), 2.22-2.06 (m, 2H), 2.01-1.90 (m, 1H), 1.88-1.75 (m, 2H), 1.68-1.36 (m, 8H), 1.34-1.23 (m, 3H), 1.23-1.12 (m, 2H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H). MS (ESI + C 45 H 56 N 13 O 10 + [M+H] + The calculated value is 938.43 and the measured value is 938.70.

[0343] Preparation of 5-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)-amino)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XT723)

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[0344] (S)-5-((4-(((ベンジルオキシ)カルボニル)アミノ)-5-メトキシ-5-オキソペンチル)アミノ)-2-ニトロbenzoic acid メチル (XT66) XT64 (1.50 g, 7.51 mmol) and XT65 (2.00 g, 7.51 mmol) were reacted in the same manner as with XT57. The reaction time for the first step was extended to 7 days. XT66 was obtained as a yellow / orange oil (2.00 g, 58%) by purification using flash chromatography (silica gel, MeOH:DCM 0:1 to 1:9). 1 H NMR (400 MHz, DMSO-d6) ppm = 7.95 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.40-7.26 (m, 5H), 6.70 (dd, J = 9.3, 2.2 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 5.03 (s, 2H), 4.85 (br s, 1H), 4.12-4.05 (m, 1H), 3.81 (s, 3H), 3.63 (s, 3H), 3.18-3.11 (m, 2H), 1.86-1.74 (m, 1H), 1.73-1.54 (m, 3H). MS (ESI + ) C 22 H 26 N3O8 + [M+H] + Calculated value: 460.17, measured value: 460.33.

[0345] (S)-5-((4-(((benzyloxy)carbonyl)(tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentyl)(tert-butoxycarbonyl)amino)-2-methyl nitrobenzoate (XT67) To a solution of aniline XT66 (2.00 g, 4.35 mmol) in THF (25 mL), Boc2O (3.03 mL, 13.06 mmol) and 4-dimethylaminopyridine (DMAP; 53 mg, 0.44 mmol) were added. The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was then concentrated, and the crude product was purified by flash chromatography (silica gel, Â1:DCM 0:1 to 1:3) to obtain XT67 protected by two Boc molecules as a yellow oil (1.90 g, 66%). MS (ESI+ ) C 22 H 26 N3O8 + [M-2xBoc+H] + Calculated value: 460.17, measured value: 460.30.

[0346] (S)-2-amino-5-((4-(((benzyloxy)carbonyl)(tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentyl)(tert-butoxycarbonyl)amino)methyl benzoate (XT68) The procedure for XT60 was the same as for XT60, except that the crude product was purified by flash chromatography (silica gel, SiO2:DCM 0:1 to 1:1). Compound XT67 (1.70 g, 2.58 mmol) was reacted with zinc powder (2.50 g, 38.7 mmol) to obtain aniline XT68 as an orange foamy substance (1.5 g, 92%). 1 H NMR (400 MHz, DMSO-d6) ppm = 7.44 (d, J = 2.3 Hz, 1H), 7.39-7.31 (m, 5H), 7.03 (dd, J = 8.7, 2.1 Hz, 1H), 6.74 (d, J = 8.9 Hz, 1H), 6.67 (br s, 2H), 5.18 (s, 2H), 4.93 (dd, J = 10.0, 4.7 Hz, 1H), 3.76 (s, 3H), 3.50-3.50 (m, 1H), 3.58 (s, 3H), 3.44-3.34 (m, 1H), 2.04-1.94 (m, 1H), 1.86-1.73 (m,1H), 1.47-1.28 (m, 2H), 1.34 (s, 18H). MS (ESI + ) C 32 H 44 N3O 10 + [M+H] + Calculated value: 630.30, measured value: 630.40.

[0347] 2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-5-(((S)-4-(((Benzyloxy)carbonyl)(tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentyl)(tert-butoxy-carbonyl)amino)methyl benzoate (XT69) Aniline XT68 (1.43 g, 2.27 mmol) and Fmoc-Ala-Cl (824 mg, 2.50 mmol) were reacted in the same manner as with XT11. The mixture was purified by flash chromatography (silica gel, siRNA:DCM 0:1 to 1:3) to obtain amide XT69 as a yellow foamy substance (2.1 g, quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 11.19 (s, 1H), 8.61-8.56 (m, 1H), 8.48 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 6.3 Hz, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.83-7.69 (m, 3H), 7.48-7.27 (m, 9H), 5.17 (s, 2H), 4.92 (dd, J = 9.8, 4.8 Hz, 1H), 4.47-4.40 (m, 1H), 4.36-4.22 (m, 2H), 4.19-4.09 (m, 1H), 3.72 (s, 3H), 3.30-3.63 (m, 1H), 3.59-3.50 (m, 1H), 3.58 (s, 3H), 2.06-1.96 (m, 1H), 1.86-1.74 (m, 1H), 1.43-1.29 (m, 5H), 1.36 (s, 9H), 1.32 (s, 9H). MS (ESI + ) C 50 H 59 N4O 13 + [M+H] + Calculated value: 923.41, measured value: 923.39.

[0348] 2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-5-(((S)-4-(((Benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentyl)amino)methyl benzoate (XT70) To a solution of XT69 (2.10 g, 2.28 mmol) protected with two Boc molecules in dioxane (2 mL), HCl in dioxane (4 M, 12 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:9) to obtain aniline XT70 as a yellow foam (1.45 g, 88%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.69 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 6.7 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.82-7.72 (m, 3H), 7.45-7.25 (m, 10H), 7.10 (d, J = 2.6 Hz, 1H), 6.83 (dd, J = 8.9, 2.7 Hz, 1H), 5.04 (s, 2H), 4.44-4.35 (m, 1H), 4.31-4.23 (m, 2H), 4.14-4.02 (m, 2H), 3.69 (s, 3H), 3.63 (s, 3H), 3.03-2.93 (m, 2H) 1.86-1.75 (m, 1H), 1.72-1.54 (m, 3H), 1.34 (d, J = 7.2 Hz, 3H). MS (ESI + ) C 40 H 43 N4O9 + [M+H] + Calculated value: 723.30, measured value: 723.51.

[0349] 5-(((S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentyl)amino)-2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)methyl benzoate (XT71) Amine XT70 (1.45 g, 2.00 mmol) protected with Fmoc was deprotected with piperidine and subsequently reacted with Boc-Val-OSu as in the case of XT43. Dipeptide XT71 was obtained as a yellow solid by purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) (600 mg, 43%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.23 (s, 1H), 8.20 (d, J = 6.8 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.42-7.26 (m, 5H), 7.04 (d, J = 2.6 Hz, 1H), 6.78 (dd, J = 8.9, 2.5 Hz, 1H), 6.67 (d, J = 9.1 Hz, 1H), 5.77 (t, J = 5.2 Hz, 1H), 5.04 (s, 2H), 4.43 (p, J = 6.9 Hz, 1H), 4.10-4.01 (m, 1H), 3.94-3.88 (m, 1H), 3.80 (s, 3H), 3.63 (s, 3H), 2.97 (q, J = 5.7 Hz, 2H), 2.09-1.97 (m, 1H), 1.86-1.75 (m, 1H), 1.72-1.52 (m, 3H), 1.38 (s, 9H), 1.31 (d, J = 7.0 Hz, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.77 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) ppm = 173.3, 171.9, 170.8, 168.0, 156.6, 156.0, 145.6, 137.4, 128.8, 128.3, 128.2, 123.8, 120.3, 117.3, 112.9, 78.5, 66.0, 59.7, 54.2, 52.6, 52.3, 50.0, 30.9, 28.8, 25.5, 19.8, 18.2, 18.1. MS (ESI + ) C 35 H 50 N5O 10 + [M+H] + Calculated value: 700.36, measured value: 700.56.

[0350] 5-(((S)-4-amino-5-methoxy-5-oxopentyl)amino)-2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)methyl benzoate (XT72) Compound XT71 (600 mg, 0.86 mmol) was dissolved in DMF (8.5 mL) under a nitrogen atmosphere. Pd / C (91 mg, 10 mol%) was added on activated carbon, and the resulting black suspension was vigorously stirred at room temperature under a hydrogen atmosphere for 90 minutes. The flask was purged with N2, and the reaction mixture was filtered through Celite®. The filtrate was concentrated to obtain XT71 as a dark green solid (quantification). MS (ESI + ) C 27 H 44 N5O8 + [M+H] + Calculated value: 566.32, measured value: 566.43.

[0351] 2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-5-(((S)-4-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-methoxy-5-oxopentyl)amino)methyl benzoate (XT721) Amine XT72 (167 mg, 0.295 mmol) and acid XT7 (100 mg, 0.295 mmol) were reacted according to general procedure XXA. The product was recrystallized from MeOH (5 mL) to obtain XT721 as an orange solid (216 mg, 83%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.23 (s, 1H), 8.80 (s, 1H), 8.72 (d, J = 7.5 Hz, 1H), 8.67 (s, 1H), 8.20 (d, J = 6.8 Hz, 1H), 7.87 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.47 (br s, 1H), 7.04 (d, J = 2.8 Hz, 1H), 6.78 (dd, J = 9.0, 2.7 Hz, 1H), 6.74 (br s, 2H), 6.67 (d, J = 9.2 Hz, 1H), 5.78 (br s, 1H), 5.23 (s, 2H), 4.47-4.39 (m, 1H), 4.33 (p, J = 7.0 Hz, 1H), 3.94-3.89 (m, 1H), 3.79 (s, 3H), 3.63 (s, 3H), 3.04-2.95 (m, 2H), 2.09-1.98 (m, 1H), 1.98-1.88 (m, 1H), 1.88-1.77 (m, 1H), 1.69-1.55 (m, 2H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.77 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 42 H 55 N 12 O 10 + [M+H] + Calculated value: 887.42, measured value: 887.90.

[0352] 2-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-5-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)amino)benzoic acid (XT722) To a solution of ester XT721 (210 mg, 0.24 mmol) in THF (1.5 mL), an aqueous solution of LiOH (28 mg, 1.2 mmol) (1.5 mL) was added. The resulting mixture was stirred for 72 hours and stored overnight at -78°C. After completion, the reaction mixture was acidified with aqueous AcOH (1.0 M, 2.5 mL), and the resulting suspension was stirred at room temperature for 60 minutes. The mixture was filtered, and the residue was washed with water (10 mL), MeOH (4 mL), and ether (10 mL). The resulting solid was air-dried overnight to obtain a yellow solid (150 mg). Next, the material was suspended in DCM (3 mL), cooled to 0°C, and TFA (3 mL) was added. The resulting solution was stirred for 15 minutes while being warmed to room temperature. The reaction mixture was concentrated, and the solvent was evaporated with DCM to obtain the TFA salt XT722 (quantified), which was used directly in the next reaction. MS (ESI + ) C 34 H 43 N 12 O7 + [M+H] + Calculated value: 731.34, measured value: 731.52.

[0353] 5-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)amino)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methyl-butanamide)propanamide)benzoic acid (XT723) Amine XT722 (132 mg, 0.18 mmol) was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (56 mg, 0.18 mmol) and DIPEA (0.314 mL, 1.80 mmol) in the same manner as with XT17. The residue was purified by trituration in MeOH, filtered, and purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%) to obtain XT723 as a yellow solid (72 mg, 43%, 3 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.67-12.00 (m, 2H), 10.77 (s, 1H), 9.35 (br s, 1H), 9.31 (br s, 1H), 8.84 (s, 1H), 8.64 (br s 1H), 8.34 (d, J = 6.5 Hz, 1H), 8.21-8.13 (m, 2H), 7.79-7.69 (m, 3H), 7.62 (br s, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.00 (s, 2H), 6.83 (dd, J = 9.0, 2.3 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 4.63 (s, 2H), 4.39-4.32 (m, 1H), 4.31-4.20 (m, 2H), 3.36 (t, J = 7.0 Hz, 2H), 3.01 (t, J = 6.8 Hz, 2H), 2.23-2.02 (m, 3H), 1.98-1.87 (m, 1H), 1.87-1.74 (m, 1H), 1.70-1.56 (m, 2H), 1.53-1.41 (m, 4H), 1.31 (d, J = 7.1 Hz, 3H), 1.23-1.13 (m, 2H), 0.83 (d, J = 6.7 Hz, 3H), 0.76 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 44 H 54 N 13 O 10 + [M+H] + Calculated value: 924.41, measured value: 924.45.

[0354] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)phenyl)amino)pentanoic acid (XT80) [ka]

[0355] (S)-2-(((benzyloxy)carbonyl)amino)-5-((4-nitrophenyl)amino)methyl pentanoate (XT74) A mixture of XT73 (795 mg, 5.63 mmol), XT65 (500 mg, 1.88 mmol), NaOH (75 mg, 1.88 mmol), and NaHCO3 (473 mg, 5.63 mmol) in water (10 mL) / EtOH (6 mL) was placed in a sealed tube and heated at 90°C for 5 days. After cooling to room temperature, the reaction mixture was diluted with water (20 mL) and washed with ether (40 mL). The aqueous solution was then adjusted to approximately 3 pH with 6 M hydrochloric acid, and the resulting mixture was extracted with 5% MeOH (2 × 75 mL) in DCM. The combined organic phase was washed with water, dried over Na2SO4, filtered, and concentrated to obtain the intermediate carboxylic acid as a yellow foam (610 mg, 84%). The material was suspended in MeOH (50 mL), cooled to 0°C, and thionyl chloride (1.26 mL, 17.3 mmol) was added dropwise. The resulting mixture was stirred at 0°C for 45 minutes, and then allowed to rise to room temperature over 1 hour. After completion, the reaction mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:9) to obtain ester XT74 as a yellow foam (670 mg, quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 7.98 (d, J = 9.2 Hz, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.39-7.25 (m, 6H), 6.62 (d, J = 9.3 Hz, 2H), 5.04 (s, 2H), 4.12-.4.04 (m, 1H), 3.10 (s, 3H), 3.17-3.08 (m, 2H), 1.87-1.74 (m, 1H), 1.72-1.56 (m, 3H). MS (ESI +) C 20 H 24 N3O6 + [M+H] + Calculated value: 402.17, measured value: 402.33.

[0356] (S)-2-amino-5-((4-nitrophenyl)amino)methyl hydrobromide pentanoate (XT75) Ester XT74 (480 mg, 1.20 mmol) was deprotected in the same manner as for XT58. The reaction mixture was then concentrated, and the solvent was evaporated with toluene. The residue was dissolved in water and freeze-dried to obtain a sticky gum-like substance. The substance was stirred in ether until a suspension was obtained, and then filtered. The solvent of the residue was evaporated with MeOH to obtain amine XT75 as a yellow foam (550 mg, quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.41 (br s, 3H), 8.00 (d, J = 9.2 Hz, 2H), 6.66 (d, J = 9.2 Hz, 2H), 4.13 (sextet, J = 5.5 Hz, 1H), 3.75 (s, 3H), 3.19 (t, J = 6.8 Hz, 2H), 1.96-1.79 (m, 2H), 1.77-1.53 ​​(m, 2H). MS (ESI + ) C 12 H 18 N3O4 + [M+H] + Calculated value: 268.13, measured value: 268.27.

[0357] (S)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-((4-nitrophenyl)-amino)pentanoate methyl (XT76) Amine XT75 (250 mg, 0.718 mmol) and acid XT7 (244 mg, 0.718 mmol) were reacted according to general procedure XXA. The product was recrystallized from MeOH (8 mL) to obtain XT76 as an orange solid (310 mg, 73%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.74 (d, J = 7.5 Hz, 1H), 8.65 (s, 1H), 7.96 (d, J = 8.9 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.66 (br s, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.34 (br s, 1H), 7.29 (t, J = 5.1 Hz, 1H), 6.62 (br s, 2H), 6.61 (d, J = 9.2 Hz, 2H), 5.21 (s, 2H), 4.51-4.41 (m, 1H), 3.63 (s, 3H) 3.20-3.12 (m, 2H), 1.99-1.75 (m, 2H), 1.73-1.57 (m, 2H). MS (ESI + ) C 27 H 29 N 10 O6 + [M+H] + Calculated value: 589.23, measured value: 589.59.

[0358] (S)-5-((4-aminophenyl)amino)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-benzamide)methyl pentanoate (XT77) Nitroaniline XT76 (340 mg, 0.578 mmol) was reduced using zinc powder (1133 mg, 17.3 mmol) in the same manner as with XT60 to obtain aniline XT77 as a gray solid (305 mg, 95%). MS (ESI + ) C 27 H 31 N 10 O4 + [M+H] + Calculated value: 559.25, measured value: 559.14.

[0359] (S)-5-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-phenyl)amino)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XT78) Aniline XT77 (300 mg, 0.54 mmol) and Boc-ValAla-OH (170 mg, 0.59 mmol) were reacted according to the general procedure XXA. XT78 was obtained as a yellow solid (240 mg, 54%) by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.55 (s, 1H), 8.81 (s, 1H), 8.78-8.68 (m, 3H), 8.52 (d, J = 8.4 Hz, 1H), 8.43 (br s, 1H), 8.12 (br s, 2H), 7.95 (d, J = 7.1 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 8.9 Hz, 1H), 6.48 (d, J = 8.8 Hz, 2H), 5.27 (s, 2H), 4.48-4.34 (m, 2H), 3.89-3.78 (m, 1H), 3.63 (s, 3H) 2.97 (t, J = 6.6 Hz, 2H), 2.03-1.74 (m, 3H), 1.69-1.51 (m, 2H), 1.38 (s, 9H), 1.31-1.19 (m, 3H), 0.90-0.78 (m, 6H). MS (ESI + ) C 40 H 53 N 12 O8 + [M+H] + Calculated value: 829.41, measured value: 829.58.

[0360] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)phenyl)amino)pentanoic acid (XT80) Carbamate XT78 (240 mg, 0.29 mmol) was suspended in DCM (3 mL), cooled to 0°C, and TFA (3 mL) was added. The resulting mixture was stirred at 0°C for 15 minutes, then concentrated, and the solvent was evaporated along with the DCM. Hydrolysis of the intermediate ester with NaOH (36 equivalents) was carried out in addition to the general procedure XXC, with the addition of lyophilization of the solution obtained after evaporation of MeOH, because the product did not solidify sufficiently after treatment with 1 M AcOH. The resulting cake was stirred in DMF (4 mL) and filtered. The filtrate containing the deprotected product was then reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (31 mg, 0.100 mmol) and DIPEA (0.105 mL, 0.600 mmol) as in the case of XT17. The crude product was tritulate with MeOH, filtered, and the solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%) to obtain XT80 as a yellow solid (37 mg, 15%, 3 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 13.53-11.98 (m, 2H), 9.63 (br s, 1H), 9.36 (br s, 1H), 9.31 (br s, 1H), 8.84 (s, 1H), 8.62 (br s, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 7.0 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.69 (br s, 1H), 7.38 (d, J = 7.4 Hz, 2H), 6.99 (s, 2H), 6.88 (br s, 1H), 6.75 (d, J = 8.6 Hz, 2H), 6.70 (br s, 2H), 4.62 (s, 2H), 4.41-4.30 (m, 2H), 4.18-4.11 (m, 1H), 3.41-3.31 (m, 2H), 3.11-2.98 (m, 2H), 2.22-2.06 (m, 2H), 2.01-1.86 (m, 2H), 1.86-1.74 (m, 1H), 1.71-1.56 (m, 2H), 1.54-1.40 (m, 4H), 1.28 (d, J = 7.0 Hz, 3H), 1.22-1.13 (m, 2H), 0.85 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 43 H 54 N 13 O8 + [M+H] + Calculated value: 880.42, measured value: 880.52.

[0361] Manufacture of 2-(((S)-5-carboxy-5-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-pentyl)amino)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)nicotinic acid (XT89) [ka]

[0362] (S)-2-((5-(((benzyloxy)carbonyl)amino)-6-methoxy-6-oxohexyl)amino)-5-methyl nitronicotinate (XT83) Amine XT82 (500 mg, 1.78 mmol) and XT81 (386 mg, 1.78 mmol) were reacted in the same manner as with XT57. The product was recrystallized from MeOH (8 mL) to obtain XT83 as a yellow solid (370 mg, 44%). MS (ESI + ) C 22 H 27 N4O8 + [M+H] + Calculated value: 475.18, measured value: 475.34.

[0363] (S)-2-((5-amino-6-methoxy-6-oxohexyl)amino)-5-methyl hydrobromide nitronicotinate (XT84) Cbz-protected amine XT83 (370 mg, 0.78 mmol) was deprotected in the same manner as for XT58. The product was dissolved in water and then lyophilized. The residue was suspended in ether and filtered to obtain XT84 as a yellow solid (quantified). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.12 (d, J = 2.7 Hz, 1H), 8.85 (t, J = 5.6 Hz, 1H), 8.70 (d, J = 2.7 Hz, 1H), 8.40 (br s, 2H), 4.12-4.02 (m, 1H), 3.90 (s, 3H), 3.75 (s, 3H), 3.60 (q, J = 6.5 Hz, 2H), 1.88-1.75 (m, 2H), 1.62 (p, J = 7.1 Hz, 2H), 1.52-1.28 (m, 2H). MS (ESI + ) C 14 H 21 N4O6 + [M+H] + Calculated value: 341.15, measured value: 341.22.

[0364] (S)-2-((5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)-5-nitronicotinate methyl (XT85) Amine XT84 (360 mg, 0.86 mmol) and acid XT7 (264 mg, 0.78 mmol) were reacted according to the general procedure XXA. The product was recrystallized from MeOH (5 mL) to obtain XT85 as an orange solid (280 mg, 55%). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.07 (d, J = 2.7 Hz, 1H), 8.84 (t, J = 5.7 Hz, 1H), 8.80 (s, 1H), 8.69-8.64 (m, 1H), 8.66 (s, 1H), 8.63 (d, J = 2. Hz, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.67 (br s, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.35 (br s, 1H), 6.64 (br s, 2H), 5.22 (s, 2H), 4.45-4.35 (m, 1H), 3.84 (s, 3H), 3.66-3.50 (m, 2H), 3.61 (s, 3H), 1.89-1.75 (m, 2H), 1.69-1.51 (m, 2H), 1.50-1.32 (m, 2H). MS (ESI + ) C 29 H 32 N 11 O8 + [M+H] + Calculated value: 662.24, measured value: 662.44.

[0365] (S)-5-amino-2-((5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)methyl nicotinate (XT86) Compound XT85 (280 mg, 0.42 mmol) was reduced using zinc powder (830 mg, 12.7 mmol) in the same manner as with XT60 to obtain aniline XT86 as a gray solid (240 mg, 90%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.68 (d, J = 7.2 Hz, 1H), 8.66 (s, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 2.7 Hz, 1H), 7.68 (br s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 2.8 Hz, 1H), 7.37 (br s, 1H), 7.22 (t, J = 5.4 Hz, 1H), 6.66 (br s, 2H), 5.22 (s, 2H), 4.47 (s, 2H), 4.42-4.34 (m, 1H), 3.77-3.57 (m, 2H) 3.74 (s, 3H), 3.61 (s, 3H), 1.86-1.75 (m, 2H), 1.59-1.48 (m, 2H), 1.47-1.32 (m, 2H). MS (ESI + ) C 29 H 34 N 11 O6 + [M+H] + Calculated value: 632.27, measured value: 632.42.

[0366] 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-(((S)-5-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-6-methoxy-6-oxohexyl)amino)methyl nicotinate (XT87) Aniline XT86 (120 mg, 0.19 mmol) and Boc-ValAla-OH (60 mg, 0.21 mmol) were reacted according to the general procedure XXA. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) yielded XT87 as a yellow solid (100 mg, 58%). MS (ESI + ) C 42 H 56 N 13 O 10 + [M+H] + Calculated value: 902.43, measured value: 902.49.

[0367] 2-(((S)-5-carboxy-5-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentyl)amino)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)nicotinic acid (XT89) Carbamate XT87 (100 mg, 0.11 mmol) was suspended in DCM (3 mL), cooled to 0°C, and TFA (3 mL) was added. The resulting mixture was stirred at 0°C for 15 minutes, then concentrated, and the solvent was evaporated along with the DCM. Hydrolysis of the intermediate ester with NaOH (18 equivalents) was carried out according to the general procedure XXC. The residual cake was stirred with DMF (3 mL) and filtered. The filtrate was treated with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (31 mg, 0.10 mmol) and DIPEA (0.105 mL, 0.600 mmol) as in the case of XT17. The mixture was concentrated and purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) to obtain XT89 as a yellow solid (12 mg, 13%, 3 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 13.60-12.03 (m, 2H), 9.79 (s, 1H), 9.35 (br s, 1H), 9.31 (br s, 1H), 8.85 (s, 1H), 8.62 (br s, 1H), 8.38 (d, J = 2.7 Hz, 1H), 8.33 (d, J = 2.7 Hz, 1H), 8.19-8.10 (m, 2H), 8.06-7.64 (m, 2H) 7.82 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 6.99 (s, 2H), 6.87 (br s, 1H), 6.74 (d, J = 8.7 Hz, 2H), 4.62 (s, 2H), 4.37-4.28 (m, 2H), 4.18-4.11 (m, 1H), 3.43-3.30 (m, 4H), 2.23-2.06 (m, 2H), 2.01-1.90 (m, 1H), 1.87-1.73 (m, 2H), 1.63-1.52 (m, 2H), 1.52-1.35 (m, 6H), 1.29 (d, J = 7.1 Hz, 3H),1.22-1.13 (m, 2H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz (3H). MS (ESI + ) C 44 H 55 N 14 O 10 + [M+H] + Calculated value: 939.42, measured value: 939.37.

[0368] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-(2H-triazole-5-yl)benzamide)Pentanoic acid (XT94) preparation [ka]

[0369] (S)-5-(4-amino-2-(2H-triazol-5-yl)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT90) Unpurified tetrazole XX21 (55 mg, 0.271 mmol) and amine XX29 (150 mg, 0.271 mmol) in DMF (1 mL) were reacted according to general procedure XXA. The product was recrystallized from MeOH (5 mL) and washed with ether (5 mL) to obtain XT90 as an orange solid (quantified). MS (ESI + ) C 29 H 32 N 13 O4 + [M+H] + Calculated value: 626.27, measured value: 626.38.

[0370] (S)-5-(4-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-2-(2H-Triazole-5-yl)benzamide)-2-(4-(2-(2,4-Diaminopteridine-6-yl)ethyl)benzamide)Methyl pentanoate (XT91) Aniline XT90 (150 mg, 0.240 mmol) and Fmoc-Ala-OH (112 mg, 0.360 mmol) were reacted according to the general procedure XXA. The crude product was purified by flash chromatography (silica gel, MeOH:DCM × 1%Et3N 0:1 to 1:1). The isolated product was dissolved in MeOH and precipitated with ether. The resulting suspension was filtered, the solid was washed with ether, and air-dried overnight to obtain amide XT91 as a yellow solid (75 mg, 34%). MS (ESI + ) C 47 H 47 N 14 O7 + [M+H] + Calculated value: 919.37, measured value: 919.79.

[0371] (S)-5-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-(2H-triazole-5-yl)benzamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT92) The Fmoc-protected amine XT91 (75 mg, 0.08 mmol) was deprotected using piperidine (0.16 mL, 1.6 mmol), and subsequently reacted with Boc-Val-OSu (34 mg, 0.11 mmol) as in the case of XT43. It was purified by precipitation from a minimal amount of methanol using ether, then filtered, and the residue was air-dried to obtain the dipeptide XT92 as a yellow solid (75 mg, 78%). MS (ESI + ) C 42 H 54 N 15 O8 + [M+H] + Calculated 896.43, Found 896.54.

[0372] (S)-5-(4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)-2-(2H-1,2,3-triazol-5-yl)benzamide)-2-(4-(2-(2,4-diaminopteridin-6-yl)ethyl)benzamide)pentaanoic acid (XT93) To a solution of the ester XT92 (75 mg, 0.084 mmol) in THF (1 mL) was added LiOH (10 mg, 0.419 mmol) in water (1 mL). The resulting solution was stirred at room temperature for 1 hour, cooled to 0 °C, and treated with AcOH (0.05 mL, 0.84 mmol). After stirring for 5 minutes, the suspension was concentrated and the solvent was evaporated with toluene. The residue was suspended in DCM (1 mL), cooled to 0 °C, and TFA (1 mL) was added. The resulting solution was stirred for 60 minutes while gradually warming to room temperature. The solvent of the reaction mixture was evaporated with DCM to obtain the crude amine XT93 (quantitative). MS (ESI + ) C 36 H 44 N 15 O6 + [M+H] + Calculated 782.36, Found 782.73.

[0373] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-(2H-triazole-5-yl)benzamide)pentanoic acid (XT94) Unpurified amine XT93 (62.5 mg) was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (25 mg, 0.080 mmol) and DIPEA (0.140 mL, 0.800 mmol) in the same manner as with XT17. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) yielded XT94 as a white solid (22 mg, 28%, 3 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.13-12.34 (m, 2H), 10.28 (s, 1H), 9.25 (br s, 1H), 9.16 (br s, 1H), 8.75 (s, 1H), 8.58 (br s, 1H), 8.50 (d, J = 7.7 Hz, 1H), 8.42 (br s, 1H), 8.23 ​​(d, J = 6.7 Hz, 1H), 7.94 (br s, 1H), 7.84-7.76 (m, 4H), 7.67-7.61 (m, 1H), 7.49 (br s, 1H) 7.36 (d, J = 8.3 Hz, 2H), 6.99 (s, 2H), 4.42-4.32 (m, 2H), 4.19-4.13 (m, 1H), 3.27-3.10 (m, 8H), 2.22-2.07 (m, 2H), 2.00-1.89 (m, 1H), 1.89-1.70 (m, 2H), 1.68-1.41 (m, 6H), 1.32 (d, J = 7.1 Hz, 3H), 1.22-1.12 (m, 2H), 0.88-0.79 (m, 6H). MS (ESI + ) C 46 H 55 N 16 O9 +[M+H] + Calculated value: 975.43, measured value: 976.02.

[0374] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)thiophene-2-carboxamide)pentanoic acid (XT97) manufacturing [ka]

[0375] (S)-5-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)thiophen-2-carboxamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT95) Amine XX29 (334 mg, 0.61 mmol) and acid XR23 (250 mg, 0.61 mmol) were reacted according to general procedure XXA. The product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain XT95 as a yellow solid (quantified). MS (ESI + ) C 39 H 52 N 11 O8S + [M+H] + Calculated value: 834.37, measured value: 834.53.

[0376] (S)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-(5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)thiophene-2-carboxamide)pentanoic acid (XT97) Hydrolysis and deprotection of ester XT95 were carried out in the same manner as for XT93. The unpurified amine was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (92 mg, 0.3 mmol) and DIPEA (388 mg, 3.00 mmol) in the same manner as for XT17. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) yielded XT97 as a white solid (43 mg, 16%, 3 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.36-12.86 (br s, 1H (TFA)), 12.83-12.26 (br s, 1H), 11.34 (s, 1H), 9.25 (br s, 1H), 9.16 (br s, 1H), 8.75 (s, 1H), 8.60 (br s, 1H), 8.55 (d, J = 7.7 Hz, 1H), 8.34-8.25 (m, 2H), 7.91-7.75 (m, 4H), 7.50 (d, J = 4.1 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H), 6.99 (s, 2H), 6.66 (d, J = 4.1 Hz, 1H), 4.43-4.33 (m, 2H), 4.19-4.12 (m, 1H), 3.29-3.15 (m, 8H) 2.23-2.06 (m, 2H), 2.01-1.71 (m, 3H), 1.69-1.41 (m, 6H), 1.31 (d, J = 7.2 Hz, 3H), 1.23-1.12 (m, 2H), 0.85 (dd, J = 16.1, 6.7 Hz, 6H). MS (ESI + ) C 43 H 53 N 12 O9S + [M+H] + Calculated value: 913.38, measured value: 913.83.

[0377] (2S,2'S)-5,5'-((5,5'-(((14S,14'S,17S,17'S)-1,1'-(((((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)pentazinoyl)bis(azandiyl))bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))bis(14-iso Manufacturing of Propyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-noyl))bis(azandiyl))bis(thiophene-5,2-diyl-2-carbonyl))bis(azandiyl))bis(2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid) (XT100) [ka]

[0378] (S)-5-(5-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amide)thiophene-2-carboxamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT98) Dipeptide XT95 (250 mg, 0.30 mmol) was dissolved in DCM (1 mL), cooled to 0°C, and TFA (1 mL) was added. The resulting solution was stirred for 15 minutes while gradually warming to room temperature. The reaction mixture was then diluted with DCM, concentrated, and the solvent was evaporated along with the DCM. The residue was suspended in ether (4 mL), filtered, washed with ether, and air-dried to obtain the intermediate amine as the TFA salt. Next, the amine was dissolved in DMF, and 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (74 mg, 0.30 mmol) was added. The mixture was cooled to 0°C, and HATU (137 mg, 0.36 mmol) and DIPEA (0.31 mL, 1.80 mmol) were added. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain azide XT98 as a yellow glassy substance (quantified). MS (ESI + ) C 43 H 59 N 14 O 10 S + [M+H] + Calculated value: 963.43, measured value: 963.56.

[0379] (S)-5-(5-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amide)thiophene-2-carboxamide)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XT99) Ester XT98 (290 mg, 0.30 mmol) was hydrolyzed with LiOH in the same manner as XT93 (the deprotection step of Boc with THA was omitted). Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) yielded XT99 as a white solid (27 mg, 9%). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.08-12.39 (m, 2H (TFA)), 11.33 (s, 1H), 9.21 (br s, 1H), 9.12 (br s, 1H), 8.75 (s, 1H), 8.55 (d, J = 7.7 Hz, 1H), 8.52 (br s, 1H), 8.31 (d, J = 6.7 Hz, 1H), 8.28 (t, J = 5.2 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 4.1 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H), 6.66 (d, J = 4.1 Hz, 1H), 4.43-4.34 (m, 2H), 4.23-4.17 (m, 1H), 3.63-4.45 (m, 14H), 3.27-3.14 (m, 6H), 2.49-2.35 (m, 2H), 2.01-1.70 (m, 3H), 1.68-1.51 (m, 2H), 1.31 (d, J = 7.1 Hz, 3H), 0.86 (dd, J = 15.9, 6.7 Hz, 6H). MS (ESI + ) C 42 H 57 N 14 O 10 S + [M+H] + Calculated value: 949.41, measured value: 949.88.

[0380] (2S,2'S)-5,5'-((5,5'-(((14S,14'S,17S,17'S)-1,1'-(((((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)pentazinoyl)bis(azandiyl))bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl))bis(14- Isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-noyl))bis(azandiyl))bis(thiophene-5,2-diyl-2-carbonyl))bis(azandiyl))bis(2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid)(XT100) A solution of XT99 (22 mg, 0.019 mmol) and XS2 (3.1 mg, 7.5 μmol) in DMF (2.5 mL) was purged with N2 for 5 minutes. Then, copper(II) sulfate (1.44 mg, 5.8 μmol) in water (60 μL) and sodium ascorbate (2.22 mg, 0.011 mmol) in water (60 μL) were added sequentially. The resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated, and the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 30%) to obtain XT100 (12.1 mg, 70%). MS (ESI + ) C 105 H 140 N 32 O 25 S2 2+ [M+2H] 2+ 1156.5, measured value [M+2H] 2+ 1157.10.

[0381] Manufacturing of (S)-5-amino-2-(N-(4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-butyl)sulfamoyl)benzoic acid (XT101) [ka]

[0382] (S)-2-(N-(4-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-methoxy-5-oxopentyl)sulfamoyl)-5-methyl nitrobenzoate (XJ22) Methyl 2-(chlorosulfonyl)-5-nitrobenzoate (0.25 g, 0.894 mmol) and amine XT48 (0.416 g, 0.770 mmol) were reacted in DMF (5 mL) at room temperature for 0.5 hours in the presence of DIPEA (0.33 mL, 1.92 mmol). After concentrating the reaction mixture, the residue was purified by flash chromatography (silica gel, DCM:MeOH 1:0 to 95:5) to obtain sulfonamide XJ22 as a yellow solid (0.159 g, 29%). MS (ESI + ) C 29 H 31 N 10 O 10 S + [M+H] + Calculated value: 711.7, measured value: 711.6.

[0383] (S)-5-amino-2-(N-(4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)sulfamoyl)benzoic acid (XT101) Aniline XT101 was prepared from sulfonamide XJ22 (160 mg, 0.225 mmol) in the same manner as the preparation of XR22. Reduction was carried out in DMF (1.5 mL) using zinc powder (442 mg, 6.75 mmol) and saturated NH4Cl aqueous solution (0.375 mL), and hydrolysis was carried out in THF (2.5 mL) and water (2.5 mL) using LiOH (54 mg, 2.25 mmol). A portion of the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%) to obtain XT101 (13 mg). 1H NMR (400 MHz, DMSO-d6) ppm = 12.46 (br s, 1H), 9.22 (br s, 1H), 9.18 (br s, 1H), 8.83 (s, 1H), 8.59-8.32 (m, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.64-7.42 (m, 1H), 7.49 (d, J = 8.8 Hz, 1H), 6.88 (br s, 1H), 6.78-6.71 (m, 3H), 6.64 (dd, J = 8.7, 2.3 Hz, 1H), 6.59-6.38 (m, 1H), 6.46 (t, J = 5.9 Hz, 1H), 6.13 (br s, 2H), 4.61 (s, 2H), 4.30-4.21 (m, 1H), 2.82-2.70 (m, 2H), 1.85-1.62 (m, 2H), 1.48-1.40 (m, 2H). MS (ESI+) C 26 H 29 N 10 O7S + [M+H] + The calculated value is 625.19 and the measured value is 625.32.

[0384] Preparation of (S)-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-methoxy-5-oxopentan-1-aminium trifluoroacetate (XX29)

change

[0385] (E)-4-(2-(2,4-ジアミノプテリジン-6-イル)ビニル)benzoic acid (XT103) A solution of PPh3 (31.5 g, 120 mmol) in cooled (0 °C) DMA (120 mL) was added dropwise with bromine (6.17 mL, 120 mmol) under a nitrogen atmosphere. After the completion of the dropwise addition, XT102 (7 g, 36.4 mmol) was added to the ice-cooled solution at once (the free base XT102 was produced as described in XX9). The resulting mixture was stirred at 0 °C for 1 hour and at room temperature for 90 minutes. Ethanol (2.4 mL) was added dropwise and the mixture was stirred at room temperature for 15 minutes. Then the solution was poured into vigorously stirred toluene (380 mL). After filtration, the solid was stirred in ether (400 mL) until a fine suspension was formed. The suspension was filtered and the residue was air-dried overnight. The resulting light brown powder was redissolved in DMA (500 mL) under a nitrogen atmosphere and PPh3 (9.55 g, 36.4 mmol) was added to the solution. The resulting mixture was heated at 65 °C for 75 minutes, cooled to 0 °C, and KOtBu (18.4 g, 164 mmol) was added. After stirring at 0 °C for 10 minutes, methyl 4-formylbenzoate (5.98 g, 36.4 mmol) was added. The mixture was stirred at room temperature for 45 minutes, and at that point additional KOtBu (12.3 g, 109 mmol) was added. After 30 minutes, the reaction mixture was cooled in a cold bath, AcOH (25 mL, 437 mmol) was added, and the mixture was stirred for 5 minutes. Then this solution was poured into ice-cold water (2.1 L). The resulting suspension was filtered, and subsequently the residue was washed with MeCN (2 × 180 mL), toluene (2 × 180 mL) and ether (2 × 180 mL) and air-dried to obtain carboxylic acid XT103 as a yellow solid (10.4 g, 93%). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.96 (br s, 1H), 8.89 (s, 1H), 8.14 (br s, 1H), 8.10 (br s, 1H), 8.03 - 7.95 (m, 3H), 7.76 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 16.1 Hz, 1H), 7.02 (br s, 2H). MS (ESI + ) C 15 H 13 N6O2 + [M + H] +Calculated value: 309.11, measured value: 309.21.

[0386] (S,E)-5-((tert-butoxycarbonyl)amino)-2-(4-(2-(2,4-diaminopteridine-6-yl)vinyl)benzamide)methyl pentanoate (XT104) Acid XT103 (9.2 g, 29.8 mmol) and H-Orn(Boc)-OMe (9.3 g, 32.8 mmol) were reacted with 10 equivalents of DIPEA according to general procedure XXA. The unpurified reaction mixture was poured into water (2.4 L), stirred for 15 minutes, and then filtered. The resulting solid residue was washed with ether (3 × 250 mL) and air-dried for 2 days to obtain ester XT104 as a yellow solid (1.8 g, 83%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.87 (s, 1H), 8.74 (d, J = 7.4 Hz, 1H), 7.97-7.90 (m, 3H), 7.88-7.77 (m, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 16.2 Hz, 1H), 6.85-6.69 (m, 3H), 4.48-4.38 (m, 1H), 3.65 (s, 3H), 3.00-2.89 (m, 2H), 1.88-1.69 (m, 2H), 1.57-1.43 (m, 2H), 1.37 (s, 9H). 13 C NMR (100 MHz, DMSO-d6) ppm = 173.2, 166.6, 163.4, 163.3, 156.0, 155.5, 150.6, 143.2, 104.1, 133.5, 131.6, 128.6, 127.1, 126.5, 122.6, 77.9, 53.0, 52.3, 39.7, 28.7, 28.3, 26.7. MS (ESI + ) C 26 H 33 N8O5 + [M+H] + Calculated value: 537.26, measured value: 537.67.

[0387] (S)-5-((tert-butoxycarbonyl)amino)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)methyl pentanoate (XT105) A solution of XT104 (11.2 g, 20.9 mmol) in AcOH (450 mL) was placed in a nitrogen-purged flask, to which Pd / C (5 g, 10% on activated carbon) was added. Hydrogen gas was added, and the mixture was stirred at room temperature under a hydrogen atmosphere. Three additional doses of Pd / C (2.5 g) were added at 3-hour intervals. After 24 hours, the flask was purged with N2, and the reaction mixture was filtered through Celite®. MnO2 (18.2 g, 209 mmol) was added to the filtrate, and the suspension was stirred for 30 minutes. After filtering through Celite®, the filtrate was concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 15:85) to obtain XT105 as a sand-colored solid (6.9 g, 61%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.61 (d, J = 7.3 Hz, 1H), 8.55 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.59 (br s, 2H), 7.35 (d, J = 8.3 Hz, 2H), 6.77 (t, J = 5.4 Hz, 1H), 6.54 (br s, 2H), 4.43-4.33 (m, 1H), 3.63 (s, 3H), 3.19-3.09 (m, 4H), 2.97-2.88 (m, 2H), 1.86-1.67 (m, 2H), 1.55-1.39 (m, 2H), 1.36 (s, 9H). MS (ESI + ) C 26 H 35 N8O5 + [M+H] + 539.27, measured value 539.62.

[0388] (S)-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)-5-methoxy-5-oxopentan-1-aminium trifluoroacetate (XX29) Compound XT105 (1.10 g, 2.04 mmol) was suspended in DCM (10 mL), and TFA (10 mL) was added at room temperature. After stirring at room temperature for 20 minutes, the mixture was concentrated. The solvent of the residue was evaporated with iPrOH (2×), and then suspended in iPrOH (10 mL). The suspension was diluted with ether (60 mL), the solid was filtered off, and dried under vacuum to obtain the TFA salt XX29 as a yellow solid (1.03 g, 88%). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.24 (s, 1H), 9.13 (s, 1H), 8.73 (s, 1H), 8.72 (s, 1H), 8.54 (br s, 1H), 8.23 ​​(br s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.78 (br s, 3H), 7.37 (d, J = 8.2 Hz, 2H), 4.46 (ddd, J = 9.5, 7.7, 5.1 Hz, 1H), 3.65 (s, 3H), 3.28-3.19 (m, 4H), 2.85-2.77 (m, 2H), 1.96-1.74 (m, 2H), 1.72-1.56 (m, 2H). MS (ESI + ) C 21 H 27 N8O3 + [M+H] + 439.22, measured value 439.56.

[0389] Manufacturing of (6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl)glycylglycyl-L-phenylalanic acid 2,5-dioxopyrrolidine-1-yl (XX30) [ka]

[0390] DCC (459 mg, 2.22 mmol) was added at room temperature to a suspension of XT53 (1.05 mg, 2.22 mmol) and 1-hydroxypyrrolidine-2,5-dione (256 mg, 2.222 mmol) in THF (40 mL). After stirring for 3.5 hours, the mixture was filtered and the residue was thoroughly washed with DCM. The filtrate was diluted with siRNA and then concentrated. The white solid was suspended in a small amount of siRNA and then filtered to obtain OSu-ester XX30 as a white solid (612 mg, 48%). MS (ESI + ) C 27 H 32 N5O9 + [M+H] + 570.22, measured value 570.43.

[0391] Preparation of 5-((S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecanamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzenesulfonate sodium (XX19) [ka]

[0392] 2-(chlorosulfonyl)-4-methyl nitrobenzoate (XX14) Aniline XX13 (2.47 g, 12.6 mmol) in SiO2 (21 mL) and concentrated HCl (22 mL) was mixed dropwise with an aqueous solution of NaNO2 (0.912 g, 13.2 mmol) (5.7 mL) at 0°C. After stirring at 0°C for 30 minutes, a suspension of copper(II) chloride dihydrate (0.966 g, 5.67 mmol), NaHSO3 (13.1 g, 126 mmol), AcOH (16 mL), and concentrated HCl (5.7 mL) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was poured onto ice, the precipitated sulfonyl chloride was filtered off, and the mixture was washed with water. The solid was dried under vacuum to obtain XX14 as a yellow solid (2.58 g, 73%). 1 H NMR (400 MHz, CDCl3) ppm = 8.99 (d, J = 2.0 Hz, 1H), 8.64-8.62 (dd, J = 8.3 Hz, 2.1 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 4.05 (s, 3H). 13 C NMR (100 MHz, CDCl3) ppm = 164.6, 148.7, 142.9, 137.3, 131.6, 129.5, 124.5, 54.0.

[0393] 2-(isobutoxysulfonyl)-4-nitrobenzoic acid (XX15) Sulfonyl chloride XX14 (0.971 g, 3.47 mmol) was dissolved in DCM (20 mL), and the yellow solution was cooled to 0°C. Isobutanol (1.61 mL, 17.4 mmol) and Et3N (0.726 mL, 5.21 mmol) were added, and the reaction mixture was stirred at 0°C for 1 hour. The solution was concentrated, redissolved in ethyl acetate, and washed with KHSO4 (0.5 M, 2×), saturated NaHCO3, and brine. The organic phase was dried over MgSO4, filtered, and concentrated. The product XX15 was purified by flash chromatography (silica gel, heptane:DCM 1:0 to 0:1) to obtain a pale yellow oily substance (911 mg, 83%). A portion of this material (791 mg, 2.49 mmol) was dissolved in dioxane (25 mL) and water (16 mL). LiOH (2M, 4.99 mL, 9.97 mmol) was added at room temperature. After 20 minutes, the reaction mixture was diluted with water and the pH was adjusted to 3 with 1M hydrochloric acid. Extraction was performed three times with ethyl acetate. The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated to obtain acid XX15 as a yellow oily substance (756 mg, quantified). 1 H NMR (400 MHz, CDCl3) ppm = 8.89 (d, J = 2.1 Hz, 1H), 8.55 (dd, J = 8.4, 2.1 Hz, 1H), 8.0 (d, J = 8.4 Hz, 1H), 4.05 (d, J = 6.4 Hz, 2H), 2.05 (sept, J = 6.7 Hz, 1H), 0.96 (d, J = 6.7 Hz, 6H). MS (ESI - ) C 11 H 12 NO7S - [MH] - 302.03, measured value 302.35.

[0394] 4-amino-2-(isobutoxysulfonyl)benzoic acid (XX16) Palladium (42 mg, 10% on activated carbon) was added to acid XX15 (104 mg, 0.342 mmol) in MeOH (4 mL) at room temperature under a nitrogen atmosphere. The mixture was then stirred under a hydrogen atmosphere for 30 minutes. The flask was purged with N2, and the mixture was filtered through Celite®. The mixture was then concentrated to obtain aniline XX16 as a white solid (83 mg, 89%). MS (ESI - ) C 11 H 14 NO5S - [MH] - 272.06, measured value 272.34.

[0395] (S)-5-(4-amino-2-(isobutoxysulfonyl)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX17) Acid XX16 (83 mg, 0.304 mmol), HATU (121 mg, 0.319 mmol), and amine XT48 (153 mg, 0.304 mmol) were reacted according to general procedure XXA. After concentrating the reaction mixture, the solvent of the residue was evaporated with MeCN (2×), resuspended in MeCN, and filtered. The solid was purified by flash chromatography (silica gel, DCM:MeOH 1:0 to 4:1) to obtain aniline XX17 as a yellow solid (136 mg, 62%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.80 (s, 1H), 8.70 (d, J = 7.4 Hz, 1H), 8.66 (s, 1H), 8.03 (t, J = 5.4 Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.74 (br s, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.43 (br s, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 2.2 Hz, 1H), 6.80 (dd, J = 8.1, 2.2 Hz, 1H), 6.70 (br s, 2H), 5.88 (br s, 2H), 5.22 (s, 2H), 4.44-4.37 (m, 1H), 3.83 (d, J = 6.4 Hz, 2H), 3.63 (s, 3H), 3.19-3.13 (m, 2H), 1.93-1.75 (m, 3H), 1.65-1.48 (m, 2H), 0.80 (d, 6.7 Hz, 6H). MS (ESI + ) C 32 H 39 N 10 O8S + [M+H] + Calculated value: 723.27, measured value: 723.45.

[0396] (S)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-(2-(isobutoxysulfonyl)-4-(2-(2,2,2-trifluoroacetamide)acetamide)benzamide)methyl pentanoate (XX18) A stock solution of unpurified XT49 (205 mg, 1.08 mmol) in THF (0.5 mL) was prepared under a nitrogen atmosphere. In a separate vial, aniline XX17 (130 mg, 0.180 mmol) and DIPEA (0.094 mL, 0.540 mmol) were dissolved in DMF (2 mL) under a nitrogen atmosphere. A portion of the stock solution (285 μL) was added to the aniline at room temperature. After 5 minutes, the remaining portion (285 μL) was added, and stirring was continued for 5 minutes. The reaction mixture was concentrated and dry-loaded onto silica gel. Purification by flash chromatography (silica gel, DCM:MeOH 1:0 to 4:1) yielded amide XX18 (106 mg, 67%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.67 (s, 1H), 9.83 (t, J = 5.8 Hz, 1H), 8.80 (s, 1H), 8.70 (d, J = 7.5 Hz, 1H), 8.65 (s, 1H), 8.34 (t, J = 5.5 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.87 (dd, J = 8.6, 6.6 Hz, 1H), 7.64 (br s, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.33 (br s, 1H), 6.61 (br s, 2H), 5.22 (s, 2H), 4.45-4.38 (m, 1H), 4.06 (d, J = 5.6 Hz, 2H), 3.87 (d, J = 6.5 Hz, 2H), 3.63 (s, 3H), 3.20 (q, J = 6.0 Hz, 2H), 1.94-1.75 (m, 3H), 1.66-1.545 (m, 2H), 0.79 (d, J = 6.6 Hz, 6H). MS (ESI + ) C 36 H 41 F3N 11 O 10 S + [M+H] + Calculated value: 876.27, measured value: 876.21.

[0397] 5-((S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaoctadecanamide)-2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)benzenesulfonate sodium (XX19) Ester XX18 (79.2 mg, 0.090 mmol) was hydrolyzed using the general procedure XXC with the following modifications: i) the reaction mixture was kept at 0°C without heating to room temperature, ii) NaOH was added in two portions at 20-minute intervals, and iii) washing of the solid with MeOH and ether was omitted. The solid was dried overnight under vacuum to obtain unpurified acid (68 mg). Next, activated ester XX30 (52.5 mg, 0.092 mmol) in DMF (1.0 mL) was added to the yellow solid at room temperature, followed by the addition of DIPEA (0.097 mL, 0.553 mmol). After stirring for 20 minutes, an additional XX30 (10.5 mg, 0.018 mmol) was added. Stirring was continued for 2 minutes, at which point the reaction mixture was concentrated. The residue was suspended in MeCN (5 mL) and stirred for 1 hour. The suspension was filtered, and the solid was washed with MeCN and ether. A yellow solid (107 mg) was suspended in acetone (11 mL), and NaI (207 mg, 1.384 mmol) was added. The vial was stoppered and heated at 60°C for 90 minutes. After cooling to room temperature, the suspension was filtered, and the solid was washed with acetone (1 mL). The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%) to obtain XX19 as a yellow solid (4.8 mg). MS (ESI + ) C 51 H 58 N 15 O 14 S + [M+H] + Calculated value: 1136.40, measured value: 1136.95.

[0398] Manufacturing of (S)-5-(4-amino-2-sulfobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX37) [ka]

[0399] (S)-5-(4-amino-2-(isobutoxysulfonyl)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)-benzamide)pentanoic acid (XX36) Unpurified ester XX17 (128 mg, 0.177 mmol) was hydrolyzed according to the general procedure XXC to obtain XX36 as a yellow solid. MS (ESI + ) C 30 H 37 N 10 O7S + [M+H] + Calculated value: 681.26, measured value: 681.46.

[0400] (S)-5-(4-amino-2-sulfobenzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX37) Alkyl sulfonic acid XX36 (35.7 mg, 0.052 mmol) and NaI (118 mg, 0.787 mmol) in acetone (6 mL) were placed in a sealed vial and heated at 60°C for 8 hours. The reaction product was concentrated, and a portion of the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 20%) to obtain XX37 as a yellow solid (4.0 mg). 1H NMR (400 MHz, DMSO-d6) ppm = 12.24 (br s, 1H), 9.28 (s, 1H), 9.22 (s, 1H), 9.20 (t, J = 5.5 Hz, 1H), 8.77 (s, 1H), 8.55 (br s, 1H), 8.07 (d, J = 7.1 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.50 (br s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 2.4 Hz, 1H), 6.80 (br s, 1H), 6.65 (d, J = 8.8 Hz, 2H), 6.46 (dd, J = 8.3, 2.2 Hz, 2H), 4.55 (s, 2H), 4.17-4.12 (m, 1H), 3.22-3.06 (m, 2H), 1.97-1.84 (m, 1H), 1.78-1.70 (m, 1H), 1.59-1.42 (m, 2H). MS (ESI + ) C 26 H 29 N 10 O7S + [M+H] + Calculated value: 625.19, measured value: 625.20.

[0401] Manufacturing of (S)-5-(4-amino-2-(2H-triazol-5-yl)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX35) [ka]

[0402] 4-amino-2-(2H-triazole-5-yl)benzoic acid (XX21) A solution of 4-amino-2-cyanobenzoic acid (XX20; 250 mg, 1.542 mmol) and NaN3 (203 mg, 3.13 mmol) in DMF (4 mL) was placed in a sealed vial under a nitrogen atmosphere and heated at 110°C for 16 hours. After cooling to room temperature, the solution was filtered, and the filtrate was used directly in the next step. MS (ESI + ) C8H8N5O2 + [M+H] +Calculated value: 206.07, measured value: 206.22.

[0403] (S)-5-(4-amino-2-(2H-triazole-5-yl)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX34) An aliquot of XX21 (0.5 mL of unpurified filtrate in DMF) was added to a solution of XT48 (100 mg, 0.185 mmol) and DIPEA (0.194 mL, 1.110 mmol) in DMF (1.0 mL). HATU (70.4 mg, 0.185 mmol) was added at room temperature, and the resulting mixture was stirred for 30 minutes. Additional XT48 (20 mg, 0.037 mmol) and HATU (14.07 mg, 0.037 mmol) were added, and after 15 minutes, the reaction was concentrated and suspended in MeCN (6 mL). After stirring for 45 minutes, the suspension was filtered, and the solid was washed with MeCN and ether to obtain unpurified XX34 as a creamy solid (126 mg). MS (ESI + ) C 29 H 31 N 14 O5 + [M+H] + Calculated value: 655.26, measured value: 655.42.

[0404] (S)-5-(4-amino-2-(2H-triazole-5-yl)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX35) Unpurified ester XX34 (58 mg, 0.089 mmol) was hydrolyzed according to the general procedure XXC. The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 25%) to obtain XX35 as a yellow solid (14.8 mg). 1H NMR (400 MHz, DMSO-d6) ppm = 12.40 (br s, 1H), 9.24 (br s, 1H), 9.20 (br s, 1H), 8.77 (s, 1H), 8.50 (br s, 1H), 8.11-8.06 (m, 2H), 7.69-7.62 (m, 3H), 7.40-7.36 (m, 1H), 6.84 (br s, 1H), 6.67 (d, J = 8.9 Hz, 2H), 6.65-6.62 (m, 2H), 4.55 (s, 2H), 4.28-4.22 (m, 1H), 3.04 (q, J = 6.4 Hz, 2H), 1.80-1.60 (m, 2H), 1.53-1.36 (m, 2H). MS (ESI + ) C 27 H 29 N 14 O4 + [M+H] + Calculated value: 613.25, measured value: 613.34.

[0405] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-(2H-triazole-5-yl)benzamide)Pentanoic acid (XX23) preparation [ka]

[0406] (S)-5-(4-((S)-2-aminopropanamide)-2-(2H-triazole-5-yl)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX22) To a solution of unpurified XX34 (245 mg, 0.374 mmol) in DMF (1.8 mL), Fmoc-Ala-OH (175 mg, 0.561 mmol), HATU (213 mg, 0.561 mmol), and DIPEA (0.392 mL, 2.25 mmol) were added at room temperature under a nitrogen atmosphere. After 30 minutes, additional Fmoc-Ala-OH (23.3 mg, 0.075 mmol) and HATU (28.5 mg, 0.075 mmol) were added, and stirring was continued for 30 minutes. The reaction mixture was concentrated and suspended in MeCN (6 mL), and stirred at room temperature for 45 minutes. After filtration, the yellow solid (355 mg) was dissolved in DMF (6 mL), and piperidine (0.593 mL, 5.99 mmol) was added at room temperature. After stirring for 5 minutes, the reaction mixture was concentrated, suspended in ether (10 mL), and stirred at room temperature for 1 hour. The solid was filtered off, washed with ether, and dried under vacuum to obtain unpurified amine XX22 as a pale yellow solid (270 mg). MS (ESI + ) C 32 H 36 N 15 O6 + [M+H] + Calculated value: 726.30, measured value: 726.27.

[0407] (S)-5-(4-((S)-2-aminopropanamide)-2-(2H-triazole-5-yl)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX31) A portion of unpurified amine XX22 (169 mg) was dissolved in DMF (0.5 mL). Boc-Val-OSu (72.1 mg, 0.229 mmol) and DIPEA (0.073 mL, 0.417 mmol) were added at room temperature. After 2 hours, additional Boc-Val-OSu (10 mg, 0.032 mmol) and DIPEA (0.025 mL, 0.146 mmol) were added at room temperature, and the mixture was stirred overnight. After concentration, the residue was suspended in MeCN (275 mL) and heated under reflux. The suspension was thermally filtered and then cooled to room temperature. The solution was concentrated to approximately 15 mL and then filtered to obtain unpurified dipeptide as a cream-colored solid (59 mg). The solid was suspended in DCM (2 mL), and TFA (2 mL) was added at room temperature. After stirring for 10 minutes, the reaction product was concentrated and purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 25%). MeCN was removed from the product fraction using a rotary evaporator, and the aqueous phase was freeze-dried to obtain amine XX31 as a white solid (22.2 mg). MS (ESI + ) C 37 H 45 N 16 O7 + [M+H] + Calculated value: 825.37, measured value: 825.50.

[0408] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-(2H-triazole-5-yl)benzamide)pentanoic acid (XX23) Amine XX31 (22.2 mg, 0.021 mmol) was dissolved in THF (0.6 mL) / water (0.15 mL). Lithium hydroxide hydrate (6.19 mg, 0.148 mmol) was added at room temperature, and the mixture was stirred for 90 minutes. After cooling to 0°C, AcOH (0.014 mL, 0.253 mmol), followed by toluene (5 mL), was added, and the mixture was concentrated. The residue was redissolved in DMF (0.5 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (6.8 mg, 0.022 mmol) and DIPEA (0.015 mL, 0.084 mmol) were added at room temperature. The mixture was stirred at room temperature for 90 minutes, and then concentrated. The residue was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20 to 50%). MeCN was removed from the product fraction using a rotary evaporator, and the aqueous phase was freeze-dried to obtain triazole XX23 as a yellow solid (9.9 mg). MS (ESI + ) C 45 H 54 N 17 O9 + [M+H] + Calculated value: 976.43, measured value: 976.48.

[0409] Manufacturing of (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX41) [ka]

[0410] 5-amino-2-((trimethylsilyl)ethynyl)benzonitrile(XX39) To a suspension of iodide XX38 (5.13 g, 18.7 mmol, prepared according to Ozaki et al, Tetrahedron, 2017, 73, 7177-7184) in EtOH (40 mL), SnCl2 dihydrate (21.1 g, 93.5 mmol) was added at room temperature under a nitrogen atmosphere. After 20 minutes, the suspension changed to a dark orange-red solution. The flask was cooled in a water bath at room temperature to remove some of the heat generated over time. After stirring for 2 hours, a new suspension was formed. The reactants were then poured into a cooled (0°C) NaOH (15.0 g) aqueous solution (120 mL). The mixture was stirred for 5 minutes and then filtered. The solid was washed with cold NaOH aqueous solution (2 M) and water. After drying under vacuum, aniline (3.94 g, 86%) was obtained as a pale yellow solid.

[0411] Aniline (3.94 g, 16.2 mmol) was placed in a 50 mL three-necked flask, and CuI (61.5 mg, 0.323 mmol) and PdCl2(PPh3)2 (227 mg, 0.323 mmol) were added. The flask was purged with N2, and Et3N (17.7 mL, 127 mmol) and ethinyltrimethylsilane (2.457 mL, 17.76 mmol) were added at room temperature. The suspension was vigorously stirred for 4 hours, during which time the suspension slowly dissolved. The reaction mixture was then concentrated and purified by flash chromatography (silica gel, heptane:DCM 1:0 to 0:1). When the product fraction was concentrated, a white flake precipitated. Additional heptane (30 mL) was added at this point, and the suspension was concentrated to approximately 35 mL. The solid was filtered off and dried in a vacuum to obtain alkyne XX39 as an off-white flake (3.06 g, 76%, 2 steps). MS (ESI + ) C 12 H 15 N2Si + [M+H] + Calculated value: 215.10, measured value: 215.15.

[0412] 5-amino-2-ethynylbenzonitrile (XX40) A solution of XX39 (2.45 g, 11.43 mmol) in MeOH (50 mL) was treated with K2CO3 (3.16 g, 22.9 mmol) for 15 minutes at room temperature. The reaction product was concentrated and dissolved in SiO2 and water. The layers were separated, and the aqueous phase was extracted with SiO2 (1 ×). The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated to obtain alkyne XX40 as a cream-colored solid (1.64 g, quantified). 1 H NMR (400 MHz, CDCl3) ppm = 7.37 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.78 (dd, J = 8.5, 2.5 Hz, 1H), 4.04 (br s, 2H), 3.30 ppm (s, 1H). 13 C NMR (100 MHz, CDCl3) ppm = 147.0, 134.2, 118.4, 117.7, 117.5, 116.8, 114.9, 80.9, 80.2. MS (ESI + ) C9H7N2 + [M+H] + Calculated value: 143.06, measured value: 143.05.

[0413] (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XX41) A solution of alkyne XX40 (29.7 mg, 0.209 mmol) and azide XS21 (94 mg, 0.209 mmol) in DMF (1.7 mL) was purged with N2 for 5 minutes. CuSO4·5H2O (39.1 mg, 0.157 mmol) in water (1.1 mL) and sodium ascorbate (62.0 mg, 0.313 mmol) in water (1.1 mL) were added sequentially, and the resulting mixture was stirred at room temperature for 40 hours. Additional DMF (1 mL), followed by CuSO4·5H2O (19.54 mg, 0.078 mmol) in water (0.5 mL), and then sodium ascorbate (31.0 mg, 0.157 mmol) in water (0.5 mL) were added, and the reaction mixture was stirred at room temperature for a further 3 days. The reaction mixture was then poured into water (25 mL), and 1 mL of aqueous AcOH solution was added. The mixture was heated with a heat gun to break up the fine suspension. After cooling to room temperature, the suspension was filtered and the solid was washed with MeCN (1 mL). A portion of the solid was purified by RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 35%) to obtain acid XX41 as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 12.53 (br s, 1H), 9.09 (s, 1H), 9.00 (s, 1H), 8.67 (s, 1H), 8.50 (d, J = 7.8 H, 1H), 8.40 (br s, 1H), 8.33 (s, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.9 Hz, 1H), 7.59 (br s, 1H), 7.28 (d, J = 8.3 Hz, 2H), 6.87 (s, 1H), 6.86 (dd, J = 9.0, 2.4 Hz, 1H), 4.39 (t, J = 6.8 Hz, 2H), 4.38-4.32 (m, 1H), 3.20-3.11 (m, 4H), 1.96-1.83 (m, 2H), 1.80-1.66 (m, 2H). MS (ESI + ) C 29 H 29 N 12 O3 + [M+H] +Calculated value: 593.25, measured value: 593.49.

[0414] Manufacturing of (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX47) [ka]

[0415] (S)-5-azido-2-(((benzyloxy)carbonyl)amino)methyl pentanoate (XX43) SOCl2 (1.73 mL, 23.9 mmol) was added at 0°C to a MeOH solution (50 mL) of azide XR19 (3.49 g, 11.9 mmol). The solution was heated at 60°C for 15 minutes. After cooling to room temperature, the mixture was concentrated, and the residual solvent was evaporated with toluene (2×). The mixture was purified by flash chromatography (silica gel, heptane:siRNA 1:0 to 2:1) to obtain methyl ester XX43 as a colorless oil (3.52 g, 96%). 1 H NMR (400 MHz, CDCl3) ppm = 7.39-7.29 (m, 5H), 5.32 (br d, J = 7.3 Hz, 1 H), 5.11 (s, 2H), 4.45-4.37 (m, 1H), 3.76 (s, 3H), 3.35-3.27 (m, 2H), 2.00-1.90 (m, 1H), 1.78-1.54 (m, 3H). MS (ESI + ) C 14 H 19 N4O4 + [M+H] + Calculated value: 307.14, measured value: 307.21.

[0416] (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(((benzyloxy)carbonyl)amino)-methylpentanoate(XX44) CuSO4·5H2O (1.05 g, 4.22 mmol) in water (52 mL) was added at room temperature to a solution of azide XX43 (1.70 g, 5.55 mmol) and alkene XX40 (0.789 g, 5.55 mmol) in THF (280 mL). The solution was purged with N2 for 20 minutes, and then sodium ascorbate (1.649 g, 8.32 mmol) in water (52 mL) was added at room temperature. After stirring for 30 minutes, DMF (40.0 mL) was added, and stirring was continued at room temperature for 88 hours. A water / brine (1:1, 220 mL) mixture was added, and then the product was extracted with siRNA / heptane (1:1, 2 × 200 mL). The combined organic phase was washed with brine (2 × 100 mL), dried over MgSO4, filtered, and concentrated. Triazole XX44 was purified by flash chromatography (silica gel, heptane:toluene 1:0 to 0:1) to obtain a yellow, sticky oily substance (2.09 g, 84%). 1 H NMR (400 MHz, CDCl3) ppm = 8.18 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.38-7.28 (m, 5H), 6.98-6.93 (m, 2H), 5.40 (br d, J = 7.8 Hz, 1H), 5.11 (s, 2H), 4.47-4.40 (m, 3H), 3.98 (s, 2H), 3.74 (s, 3H), 2.10-1.89 (m, 3H), 1.76-1.66 (m, 1H). MS (ESI + ) C 23 H 25 N6O4 + [M+H] + Calculated value: 449.19, measured value: 449.31.

[0417] (S)-2-amino-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)methyl pentanoate (XX45) Triazole XX44 (1.12 g, 2.497 mmol) and Pd / C (10% palladium on activated carbon, 0.133 g, 0.125 mmol) were reacted in siRNA (22 mL) under a hydrogen atmosphere at room temperature. After 16 hours, the flask was purged with N2, and additional Pd / C (10% palladium on activated carbon, 0.200 g, 0.188 mmol) was added at room temperature. Hydrogen gas was added again, and the mixture was stirred at room temperature for a further 4.5 hours. The flask was purged with N2, and the mixture was filtered through Celite®. Then, HCl (4.0 N, 2.0 mL) in dioxane was added to the filtrate. The volume of the filtrate was reduced to approximately 50 mL using a rotary evaporator, and the suspension was then filtered, and the solid was washed with siRNA and ether. The white solid was dried in a vacuum to obtain amine XX45 as its dihydrochloride salt (849 mg, 88%). 1 H NMR (400 MHz, methanol-d4) ppm = 8.66 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.71 (dd, J = 8.5, 2.4 Hz, 1H), 4.63 (t, J = 6.6 Hz, 2H), 4.16 (t, J = 6.4 Hz, 1H), 3.87 (s, 3H), 2.27-1.91 (m, 4H). MS (ESI + ) C 15 H 19 N6O2 + [M+H] + Calculated value: 315.16, measured value: 315.25.

[0418] (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX46) Amine XX45 dihydrochloride (498 mg, 1.29 mmol) and amine XT48 (397 mg, 1.17 mmol) in DMF (10 mL) were reacted according to general procedure XXA. After concentration, the crude product was stirred in MeCN (10 mL) at room temperature for 3 days and then filtered. The solid was washed with MeCN and ether and finally dried under vacuum to obtain amide XX46 as an orange solid (688 mg, 93%). MS (ESI + ) C 30 H 30 N 13 O4 + [M+H] + Calculated value: 636.25, measured value: 636.45.

[0419] (S)-5-(4-(4-amino-2-cyanophenyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XX47) Ester XX46 (330 mg, 0.519 mmol) was hydrolyzed according to the general procedure XXC. A portion of the crude product was purified by RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 30%) to obtain acid XX47 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) ppm = 12.42 (br s, 1H), 9.22 (s, 1H), 9.18 (s, 1H), 8.77 (s, 1H), 8.51 (br s, 1H), 8.32 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.81 (br s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.9 Hz, 1H), 6.87 (s, 1H), 6.88 (dd, J = 9.0, 2.4 Hz, 1H), 6.84 (br s, 1H), 6.67 (d, J = 8.8 Hz, 2H), 4.55 (s, 2H), 4.39 (t, J = 6.9 Hz, 2H), 4.36-4.30 (m, 1H), 1.96-1.81 (m, 2H), 1.79-1.65 (m, 2H). MS (ESI + ) C 28 H 28 N 13 O3 + [M+H] + Calculated value: 594.24, measured value: 594.39.

[0420] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-hydroxybenzamide)Pentanoic acid (XX28) preparation [ka]

[0421] (S)-4-(2-aminopropanamide)-2-(benzyloxy)benzylbenzoate(XX25) To a stirred solution of 4-amino-2-hydroxybenzoic acid (XX24; 5.00 g, 32.7 mmol) in DMF (150 mL), KOtBu (4.03 g, 35.9 mmol) was added at 0°C. After 15 minutes, BnBr (4.27 mL, 35.9 mmol) was added dropwise, and the suspension was stirred at room temperature for a further 4 hours, after which the reaction vessel was cooled again to 0°C. At this point, additional KOtBu (4.03 g, 35.9 mmol) and BnBr (4.27 mL, 35.9 mmol) were added. The reaction mixture was stirred overnight, followed by quenching with water and extraction with SiO(3×). The combined organic phases were diluted 2:1 with heptane, then washed with water(2×) and brine, dried over Na₂SO₄, and concentrated using silica gel. The benzyl ester was obtained by purification using flash chromatography (silica gel, heptane, 0-60% acetyl) (5.30 g).

[0422] This product was reacted with Fmoc-Ala-Cl in the same manner as in the case of XT11. After concentrating the reaction mixture, the crude product was dissolved in HCl and washed with HCl (0.1 M) until the aqueous phase became acidic. The combined aqueous phase was back-extracted with HCl, and the combined organic phase was washed with saturated NaHCO3 aqueous solution and brine, dried over MgSO4, filtered, and concentrated to obtain a pale yellow, sticky, gum-like substance (11.08 g). MS (ESI + ) C 39 H 34 N2NaO6[M+Na] + 649.23, measured value 649.44.

[0423] The crude product was dissolved in DMF (122 mL), and piperidine (49 mL) was added at room temperature. After 3 minutes, the reaction mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, MeOH in 0-20% DCM) to obtain amine XX25 (4.45 g, 34%, 3 steps). 1 H NMR (400 MHz, CDCl3) ppm = 9.73 (br s , 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 1.6 Hz, 1H), 7.48 (d, J = 7.5 Hz, 2H), 7.41-7.37 (m, 2H), 7.36-7.28 (m, 6H), 6.90 (dd, J = 8.5, 1.8 Hz, 1H), 5.33 (s, 2H), 5.19 (s, 2H), 3.63 (q, J = 7.0 Hz, 1H), 1.65 (br s, 2H), 1.44 (d, J = 7.0 Hz, 3H). MS (ESI + ) C 24 H 25 N2O4[M+H] + Calculated value: 405.18, measured value: 405.29.

[0424] 2-(benzyloxy)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)benzoic acid (XX26) DIPEA (4.81 mL, 27.5 mmol) and Boc-Val-OSu (3.81 g, 12.1 mmol) were added to a solution of amine XX25 (4.45 g, 11.0 mmol) in DMF (50 mL) at room temperature. The mixture was stirred for 5 hours and then poured into water. The mixture was extracted with SiO2 / heptane (1:1, 3×). The combined organic phase was washed with water (2×) and brine, dried over MgSO4, filtered, and concentrated. The dipeptide was purified by flash chromatography (0-100% SiO2 in heptane) (6.57 g).

[0425] The crude product was dissolved in THF (39 mL) / MeOH (8 mL), and lithium hydroxide hydrate (1.69 g, 40.3 mmol) in water (15.7 mL) was added at room temperature. The mixture was stirred for 19 hours, after which the organic matter was removed using a rotary evaporator. Water (300 mL) was added, the mixture was filtered, washed with water (100 mL), and the solid was washed with ether (2 × 150 mL). The aqueous phase was washed separately using these two ether fractions. Depositphotos (250 mL) was added to the aqueous phase, followed by HCl (1.0 M, 41 mL), and then the solid. After mixing, the product was dissolved in the organic phase. The aqueous phase was washed with Depositphotos (2 × 100 mL), the combined organic phase containing the ether fraction was washed with brine, dried over Na₂SO₄, filtered, and concentrated. A white solid was formed, which was suspended in approximately 1:1 siRNA / heptane (approximately 20 mL) and filtered to obtain acid XX26 as a white solid (3.78 g, 67%, 2 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.35 (br s, 1H), 10.23 (s, 1H), 8.12 (d, J = 6.7 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 1.2 Hz, 1H), 7.52 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.34-7.30 (m, 1H), 7.23 (dd, J = 8.5, 1.4 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 5.14 (s, 2H), 4.42 (quint, J = 7.0 Hz, 1H), 3.85 (t, J = 7.8 Hz, 1H), 2.00-1.90 (m, 1H), 1.39 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 27 H 36 N3O7[M+H] + Calculated value: 514.26, measured value: 514.61.

[0426] (S)-5-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-hydroxybenzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XX27) Acid XX26 (421 mg, 0.820 mmol) was reacted with HATU (374 mg, 0.983 mmol) and XT48 (413 mg, 0.820 mmol) according to general procedure XXA. The mixture was purified by flash chromatography (silica gel, DCM:MeOH 1:0 to 4:1) to obtain a yellow solid, which was suspended in hot MeCN (6 mL). After cooling to room temperature, the solid was filtered off and stirred in water for 10 minutes. After filtration and drying, unpurified benzyl ether (200 mg, 0.208 mmol) was reacted with Pd(OH)2 / C (20%, 100 mg on activated carbon) in AcOH (2.0 mL) under a hydrogen atmosphere at room temperature for 2 hours and 20 minutes. The solution was then purged with N2 and filtered through Celite®. After concentration, the solvent in the material was evaporated with toluene. The solid was dissolved in DMF (1 mL) / DCM (3 mL), and MnO2 (300 mg) was added at room temperature. After stirring for 3 hours, the reaction product was filtered and concentrated. Purification by flash chromatography (silica gel, 0-20% MeOH in DCM) yielded phenol XX27 (79 mg, 11%, 2 steps). MS (ESI + ) C 41 H 53 N 12 O 10 [M+H] + Calculated value: 873.40, measured value: 873.90.

[0427] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)-2-hydroxybenzamide)pentanoic acid (XX28) Phenol XX27 (79 mg, 0.090 mmol) was reacted with NaOH according to the general procedure XXC to obtain an acid (75 mg) as a green / gray solid. The material was suspended in DCM (2.0 mL) and TFA (2.0 mL) was added at 0°C. After 30 minutes, the reaction product was concentrated. The brown oily substance was dissolved in DMF (3 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (29.1 mg, 0.094 mmol) and DIPEA (0.063 mL, 0.360 mmol) were added at room temperature. Additional DIPEA (0.031 mL, 0.180 mmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (5.55 mg, 0.018 mmol) were added after 20 minutes and 90 minutes, respectively, and the reaction was stopped after 2 hours. The mixture was concentrated, and the residue was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The organic solvent was removed using a rotary evaporator, and the aqueous solution was then freeze-dried. The solid was purified again by preparative RP-HPLC (water × 0.1% TFA / MeOH:MeCN (1:1) × 0.1% TFA, gradient 20% to 50%) using a different eluent. The organic solvent was removed using a rotary evaporator, and the aqueous solution was then freeze-dried to obtain phenol XX28 as a yellow solid (3.1 mg). MS (ESI + ) C 44 H 54 N 13 O 10 [M+H] + Calculated value: 924.41, measured value: 924.91.

[0428] Preparation of (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminoquinazoline-6-yl)ethyl)benzamide)pentanoic acid (XJ4) [ka]

[0429] (S)-5-(4-azidobenzamide)-2-(4-(2-(2,4-diaminoquinazoline-6-yl)ethyl)benzamide)methyl pentanoate (XJ2) Acid XJ1 (514 mg, 1.67 mmol, synthesized as described in U.S. Patent Application Publication No. 2005 / 0020833 and U.S. Patent Application Publication No. 2009 / 0253719) was reacted with amine XX3 (534 mg, 1.83 mmol) according to general procedure XXA. Purification by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) yielded ester XJ2 as a yellow solid (474 ​​mg, 49%). MS (ESI + ) C 30 H 32 N9O4 + [M+H] + Calculated value: 582.25, measured value: 582.62.

[0430] (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminoquinazolin-6-yl)ethyl)benzamide)methyl pentanoate (XJ3) Azide XJ2 (474 ​​mg, 0.815 mmol) was reduced according to the general procedure XXB, and aniline XJ3 was obtained as a yellow solid (453 mg, quantified). MS (ESI + ) C 30 H 34 N7O4 + [M+H] + Calculated value: 556.26, measured value: 556.62.

[0431] (S)-5-(4-aminobenzamide)-2-(4-(2-(2,4-diaminoquinazolin-6-yl)ethyl)benzamide)pentanoic acid (XJ4) Ester XJ3 (45 mg, 0.081 mmol) was hydrolyzed according to the general procedure XXC to obtain acid XJ4 as a yellow solid (9 mg, 20%). 1H NMR (400 MHz, DMSO-d6) ppm = 8.40 (br d, J = 7.5 Hz, 1H), 8.00 (br t, J = 5.4 Hz, 1H), 7,91 (s, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.55 (br s, 2H), 7.43 (br d, J = 8.7 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.5 Hz, 1H), 6.52 (d, J = 8.5 Hz, 2H), 6.51 (br s, 2H), 5.56 (br s, 2H), 4.35-4.29 (m, 1H), 3.24-3.19 (m, 2H), 3.00-2.93 (m, 4H), 1.88-1.71 (m, 2H), 1.62-1.53 ​​(m, 2H). MS (ESI + ) C 29 H 32 N7O4 + [M+H] + Calculated value: 542.24, measured value: 542.62.

[0432] (S)-2-(4-(2-(2,4-diaminoquinazoline-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide) preparation of pentanoic acid (XJ9) [ka]

[0433] (S)-5-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)benzamide)-2-(4-(2-(2,4-diaminoquinazoline-6-yl)ethyl)benzamide)methyl pentanoate (XJ6) Aniline XJ3 (405 mg, 0.729 mmol) and Fmoc-Ala-Cl (481 mg, 1.45 mmol) were reacted in the same manner as in the case of XT11 to obtain amide XJ5 (quantified) as a yellow solid. MS (ESI + ) C 48 H 49 N8O7 + [M+H]+ Calculated value: 849.36, measured value: 849.49.

[0434] Amine XJ5 (619 mg, 0.729 mmol) protected with Fmoc was deprotected using TBAF·3H2O and decanethiol, and subsequently reacted with Boc-Val-OSu, similar to the conversion of XT11 to XT12. The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain dipeptide XJ6 containing the tetrabutylamine salt (317 mg, 52%). MS (ESI + ) C 43 H 56 N9O8 + [M+H] + Calculated value: 826.42, measured value: 826.83.

[0435] (S)-5-(4-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)benzamide)-2-(4-(2-(2,4-diaminoquinazoline-6-yl)ethyl)benzamide)pentanoic acid (XJ8) Dipeptide XJ6 (310 mg, 0.375 mmol) was deprotected in the same manner as for XT13. The crude product (272 mg) was hydrolyzed with NaOH according to the general procedure XXC to obtain XJ8 as an off-white solid (84 mg, 32%). MS (ESI + ) C 37 H 46 N9O6 + [M+H] + Calculated value: 712.35, measured value: 712.80.

[0436] (S)-2-(4-(2-(2,4-diaminoquinazolin-6-yl)ethyl)benzamide)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)pentanoic acid (XJ9) Amine XJ8 (76 mg, 0.107 mmol) was reacted with 6-maleimidohexanoic acid N-hydroxysuccinimide ester (32.9 mg, 0.107 mmol) and DIPEA (0.112 mL, 0.64 mmol) in the same manner as with XT17. The crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 95%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain XJ9 as a white solid (21.6 mg, 22%). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.36 (br s, 1H), 10.10 (br s, 1H), 8.88 (br s, 1H), 8.75 (br s, 1H), 8.54 (d, J = 7.7 Hz, 1H), 8.35 (t, J = 5.6 Hz, 1H), 8.18 (d, J = 6.8 Hz, 1H), 8.13 (d, J = 1.0 Hz, 1H), 7.82-7.78 (m, 7H), 7.68-7.64 (m, 3H), 7.35 (d, J = 8.4 Hz, 1H), 7.31 (br d, J = 8.3 Hz, 2H), 6.99 (s, 2H), 4.42-4.35 (m, 2H), 4.17 (dd, J = 8.5, 7.0 Hz, 1H), 3.29-3.25 (m, 4H), 3.01 (br s, 4H), 2.22-2.07 (m, 2H), 2.00-1,89 (m, 1H), 1.89-1.74 (m, 2H), 1.69-1.58 (m, 2H), 1.51-1.44 (m, 4H), 1.31 (d, J = 7.1 Hz, 3H), 1.22-1.14 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 47 H 57 N 10 O9 + [M+H] + Calculated value: 905.42, measured value: 905.94.

[0437] Manufacturing of (S)-5-amino)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XJ11) and (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecanamide)benzyl)oxy)carbonyl)amino)pentanoic acid (XJ13) [ka]

[0438] (S)-5-amino-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XJ11) Formamide XT47 (270 mg, 0.476 mmol) was hydrolyzed with NaOH (12 equivalents) according to the general procedure XXC. The product (178 mg) was then deprotected using TFA as in the case of XT13. A portion of the crude product (40 mg) was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 5% to 35%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain XJ11 as a yellow solid (6.7 mg). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.6 (br s, 1H), 9.19 (br s, 1H), 8.86 (s, 1H), 8.45 (br s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.90 (br s, 1H),7.78-7.73 (m, 4H), 6.95 (br s, 1H), 6.79 (d, J = 8.7 Hz, 2H), 4.65 (s, 2H), 4.44-4.38 (m, 1H), 2.88-2.79 (m, 2H), 1.95-1.88 (m, 2H), 1.84-1.76 (m, 2H), 1.71-1.61 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 174.2, 166.7, 163.3, 151.0, 129.5, 122.0, 121.9, 111.8, 62.4, 52.2, 46.0, 28.1, 25.9, 24.6. MS (ESI + ) C 19 H24 N9O3 + [M+H] + Calculated value: 426.19, measured value: 426.31.

[0439] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecanamide)benzyl)oxy)carbonyl)amino)pentanoic acid (XJ13) Amines XJ11 (234 mg, 0.358 mmol) and XJ12 (271 mg, 0.358 mmol) were reacted in DMF (4 mL) at 0°C for 1.5 hours in the presence of Et3N (0.150 mL, 1.07 mmol) (see Elgersma et al, Mol. Pharm. 2015, 12, 1813-35), during which time the temperature was gradually increased to room temperature. After concentration, the crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 25% to 75%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain XJ13 as a yellow solid (99 mg, 27%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.1, (br s, 1H), 12.49 (br s, 1H), 10.02 (s, 1H), 9.35 (s, 1H), 9.31 (s, 1H), 8.84 (s, 1H), 8.63 (br s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 7.3 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.21-7.17 (m, 2H), 7.02 (s, 2H), 6.75 (m, 3H), 6.00 (br s, 2H), 4.93 (s, 2H), 4.62 (s, 2H), 4.43-4.39 (m, 1H), 4.34-4.28 (m, 1H), 4.06-3.97 (m, 2H), 3.90-3.87 (m, 1H), 3.57-2.52 (m, 6H), 3.03-2.93 (m, 4H), 2.00-1.91 (m, 1H), 1.83-1.78 (m, 1H), 1.73-1.66 (m, 1H), 1.63-1.34 (m, 5H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H). MS (ESI + C 47 H 60 N 15 O 13 + [M+H] + The calculated value is 1042.44 and the measured value is 1042.92.

[0440] Preparation of (S)-2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)pentanamide)-N1,N5-di(propa-2-in-1-yl)pentanediamide (XS2)

change

[0441] (S)-(1,5-ジオキソ-1,5-ビス(プロパ-2-イン-1-イ(XS1) To a suspension of Boc-Glu-OH (769 mg, 3.11 mmol) and propargylamine (438 μL, 6.85 mmol) in DCM (15 mL), Et3N (1.74 mL, 12.5 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.49 g, 7.78 mmol) were added at 0°C. The reaction mixture was allowed to cool to room temperature and stirred overnight. The reaction mixture was concentrated, dissolved in HCl (30 mL), and washed with water (30 mL). The aqueous phase was extracted with HCl (2 × 30 mL), and the combined organic phase was washed with saturated NaHCO3 aqueous solution (2 × 30 mL) and brine (30 mL), dried over Na2SO4, and concentrated. In UPLC-MS, the aqueous phase still containing the product was extracted with DCM (2 × 20 mL), the combined DCM phase was washed with brine (30 mL), dried over Na₂SO₄, and combined with the crude product from the siRNA extraction step, was evaporated under reduced pressure. The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain dialkyne XS1 as a pale yellow solid (440 mg, 44%). 1 H NMR (400 MHz, CDCl3) ppm = 7.05 (br s, 1H), 6.40 (br s, 1H), 5.67 (d, J = 7.4 Hz, 1H), 4.25-4.13 (m, 1H), 4.12-3.99 (m, 4H), 2.45-2.29 (m, 2H), 2.24 (dt, J = 7.5, 2.6 Hz, 2H), 2.16-2.05 (m, 1H), 2.05-1.94 (m, 1H), 1.44 (s, 9H). 13 C NMR (100 MHz, CDCl3) ppm = 172.4, 171.4, 79.4, 79.3, 71.7, 53.5, 32.4, 29.3, 29.1, 28.3. MS (ESI + ) C 16 H 24 N3O4 + [M+H] + Calculated value: 322.18, measured value: 322.44.

[0442] (S)-2-(5-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)pentanamide)-N1,N5-di(propa-2-in-1-yl)pentanediamide(XS2) Dialkyne XS1 (430 mg, 1.34 mmol) was dissolved in DCM (5.0 mL) and cooled to 0°C. TFA (5.0 mL) was added, and the reaction mixture was allowed to rise to room temperature and stirred for 1 hour. The solvent in the reaction mixture was evaporated with toluene (5.0 mL) and toluene:DCM (6 mL, 5:1), and the mixture was dried under high vacuum to obtain the amine as a reddish-brown oil. The material was dissolved in DMF (10 mL), and then 6-maleimidohexanoic acid N-hydroxysuccinimide ester (495 mg, 1.61 mmol) and DIPEA (1.40 mL, 8.03 mmol) were added at room temperature. After 18 hours, the reaction mixture was concentrated, and the solvent was evaporated with DCM:toluene (15 mL, 2:1). The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain maleimide XS2 as a white solid (307 mg, 55%, 2 steps). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.30 (t, J = 5.5 Hz, 1H), 8.24 (t, J = 5.8 Hz, 1H), 7.96-7.88 (m, 1H), 7.00 (s, 2H), 4.23-4.14 (m, 1H), 3.83 (td, J = 2.8, 2.4 Hz, 4H), 3.41-3.35 (m, 2H), 3.08 (dt, J = 7.1, 2.5 Hz, 2H), 2.16-2.01 (m, 4H), 1.90-1.79 (m, 1H), 1.74-1.62 (m, 1H), 1.48 (quint, J = 7.4 Hz, 4H), 1.27-1.13 (m, 2H). 13C NMR (100 MHz, DMSO-d6) ppm = 172.6, 171.7, 171.6, 171.6, 134.9, 81.7, 81.5, 73.4, 73.3, 52.4, 37.5, 35.4, 32.0, 28.4, 28.3, 28.2, 26.3, 25.7, 25.1. MS (ESI + ) C 21 H 27 N4O5 + [M+H] + Calculated value: 415.20, measured value: 415.34.

[0443] (S)-5-(2-chloro-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)Pentanoic acid (XS6) preparation [ka]

[0444] (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-2-methyl chlorobenzoate (XS3) Methyl 4-amino-2-chlorobenzoate (1.00 g, 5.39 mmol) and Fmoc-Ala-Cl were reacted in the same manner as in the case of XT11. The quenched reaction mixture was concentrated, the solvent was evaporated with toluene (20 mL), and the mixture was partially dissolved in toluene (40 mL) and extracted with 0.1 M hydrochloric acid (2 × 15 mL). The aqueous phase was extracted with toluene (20 mL), and the combined organic phase was washed with aqueous NaHCO3 solution (2 × 15 mL) and brine (2 × 15 mL), dried over MgSO4, filtered, and concentrated to obtain amide XS3 as a pale yellow solid (2.63 g, quantitative analysis, 2 steps). MS (ESI + ) C 26 H 24 ClN2O5 + [M+H] + Calculated value: 479.14, measured value: 479.30.

[0445] 4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-methylchlorobenzoate (XS4) Carbamate XS3 (2.63 g, 5.49 mmol) was mixed with piperidine (39.0 mL, 395 mmol), and the resulting solution was stirred for 15 minutes. The reaction mixture was concentrated and purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain a deprotected product as a light brown oily substance (1.17 g, 83%). A portion of the material (1.15 g, 4.48 mmol) was dissolved in DMF (20 mL). Then, DIPEA (1.96 mL, 11.2 mmol) and Boc-Val-OSu (1.55 g, 4.93 mmol) were added at room temperature, and the resulting solution was stirred at room temperature for 3 hours. The reaction mixture was poured into water (240 mL), and the aqueous phase was extracted with toluene:heptane (1:1, 3 × 80 mL). The combined organic phases were washed with water (100 mL) and brine (2 × 50 mL), dried over MgSO4, filtered, and concentrated. The crude product was purified by flash chromatography (silica gel, Â1:Heptane 0:1 to 1:1) to obtain amide XS4 as an off-white solid (1.51 g, 74%). 1 H NMR (400 MHz, CDCl3) ppm = 9.16 (br s, 1H), 7.86-7.80 (m, 2H), 7.73-7.55 (m, 1H), 6.65 (d, J = 6.3 Hz, 1H), 4.98 (d, J = 6.1 Hz, 1H), 4.67 (quint, J = 7.2 Hz, 1H), 3.96 (t, J = 5.5 Hz, 1H), 3.90 (s, 3H), 2.27-2.17 (m, 1H), 1.49-1.46 (m, 3H), 1.44 (s, 9H), 1.01 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) ppm = 172.1, 170.5, 165.5, 156.5, 142.0, 134.9, 132.6, 124.4, 121.6, 117.2, 81.2, 60.8, 52.2, 49.8, 30.2, 28.3, 24.8, 19.3, 17.7, 17.3. MS (ESI + ) C 21 H 31 ClN3O6 + [M+H] + Calculated value: 456.19, measured value: 456.56.

[0446] (S)-5-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-chlorobenzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XS5) Ester XS4 (0.750 g, 1.65 mmol) was suspended in dioxane (17 mL) and water (10 mL). LiOH aqueous solution (2.0 M, 3.29 mL, 6.58 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. An additional 10 mL of dioxane was added, and stirring continued for 1 hour. Water (25 mL) was added, and 1 M hydrochloric acid was added to the reaction mixture to adjust the pH to 3. The product was extracted with SiO2 (3 × 35 mL), the combined organic phase was washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and concentrated. Unpurified acid (0.125 g, 0.284 mmol) and amine XT48 (0.146 g, 0.270 mmol) were reacted according to general procedure XXA. The amide XS5 was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain a yellow, sticky solid (0.159 g, 66%, 2 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 10.30 (s, 1H), 8.80 (s, 1H), 8.74 (d, J = 7.5 Hz, 1H), 8.66 (s, 1H), 8.61-8.58 (m, 1H), 8.44-8.36 (m, 1H), 8.36-8.29 (m, 1H), 8.16 (d, J = 6.8 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.86-7.76 (m, 1H), 7.74-7.62 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.42-7.34 (m, 2H), 6.72 (d, J = 8.8 Hz, 1H), 6.68-6.63 (m, 1H), 5.25-5.18 (m, 2H), 4.46-4.36 (m, 2H), 3.90-3.78 (m, 1H), 3.63 (s, 3H), 3.27-3.19 (m, 2H), 3.15-3.07 (m, 2H), 1.96-1.78 (m, 2H), 1.68-1.49 (m, 2H), 1.38 (s, 9H), 1.31 (d, J = 7.0 Hz, 3H), 1.26 (d, J = 7.1 Hz, 3H). MS (ESI + ) C 41 H 52 ClN 12 O9 + [M+H] + Calculated value: 891.37, measured value: 891.83.

[0447] (S)-5-(2-chloro-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XS6) To a suspension of methyl ester XS5 (0.159 g, 0.178 mmol) in DMSO (0.16 mL) and MeOH (0.80 mL), NaOH (2.0 M, 0.535 mL, 1.07 mmol) was added dropwise at 10°C. The reaction mixture was brought to room temperature and stirred for 15 minutes. Additional NaOH (2.0 M, 0.535 mL, 1.07 mmol) was added, and after 2 hours, the reaction mixture was diluted with DMSO (0.32 mL) and MeOH (1.6 mL). The reaction mixture was stirred for 2 hours, the final NaOH (2.0 M, 0.535 mL, 1.07 mmol) was added, and the mixture was stirred for 4 hours. AcOH (1.0 M, 6.0 mL) was then added to the product to precipitate it, filtered, and washed with water (2.0 mL), MeCN (2.0 mL), and Et2O (2.0 mL). The unpurified material was suspended in DCM (5.0 mL) and the mixture was cooled to 0°C. TFA (5.0 mL) was added dropwise, and the reaction mixture was stirred for 30 minutes. The reaction mixture was concentrated, and the solvent was subsequently evaporated with toluene (10 mL) to obtain the unpurified amine as a yellow oil. The material was dissolved in DMF (6.0 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (55.1 mg, 0.179 mmol) and DIPEA (0.125 mL, 0.715 mmol) were added at 0°C. After 15 minutes, an additional DIPEA (0.125 mL, 0.715 mmol) was added, followed by 6-maleimidohexanoic acid N-hydroxysuccinimide ester (11.0 mg, 0.036 mmol) after 2.5 hours. After a total reaction time of 3.5 hours, the reaction mixture was concentrated, and the solvent was subsequently evaporated with toluene (5 mL). The crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain maleimide XS6 as a yellow solid (22.3 mg, 13%, 3 steps). MS (ESI + ) C 44 H 53 ClN 13 O9 + [M+H] + Calculated value: 942.38, measured value: 942.90.

[0448] Preparation of 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)-2-fluorobenzamide)butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XS12) [ka]

[0449] 4-(((2,4-diaminopteridine-6-yl)methyl)amino)-2-fluorobenzoic acid (XS7) Bromine (0.804 mL, 15.6 mmol) was added dropwise to a suspension of PPh3 (4.09 g, 15.6 mmol) in DMA (14 mL) over 10 minutes at 0°C. After stirring for 30 minutes, (2,4-diaminopteridine-6-yl)methanol (1.00 g, 5.20 mmol, prepared as described in XX9) was added, and the resulting mixture was stirred at room temperature for 18 hours. Finally, 4-amino-2-fluorobenzoic acid (1.07 g, 6.90 mmol) and BaO (1.11 g, 6.50 mmol) were added, and the suspension was heated at 55°C for 24 hours. After cooling to room temperature, the reaction mixture was poured into DCM:MeOH (150 mL, 29:1), and the solid was filtered off. The residue was stirred in water (50 mL), filtered, stirred in hot MeCN (60 mL), cooled to room temperature, and filtered again to obtain aniline XS7 (1.30 g, 76%) as a brown solid. MS (ESI + ) C 14 H 13 FN7O2 + [M+H] + Calculated value: 330.11, measured value: 330.35.

[0450] 4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-2-fluorobenzoic acid (XS8) A solution of acetic anhydride (11.1 mL, 118 mmol) in formic acid (48.3 mL, 1.26 mol) was stirred at room temperature for 40 minutes, and then aniline XS7 (1.30 g, 3.94 mmol) was added. The resulting suspension was stirred at reflux temperature for 2.5 hours. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (88 mL) / NH4OH aqueous solution (35%, 12.6 mL) and then heated at 70°C for 30 minutes. The mixture was filtered, and acetic acid was added to the filtrate to adjust the pH to approximately 5.5. The precipitate was filtered off and subsequently stirred in water / AcOH (55 mL, 10:1) at 50°C for 45 minutes. After filtration, the solid was washed with water (2 × 10 mL), EtOH (2 × 5 mL) and Et2O (2 × 5 mL), and dried under vacuum to obtain formamide XS8 as a light brown powder (0.788 g, 55%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.90 (s, 1H), 8.69 (s, 1H), 7.86 (t, J = 8.5 Hz, 1H), 7.72 (br s, 1H), 7.55 (dd, J = 12.6, 2.0 Hz, 1H), 7.47-7.36 (m, 2H), 6.75 (br s, 2H), 5.23 (s, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 165.1, 163.2, 163.1, 160.8, 155.3, 150.0, 146.8, 143.8, 133.3, 121.6, 117.3, 116.3, 110.2, 109.9, 46.5. MS (ESI + ) C 15 H 13 FN7O3 + [M+H] + Calculated value: 358.11, measured value: 358.38.

[0451] (S)-5-(5-amino-1,3-dioxoisoindolin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-2-fluorobenzamide)methyl pentanoate (XS9) Acid XS8 (760 mg, 2.13 mmol) and amine XT6 (871 mg, 2.66 mmol) were reacted according to general procedure XXA. The amide XS9 was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:3) to obtain a yellow solid (406 mg, 30%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.83 (s, 1H), 8.69 (br s, 1H), 8.59 (d, J = 7.3 Hz, 2H), 7.72 (br s, 1H), 7.58 (s, 1H), 7.57-7.53 (m, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.42 (dd, J = 8.4, 1.8Hz, 1H), 6.91 (d, J = 2.0 Hz, 1H), 6.78 (dd, J = 8.3, 2.0 Hz, 2H), 6.70 (br s, 1H), 6.45 (br s, 2H), 5.23 (s, 2H), 4.44-4.37 (m, 1H), 3.62 (s, 3H), 3.50 (t, J = 6.3 Hz, 2H), 1.80-1.68 (m, 2H), 1.66-1.57 (m, 2H). MS (ESI + ) C 29 H 28 FN 10 O6 + [M+H] + Calculated value: 631.22, measured value: 631.66.

[0452] (S)-5-(5-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-1,3-Dioxoisoindoline-2-yl)-2-(4-(N-((2,4-Diaminopteridine-6-yl)methyl)formamide)-2-Fluorobenzamide)Methyl pentanoate (XS10) Aniline XS9 (315 mg, 0.500 mmol) and Fmoc-Ala-Cl were reacted in the same manner as for XT11. The quenched reaction mixture was concentrated, and the solvent was evaporated with MeOH:toluene (30 mL, 1:2). The material was then purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain amide XS10 as a yellow solid (0.352 g, 76%). 1 H NMR (400 MHz, DMSO-d6) ppm = 10.68 (s, 1H), 8.83 (s, 1H), 8.66 (s, 1H), 8.59 (d, J = 7.1 Hz, 1H), 8.20 (s, 1H), 7.93-7.88 (m, 3H), 7.81 (d, J = 8.3 Hz, 2H), 7.74 (t, J = 7.1 Hz, 2H), 7.70-7.64 (m, 1H), 7.60-7.57 (m, 1H), 7.56-7.53 (m, 1H), 7.45-7.37 (m, 4H), 7.37-7.31 (m, 2H), 6.67 (br s, 2H), 5.22 (s, 2H), 4.46-4.37 (m, 1H), 4.34-4.27 (m, 3H), 4.23 (t, J = 6.8 Hz, 1H), 3.62 (s, 3H), 3.59-3.54 (m, 2H), 1.83-1.72 (m, 2H), 1.72-1.61 (m, J = 8.5 Hz, 2H), 1.35 (d, J = 7.1 Hz, 3H). MS (ESI + ) C 47 H 43 FN 11 O9 + [M+H] + Calculated value: 924.32, measured value: 924.86.

[0453] (S)-5-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-1,3-dioxoisoindorin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)-2-fluorobenzamide)methyl pentanoate (XS11) Amine XS10 (0.150 g, 0.162 mmol) protected with Fmoc was deprotected using TBAF·3H2O and decanethiol, and subsequently reacted with Boc-Val-OSu, similar to the conversion of XT11 to XT12. The amide XS11 was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4) to obtain a yellow film (85 mg, 46%, 2 steps). MS (ESI + ) C 42 H 50 FN 12 O 10 + [M+H] + Calculated value: 901.38, measured value: 901.90.

[0454] 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)-2-fluorobenzamide)butyl)carbamoyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XS12) To a suspension of carbamate XS11 (85 mg, 0.094 mmol) in DCM (3.0 mL), TFA (3.0 mL) was added dropwise at 0°C. The reaction mixture was allowed to cool to room temperature and stirred for 1 hour. The solution was concentrated, and the solvent was evaporated with DCM:toluene (7 mL, 2:5) and toluene (5 mL) to obtain the unpurified amine as a dark yellow film. The material was dissolved in MeOH / DMSO (0.60 mL, 5:1), and aqueous NaOH solution (2.0 M, 0.285 mL, 0.569 mmol) was added dropwise at 10°C. The mixture was removed from the cold bath and stirred at room temperature for 1 hour. Additional aqueous NaOH solution (0.285 mL, 0.569 mmol) was added, and the mixture was stirred for 5 hours. The reaction mixture was cooled to 0°C, and aqueous AcOH solution (1.0 M, 1.0 mL), followed by water (2.0 mL), was added. The solid was filtered off, then washed with water (2 mL) and MeCN (2 mL), and dissolved in DMF (15 mL). Next, 6-maleimidehexanoic acid N-hydroxysuccinimide ester (0.035 g, 0.11 mmol) and DIPEA (0.100 mL, 0.572 mmol) were added to this solution at room temperature. The reaction was stirred for 4 hours, then the reaction was concentrated, the solvent was evaporated with DCM:toluene (10 mL, 1:4), and the mixture was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain maleimide XS12 as a pale yellow solid (19.6 mg, 21%, 3 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 13.50-12.42 (m, 2H), 9.29 (br s, 2H), 8.84 (s, 1H), 8.59 (br s, 1H), 8.27-8.18 (m, 2H), 7.97 (d, J = 2.1 Hz, 1H), 7.86-7.65 (m, 4H), 7.63-7.51 (m, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.16 (br s, 1H), 7.00 (s, 2H), 6.63 (dd, J = 2.0, 8.6 Hz, 1H), 6.53 (dd, J = 1.8, 14.4 Hz, 1H), 4.62 (br s, 2H), 4.43-4.32 (m, 2H), 4.20-4.13 (m, 1H), 3.37-3.33 (m, 2H), 3.21-3.14 (m, 2H), 2.23-2.06 (m, 2H), 2.01-1.89 (m, 2H), 1.88 (br s, 1H), 1.84-1.71 (m, 1H), 1.64-1.53 ​​(m, 2H), 1.53-1.43 (m, 4H), 1.33-1.30 (m, 2H), 1.24-1.13 (m, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 45 H 53 FN 13 O 11 + [M+H] + Calculated value: 970.40, measured value: 971.00.

[0455] Preparation of (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-hydroxybenzamide)pentanoic acid (XS14) [ka]

[0456] (S)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-(4-hydroxybenzamide)methyl pentanoate (XS13) Amine XT48 (97 mg, 0.18 mmol) and 4-hydroxybenzoic acid were reacted according to general procedure XXA. After concentrating the reaction mixture, the solvent in the crude product was evaporated with DCM / toluene (4 mL, 1:1), toluene (5 mL), and MeCN (2 × 2 mL). The residue was stirred in MeOH / MeCN (3 mL, 1:1) for 5 minutes and then filtered. The solid was washed with MeCN (2 mL) and Et2O (2 mL), dried under vacuum, and amide XS13 was obtained as a yellow solid (44 mg, 44%). MS (ESI + ) C 28 H 30 N9O6 + [M+H] + Calculated value: 588.23, measured value: 588.66.

[0457] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-hydroxybenzamide)pentanoic acid (XS14) A suspension of ester XS13 (44 mg, 0.074 mmol) in MeOH / DMSO (0.48 mL, 5:1) was cooled to 10°C, and then aqueous NaOH (2.0 M, 0.223 mL, 0.446 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 5 hours. The mixture was then cooled to 0°C, and aqueous AcOH (1.0 M, 0.6 mL) was added, followed by water (1.3 mL). The suspension was stirred for 20 minutes. The solid was filtered and washed with water (1 mL), MeCN (1.0 mL), and Et2O (2 × 1 mL), and dried under high vacuum. The residue was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 55%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain acid XS14 as a pale yellow solid (21.2 mg, 52%). 1H NMR (400 MHz, DMSO-d6) ppm = 13.51-12.76 (m, 1H), 12.57-12.27 (m, 1H), 9.87 (s, 1H), 9.07-8.92 (m, 2H), 8.75 (s, 1H), 8.16-8.09 (m, 2H), 7.65 (d, J = 8.7 Hz, 4H), 7.59-7.37 (m, 1H), 6.82 (t, J = 5.8 Hz, 1H), 6.73-6.64 (m, 4H), 4.58-4.48 (m, 2H), 4.33-4.23 (m, 1H), 3.20-3.14 (m, 2H), 1.82-1.63 (m, 2H), 1.62-1.40 (m, 2H). MS (ESI + ) C 26 H 28 N9O5 + [M+H] + Calculated value: 546.22, measured value: 546.63.

[0458] Manufacturing of (S)-5-(5-aminothiophen-2-carboxamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XS16) [ka]

[0459] (S)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-(5-nitrothiophene-2-carboxamide)methyl pentanoate (XS15) A solution of 5-nitrothiophene-2-carboxylic acid (67 mg, 0.389 mmol) in thionyl chloride (0.621 mL, 8.51 mmol) was heated under reflux for 4 hours. The reaction mixture was cooled to room temperature, and the solvent was evaporated with heptane (2 × 1 mL). The residue was dissolved in DMF (0.48 mL) and added at 0°C to a stirred solution of amine XT48 (0.200 g, 0.370 mmol) and Et3N (0.103 mL, 0.740 mmol) in DMF (0.48 mL). The reaction mixture was stirred at room temperature for 20 minutes, and then quenched with MeOH (5 mL). The mixture was concentrated, and the solvent was evaporated with toluene (2 × 5 mL). The residue was dissolved in MeOH (10 mL), and MeCN (20 mL) was added to induce precipitation. Part of the MeOH was evaporated, and the remaining solution was allowed to stand overnight at 4°C. The solid was filtered and washed with MeCN (1 mL) and Et2O (2 mL). The filtrate was concentrated, and the residue was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 1:4). The isolated product was combined with the residue to obtain amide XS15 (0.122 g, 53%). 1 H NMR (400 MHz, DMSO-d6) ppm = 8.98 (t, J = 5.6 Hz, 1H), 8.80 (s, 1H), 8.76-8.70 (m, 1H), 8.65 (s, 1H), 8.13 (d, J = 4.4 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 4.4 Hz, 1H), 7.64 (br s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.32 (br s, 1H), 6.61 (br s, 2H), 5.22 (s, 2H), 4.49-4.42 (m, 1H), 3.63 (s, 3H), 3.30-3.26 (m, 2H), 1.89-1.74 (m, 2H), 1.71-1.53 ​​(m, 2H). MS (ESI + ) C 26 H 27 N 10 O7S + [M+H] + Calculated value: 623.18, measured value: 623.52.

[0460] (S)-5-(5-aminothiophen-2-carboxamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XS16) Amide XS15 (0.122 g, 0.196 mmol) was dissolved in DMF (2.0 mL) / NH4Cl aqueous solution (7.2 M, 0.383 mL, 2.74 mmol). Zinc powder (0.128 g, 1.96 mmol) was added at room temperature, and the reaction mixture was stirred for 3 hours. The reaction mixture was filtered through Celite®, and the filtrate was stirred in air for 18 hours. The mixture was concentrated, and the solvent was evaporated with MeCN (2 mL). The residue was suspended in MeCN (10 mL), and the precipitate was filtered off. The solid was washed with MeCN (1 mL) and Et2O (2 mL), dried under vacuum, and obtained as a brown solid. The material was suspended in MeOH / DMSO (0.48 mL, 5:1), NaOH (2.0 M, 0.187 mL, 0.375 mmol) was added dropwise, and the reaction mixture was stirred for 5 hours. Add 1.0 M AcOH aqueous solution (0.40 mL) and stir the mixture for 15 minutes. Add water (1.6 mL) and stir for 30 minutes, then allow the mixture to stand overnight at 4°C. Filter off the precipitate and wash the solid with water (0.5 mL), MeCN (0.5 mL), and Et2O (1 mL). Purify by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 10% to 25%). Collect the product fraction, remove MeCN with a rotary evaporator, and freeze-dry the aqueous solution to obtain acid XS16 as a pale yellow cottony substance (4.7 mg, 4%, 2 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 12.46 (br s, 1H), 9.29-9.00 (m, 2H), 8.83 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.87 (t, J = 5.7 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.70 (br s, 1H), 7.64-7.45 (m, 1H), 7.25 (d, J = 4.0 Hz, 1H), 6.89 (br s, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.53 (br s, 1H), 6.29-6.01 (m, 1H), 5.79 (d, J = 4.0 Hz, 1H), 4.61 (br s, 2H), 4.38-4.29 (m, 1H), 3.23-3.10 (m, 2H), 1.87-1.66 (m, 2H), 1.63-1.44 (m, 2H). MS (ESI + ) C 24 H 27 N 10 O4S + [M+H] + Calculated value: 551.19, measured value: 551.37.

[0461] Preparation of 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-ethynylbenzoate methyl (XS19) [ka]

[0462] (S)-5-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)-2-iodobenzoate methyl (XS17) Amine XS99 (0.800 g, 2.89 mmol, prepared as described in Ozaki et al, Tetrahedron, 2017, 73, 7177-7184) and Fmoc-Ala-OH (0.944 g, 3.03 mmol) were dissolved in DMF (9.6 mL). DIPEA (2.02 mL, 11.6 mmol) was added, followed by HATU (1.59 g, 4.18 mmol) over 1 hour. The reaction mixture was then stirred at room temperature for 1 hour. The reaction mixture was concentrated and dissolved in siRNA (50 mL). The organic phase was washed with aqueous KHSO4 (0.5 M, 2 × 25 mL), saturated aqueous NaHCO3 (25 mL), and brine (100 mL), dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, ԅ:DCM 0:1 to 30:70) to obtain amide XS17 as a pale yellow solid (1.17 g, 71%). 1 H NMR (400 MHz, CDCl3) ppm = 8.48 (br s, 1H), 7.99-7.91 (m, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.60-7.51 (m, 2H), 7.43-7.32 (m, 3H), 7.30-7.23 (m, 2H), 5.30 (br s, 1H), 4.49 (d, J = 6.6 Hz, 2H), 4.36 (br s, 1H), 4.21 (t, J = 6.7 Hz, 1H), 3.89 (s, 3H), 1.45 (d, J = 6.9 Hz, 3H). 13 C NMR (100 MHz, CDCl3) ppm = 170.4, 166.4, 143.5, 143.4, 141.7, 141.3, 137.8, 135.3, 127.9, 127.2, 127.1, 124.9, 124.0, 122.2, 120.1, 87.3, 67.4, 52.6, 47.1, 17.5. MS (ESI + ) C 26 H 24 IN2O5 + [M+H]+ Calculated value: 571.07, measured value: 571.27.

[0463] 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-iodobenzoate methyl(XS18) Piperidine (0.989 mL, 9.99 mmol) was added to a stirred solution of amide XS17 (1.14 g, 2.00 mmol) in DMF (10 mL). After stirring for 15 minutes, the reaction mixture was concentrated, the solvent was evaporated with toluene (2 × 20 mL), and the mixture was dried under vacuum. The crude product was purified by flash chromatography (silica gel, MeOH:DCM 0:1 to 20:80) to obtain a deprotected product (0.740 g, quantified). The product and Boc-Val-OSu (0.735 g, 2.34 mmol) were dissolved in DMF (9.7 mL). DIPEA (0.928 mL, 5.31 mmol) was added at room temperature, and the reaction mixture was stirred overnight. Water (100 mL) was added to the reaction mixture, and the aqueous phase was extracted with ELISA:heptane (1:1, 3 × 50 mL). The combined organic phases were washed with water (2 × 75 mL) and ice-cold brine (2 × 100 mL), dried over Na₂SO₄, and concentrated. The crude product was purified by flash chromatography (silica gel, siRNA:heptane 20:80 to 70:30) to obtain amide XS18 as an off-white solid (0.790 g, 68%). 1H NMR (400 MHz, CDCl3) ppm = 9.02 (br s, 1H), 8.09 (br s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 7.3 Hz, 1H), 6.55 (d, J = 7.0 Hz, 1H), 5.07-4.92 (m, 1H), 4.67 (quint, J = 7.2 Hz, 1H), 3.96-3.92 (m, 1H), 3.91 (s, 3H), 2.27-2.16 (m, 1H), 1.48 (d, J = 8.1 Hz, 3H), 1.43 (s, 9H), 1.01 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H). 13 C NMR (100 MHz, CDCl3) ppm = 171.9, 170.3, 166.5, 141.6, 138.3, 135.1, 124.0, 122.2, 87.0, 81.1, 60.9, 52.5, 49.7, 30.1, 28.2, 19.4, 17.7, 17.5. MS (ESI + ) C 21 H 31 IN3O6 + [M+H] + Calculated value: 548.13, measured value: 548.32.

[0464] 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-ethynylbenzoate methyl (XS19) Iodoarene XS18 (0.720 g, 1.32 mmol), Pd(PPh3)2Cl2 (18 mg, 0.026 mmol), and copper(I) iodide (13 mg, 0.066 mmol) were placed in a microwave vial. The vial was purged with N2, and DMF (4.5 mL), ethynyltrimethylsilane (0.273 mL, 1.97 mmol), and Et3N (0.183 mL, 1.32 mmol) were added sequentially. The reaction mixture was heated in a microwave at 80°C for 3.5 hours. The reaction mixture was concentrated, and the solvent was evaporated with toluene (10 mL). The crude product was purified by flash chromatography (silica gel, Â:DCM 0:1 to 1:1) to obtain a protected alkyne as a yellow solid (0.466, 68%). A portion of the intermediate (0.450 g, 0.869 mmol) was dissolved in MeOH (4.4 mL), and K2CO3 (12 mg, 0.087 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then diluted with ELISA (25 mL), followed by washing with saturated NaHCO3 aqueous solution (2 × 20 mL), water (20 mL), and brine (20 mL). The mixture was dried over Na2SO4, filtered, concentrated, and the alkyne XS19 was obtained as a yellow film (0.365 g, 94%). 1 H NMR (400 MHz, CDCl3) ppm = 9.06 (br s, 1H), 8.16 (br s, 1H), 7.94 (br d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 6.51 (d, J = 6.8 Hz, 1H), 5.00 (br s, 1H), 4.69 (quint, J = 7.2 Hz, 1H), 3.98-3.92 (m, 1H), 3.91 (s, 3H), 3.34 (s, 1H), 2.28-2.18 (m, 1H), 1.49 (d, J = 7.1 Hz, 3H), 1.43 (s, 9H), 1.01 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) ppm = 170.3, 166.0, 138.4, 135.8, 133.0, 122.7, 121.2, 117.9, 81.4, 60.9, 52.2, 49.6, 30.1, 28.2, 19.4, 17.7, 17.5. MS (ESI + ) C 23 H 32 N3O6 + [M+H] + Calculated value: 446.23, measured value: 446.35.

[0465] Preparation of 2-(1-((S)-4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)-1H-1,2,3-triazole-4-yl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XS23) [ka]

[0466] (S)-5-((tert-butoxycarbonyl)amino)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XS20) Ester XT105 (0.300 g, 0.557 mmol) was suspended in DMSO (0.33 mL) and MeOH (1.65 mL). The mixture was cooled to 10°C, and 2 M NaOH (2.23 mL, 4.46 mmol) was added dropwise. The reaction mixture was allowed to rise to room temperature and stirred for 5 hours. The reaction mixture was cooled to 0°C, quenched with 1 M AcOH aqueous solution (6 mL), and diluted with water (10 mL). The mixture was warmed to room temperature and subsequently filtered. The residue was washed with MeCN (5 mL) and Et2O (2 × 5 mL) to obtain unpurified acid. The material was dissolved in ice-cold DCM (5 mL). TFA (5 mL) was added, and the mixture was stirred at 0°C for 15 minutes. The reaction mixture was concentrated, and the solvent was evaporated with toluene (2 × 10 mL) to obtain amine XS20 as a brown oily substance (0.292 g, quantitative analysis, 2 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 12.99-12.41 (m, 1H), 9.14-8.89 (m, 2H), 8.71 (s, 1H), 8.56 (d, J = 7.9 Hz, 1H), 8.46-7.90 (m, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.76-7.67 (m, 2H), 7.36 (d, J = 8.3 Hz, 2H), 4.47-4.33 (m, 1H), 3.28-3.17 (m, 4H), 2.89-2.74 (m, 2H), 1.96-1.85 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.56 (m, 2H). 13 C NMR (100 MHz, DMSO-d6) ppm = 173.9, 166.8, 163.3, 158.9, 158.6, 153.1, 150.6, 145.1, 132.2, 128.8, 128.1, 122.3, 52.4, 39.0, 35.8, 34.0, 28.0, 24.6. MS (ESI + ) C 20 H 25 N8O3 + [M+H] + Calculated value: 425.20, measured value: 425.41.

[0467] (S)-5-azido-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XS21) To a solution of NaN3 (0.361 g, 5.56 mmol) in ice-cold water (0.95 mL) / DCM (1.6 mL), trifluic anhydride (0.188 mL, 1.11 mmol) was added dropwise. The resulting mixture was stirred at 0°C for 4 hours. The organic phase was separated, and the aqueous phase was extracted with DCM (2 × 1 mL). The combined organic phase was washed with 1 M Na2CO3 (1 mL), and a solution of K2CO3 (0.123 g, 0.890 mmol), CuSO4·5H2O (2.8 mg, 0.011 mmol), and amine XS20 (0.236 g, 0.556 mmol) in room temperature water (1.8 mL) / MeOH (3.7 mL) was added. The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with water, and the pH was adjusted to approximately 5 with acetic acid (1.0 M). The mixture was gently heated to induce precipitation and cooled to room temperature. The solid was filtered, washed with water (1 mL), MeCN (1 mL), and Et2O (5 mL), and dried in vacuum to obtain azide XS21 as a greenish-brown solid (0.103 g, 41%). 1 H NMR (400 MHz, DMSO-d6) ppm = 13.60-11.50 (m, 1H), 8.56 (br s, 1H), 8.54-8.49 (m, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.68-7.48 (m, 2H), 7.35 (d, J = 8.0 Hz, 2H), 6.53 (br s, 2H), 4.45-4.28 (m, 1H), 3.38-3.33 (m, 2H), 3.15 (br s, 4H), 1.94-1.71 (m, 2H), 1.71-1.54 (m, 2H). MS (ESI + ) C 20 H 23 N 10 O3 + [M+H] + Calculated value: 451.19, measured value: 451.50.

[0468] (S)-5-(4-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)-2-(methoxycarbonyl)phenyl)-1H-1,2,3-triazole-1-yl)-2-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)pentanoic acid (XS22) A solution of alkyne XS19 (0.070 g, 0.16 mmol) and azide XS21 (0.078 g, 0.17 mmol) in DMF (1.7 mL) was purged with N2 for 5 minutes. Then, copper(II) sulfate (33 mg, 0.13 mmol) in water (889 μL) and sodium ascorbate (51 mg, 0.26 mmol) in water (942 μL) were added sequentially. The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was added to water (25 mL). AcOH aqueous solution (1.0 M, 0.5 mL) was added, and the mixture was stirred for 30 minutes. The mixture was gently heated to induce precipitation and cooled to room temperature. The solid was filtered and washed with MeCN (20 mL), SiO (2 × 20 mL), and Et2O (10 mL), stirred in SiO (15 mL), filtered, and washed with Et2O (10 mL). The solid was then dried in a vacuum to obtain triazole XS22 as a brown solid (0.103 g, 66%). MS (ESI + ) C 43 H 54 N 13 O9 + [M+H] + Calculated value: 896.42, measured value: 896.65.

[0469] 2-(1-((S)-4-carboxy-4-(4-(2-(2,4-diaminopteridine-6-yl)ethyl)benzamide)butyl)-1H-1,2,3-triazole-4-yl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)benzoic acid (XS23) Ester XS22 (0.100 g, 0.112 mmol) was suspended in THF (2.0 mL) and cooled to 10°C. LiOH (0.4 M, 1.40 mL, 0.558 mmol) was added dropwise, and the reaction mixture was stirred for 4.5 hours. Additional LiOH (0.4 M, 0.698 mL, 0.279 mmol) was added, and stirring continued for 1 hour. The reaction mixture was cooled to 0°C, and AcOH (96 μL, 1.67 mmol) was added. The mixture was concentrated, and the solvent was evaporated with toluene (2 × 5 mL). The unpurified material was then dissolved in a solution of TFA (3.0 mL) in DCM (3.0 mL) at 0°C. The reaction mixture was stirred at 0°C for 15 minutes, then concentrated, and the solvent was evaporated with toluene (2 × 5 mL). The mixture was dried under vacuum to obtain the unpurified amine as a brown oily substance. The materials were dissolved in DMF (5.0 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (38 mg, 0.12 mmol) and DIPEA (0.117 mL, 0.668 mmol) were added at 0°C. After 15 minutes, DIPEA (0.117 mL, 0.668 mmol) was added, and after 3 hours, 6-maleimidohexanoic acid N-hydroxysuccinimide ester (15 mg, 0.049 mmol) was added. After 24 hours, the reaction mixture was concentrated to 1 mL and stirred for 192 hours. Additional DIPEA (0.117 mL, 0.668 mmol) was added, and after a total reaction time of 240 hours, the reaction mixture was concentrated and the solvent was evaporated with toluene (5 mL). A portion of the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 40%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain maleimide XS23 as a pale yellow solid (13.9 mg, 13%, 3 steps). 1H NMR (400 MHz, DMSO-d6) ppm = 12.99-12.41 (m, 1H), 9.14-8.89 (m, 2H), 8.71 (s, 1H), 8.56 (d, J = 7.9 Hz, 1H), 8.46-7.90 (m, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.76-7.67 (m, 2H), 7.36 (d, J = 8.3 Hz, 2H), 4.47-4.33 (m, 1H), 3.28-3.17 (m, 4H), 2.89-2.74 (m, 2H), 1.96-1.85 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.56 (m, 2H). MS (ESI + ) C 47 H 55 N 14 O 10 + [M+H] + Calculated value: 975.42, measured value: 975.60.

[0470] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-methyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecanamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)pentanoic acid (XR4) manufacturing [ka]

[0471] 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl benzoate (XR1) To a solution of methyl 4-hydroxybenzoate (1.00 g, 6.57 mmol) in THF (25 mL), Et3N (2 mL, 14.5 mmol) was added at 0°C. 4-nitrophenyl chloroformic acid (1.46 g, 7.23 mmol) was added. The resulting suspension was diluted with THF (30 mL), and the mixture was warmed to room temperature. After 1 hour, methyl (2-(methylamino)ethyl)-tert-butyl carbamate (1.24 g, 6.57 mmol) was added, and the mixture was stirred for 1 hour. The mixture was concentrated and dissolved in ethyl acetate. The ethyl acetate phase was washed with KHSO4 (0.5 M, 2 ×), saturated aqueous NaHCO3 (2 ×), aqueous Na2CO3 (1 M), and brine. The organic phase was dried over MgSO4 and concentrated. The crude product was purified by flash chromatography (silica gel, heptane:alkyl, 1:0 to 3:7) to obtain carbamate XR1 (2.07 g, 85%). 1 H NMR (400 MHz, CDCl3) ppm = 8.05 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 3.91 (s, 3H), 3.62-3.42 (m, 4H), 3.13-3.05 (s, 3H), 2.93 (m, 3H), 1.44 (m, 9H). MS (ESI + ) C 18 H 27 N2O6 + [M+H] + Calculated value: 367.19, measured value: 367.35.

[0472] 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzoic acid (XR2) Carbamate XR1 (2.07 g, 5.66 mmol) was dissolved in THF / water (1:1, 30 mL), and NaOH (0.293 g, 7.33 mmol) was added. The reaction mixture was heated at 45°C for 2 hours, cooled to room temperature, and acidified with KHSO4 aqueous solution (0.5 M, approximately 50 mL). The reaction mixture was extracted three times with toluene, the combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated to obtain acid XR2. MS (ESI + ) C 17 H 25 N2O6 + [M+H] + Calculated value: 353.17, measured value: 353.27.

[0473] (S)-5-(4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XR3) Amine XT48 (40 mg, 0.079 mmol) and acid XR2 (28.0 mg, 0.079 mmol) were reacted according to the general procedure XXA. Amide XR3 was obtained by purification by flash chromatography (silica gel, DCM:MeOH, 1:0 to 1:1) (quantified). MS (ESI + ) C 38 H 48 N 11 O9 + [M+H] + Calculated value: 802.36, measured value: 802.80.

[0474] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(4-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-methyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecaneamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)pentanoic acid (XR4) Ester XR3 (38 mg, 0.047 mmol) was dissolved in water / THF (1:1, 1 mL), treated with LiOH (5.7 mg, 0.238 mmol) for 3 hours, and the reaction was quenched with AcOH (10 equivalents). The mixture was concentrated, the solvent was evaporated with toluene (2×), and the mixture was dried under reduced pressure. MS (ESI + ) C36 H 46 N 11 O8 + [M+H] + Calculated value: 760.35, measured value: 760.77.

[0475] The obtained intermediate was suspended in HCl / dioxane (2 mL, 4 M) and stirred for 30 minutes. The reaction mixture was concentrated, the solvent was evaporated with chloroform, and the mixture was dried under reduced pressure. Next, the deprotected intermediate and XJ12 (45 mg, 0.06 mmol) were dissolved in DMF (1 mL), cooled on an ice bath, and DIPEA (0.082 mL, 0.47 mmol) was added. After 15 minutes, the reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was concentrated, and the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA). The fraction containing the product was collected, acetonitrile was evaporated, and the remaining solution was freeze-dried to obtain XR4 (25 mg, 42%). MS (ESI + ) C 59 H 75 N 17 O 16 + [M+H] + Calculated value: 1277.56, measured value: 1277.12.

[0476] Manufacture of 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecanamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzoic acid (XR8-p and XR8-m) [ka]

[0477] 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)dimethyl phthalate (XR6) To a solution of dimethyl 4-hydroxyphthalate (XR5; 500 mg, 2.38 mmol) in cooled (0°C) DCM (20 mL), 4-nitrophenyl chloroformate (527 mg, 2.62 mmol) was added, followed by Et3N (0.66 mL, 4.76 mmol). The reaction mixture was stirred at 0°C for 2 hours. Then, tert-butyl methyl(2-(methylamino)ethyl)carbamate (537 mg, 2.85 mmol) in DCM (2 mL) was added all at once. The mixture was stirred at room temperature for 1 hour, and then concentrated. The crude product was purified by flash chromatography (silica gel, heptane:ethyl, 1:0 to 3:7) to obtain carbamate XR6 (57%, 580 mg). 1 H NMR (400 MHz, CDCl3) ppm = 7.76 (d, J = 8.4 Hz, 1H), 7.46 (m, 1H), 7.32 (dd, J = 8.5 Hz, 1.9 Hz, 1H), 3.90 (s, 6H), 3.62-3.41 (m, 4H), 3.12 (s, 1.5H, rotamer), 3.05 (s, 1.5H, rotamer), 2.92 (s, 3H), 1.44 (m, 9H). (For clarity, singlets derived from carbamate rotational isomers that are divided but partially overlapping are shown as singlets.) MS (ESI + ) C 20 H 28 N2NaO8 + [M+Na] + 447.17, measured value 447.50.

[0478] (S)-5-(5-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1,3-dioxoisoindorin-2-yl)-2-(4-(N-((2,4-diaminopteridine-6-yl)methylformamide)benzamide)methyl pentanoate (XR7) NaOH (269 mg, 6.71 mmol) in water (4 mL) was added to a solution of carbamate XR6 (570 mg, 1.34 mmol) in THF (5 mL) at room temperature. After 20 hours, the reaction mixture was acidified with an aqueous solution of KHSO4 (0.5 M), and the reaction mixture was then extracted with siRNA (2 × 25 mL). The combined organic phase was dried over MgSO4, filtered, and concentrated. A portion of the unpurified diacid obtained (34.7 mg, 0.087 mmol) and amine XT48 (52 mg, 0.096 mmol) were reacted in DMF (0.5 mL) with HATU (83 mg, 0.219 mmol) and DIPEA (0.061 mL, 0.350 mmol) for 20 hours, as described in general procedure XXA. After concentration, the crude product was purified by flash chromatography (silica gel, DCM:MeOH, 1:0 to 1:1) to obtain phthalimide XR7 (58.2 mg, 80%). MS (ESI + ) C 39 H 46 N 11 O 10 + [M+H] + Calculated value: 828.34, measured value: 828.83.

[0479] 2-(((S)-4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)carbamoyl)-5-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecanamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzoic acid (XR8-p and XR8-m) Phthalimide XR7 (58.2 mg, 0.070 mmol) was dissolved in water / THF (1:1, 1 mL) and treated with LiOH (13.5 mg, 0.56 mmol) for 3 hours at room temperature. AcOH was added to the product to precipitate it, and the solid was subsequently isolated by filtration to obtain the unpurified material (18 mg, 32%). MS (ESI+ ) C 37 H 46 N 11 O 10 + [M+H] + Calculated value: 804.34, measured value: 804.48.

[0480] The saponified intermediate (18 mg) was suspended in HCl / dioxane (4 M, 0.5 mL), the orange suspension was stirred for 30 minutes, then concentrated and dried under reduced pressure. The deprotected intermediate with Boc and XJ12 (16.9 mg, 0.022 mmol) were dissolved in DMF (0.5 mL), followed by the addition of DIPEA (0.023 mL, 0.134 mmol). The mixture was stirred at room temperature for 2 hours and then concentrated. Purification by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA) yielded a mixture of para and meta-ring-opening phthalimide linkers-drugs, XR8-p and XR8-m, as a yellow solid. MS (ESI + ) C 60 H 74 N 17 O 18 + [M+H] + Calculated value: 1320.54, measured value: 1320.74.

[0481] (S)-5-(2-(benzyloxy)-4-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-methyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecaneamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)Pentanoic acid (XR12) [ka]

[0482] 4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-2-hydroxybenzoate methyl (XR9) Et3N (1.71 mL, 12.3 mmol) and 4-nitrophenyl chloroformate (1.36 g, 6.74 mmol) were sequentially added to a solution of methyl 2,4-dihydroxybenzoate (1.03 g, 6.13 mmol) in cooled (0°C) THF (50 mL). After 1 hour, methyl (2-(methylamino)ethyl)-tert-butyl carbamate (1.27 g, 6.74 mmol) in THF (10 mL) was added at 0°C, and the mixture was stirred at room temperature for 1 hour. After concentrating the reaction mixture, the crude product was purified by flash chromatography (silica gel, heptane:SiO, 1:0 to 1:1) to obtain carbamate XR9 (1.64 g, 70%). 1 H NMR (400 MHz, CDCl3) ppm = 10.85 (s, 1H), 7.82 (dd, J = 8.8 Hz, J = 1.8 Hz, 1H), 6.75 (d, J = 2.1 Hz, 1H), 6.69 (d, J = 8.9 Hz, 1H), 3.94 (s, 3H), 3.61-3.42 (m, 4H), 3.11 (s, 1.5H, rotamer), 3.04 (s, 1.5H, rotamer), 2.92 (s, 3H), 1.45 (s, 9H). (For clarity, singlets derived from carbamate rotational isomers that are divided but partially overlapping are shown as singlets.) MS (ESI + ) C 18 H 27 N2O7 + [M+H] + Calculated value: 383.18, measured value: 383.27.

[0483] 2-(benzyloxy)-4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)methyl benzoate (XR10) BnBr (0.253 mL, 2.13 mmol) and K2CO3 (321 mg, 2.32 mmol) were added to a solution of carbamate XR9 (740 mg, 1.935 mmol) in DMF (10 mL) at room temperature, and the mixture was stirred for 16 hours. The mixture was then concentrated and dissolved in toluene (approximately 75 mL). The organic phase was washed with water, aqueous KHSO4 (0.5 M) and brine. The toluene phase was dried over MgSO4, filtered, and concentrated. The crude product was purified by flash chromatography (twice, 1; silica gel, heptane:toluene, 1:0 to 1:1, 2; DCM:MeOH, 1:0 to 4:1) to obtain benzyl ether XR10 (585 mg, 64%). MS (ESI + ) C 25 H 32 N2NaO7 + [M+Na] + 495.21, measured value 495.55.

[0484] (S)-5-(2-(benzyloxy)-4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XR11) LiOH (148 mg, 6.2 mmol) was added to a solution of XR10 (585 mg, 1.24 mmol) in THF (10 mL) at room temperature, and the mixture was stirred overnight. Additional LiOH (59.4 mg, 2.48 mmol) was added, and the reaction was continued for 24 hours. The reaction product was acidified with aqueous KHSO4 (0.5 M) and extracted with SiO4 (2 × 25 mL). The combined organic phase was dried over MgSO4, filtered, concentrated, and the unpurified acid (518 mg) was obtained. 1H NMR (400 MHz, CDCl3) ppm = 10.55 (br s, 1H), 8.18 (d, J = 8.6 Hz, 1H), 7.48-7.37 (m, 5H), 7.15-7.05 (m, 1H), 6.95-6.86 (m, 1H), 5.25 (s, 2H), 3.59-3.54 (m, 1H), 3.52-3.44 (m, 3H), 3.13 (s, 1.3H, rotamer), 3.06 (s, 1.7H, rotamer), 2.92 (s, 3H), 1.45 (s, 9H). (For clarity, singlets derived from carbamate rotetromyomers that are divided but partially overlapping are shown as singlets.)

[0485] A portion of this material (52.5 mg, 0.115 mmol) was reacted with amine XT48 (57.7 mg, 0.115 mmol), HATU (52.2 mg, 0.137 mmol), and DIPEA (0.123 mL, 0.687 mmol) in DMF (1 mL) according to the general procedure XXA. The crude product was purified by flash chromatography (silica gel, DCM:MeOH, 1:0 to 1:1) to obtain amide XR11 (100 mg, 87%). MS (ESI + ) C 45 H 54 N 11 O 10 + [M+H] + Calculated value: 908.40, measured value: 908.55.

[0486] (S)-5-(2-(benzyloxy)-4-(((2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-methyl-4,7-dioxo-2-(3-ureidopropyl)-8,11-dioxa-3,6-diazatridecaneamide)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)benzamide)-2-(4(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)pentanoic acid (XR12) Maleimide XR12 was synthesized in the same manner as the conversion from XR3 to XR4. The product was obtained as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 13.10 (br s, 1H), 12.41 (br s, 1H), 11.01 (s, 1H), 9.96 (s, 1H), 9.19 (s, 1H), 9.14 (s, 1H),8.77 (s, 1H), 8.14-7.96 (m, 5H), 7.68 (d, J = 8.7 Hz, 2H), 7.63 (t, J = 8.1 Hz, 1H), 7.50 (m, 2H), 7.39 (m, 2H), 7.29-7.08 (m, 6H), 6.94 (s, 2H), 6.92-6.80 (m, 4H), 6.69 (m, 3H), 5.91 (t, J = 5.4 Hz, 1H), 5.35 (br s, 2H), 5.09 (m, 2H), 4.94 (m, 2H), 4.55 (s, 2H), 4.39-4.20 (m, 2H), 3.94 (m, 2H), 3.82 (t, J = 7.4 Hz, 1H), 3.52-3.36 (m, 11H), 3.17 (m, 2H), 3.00-2.75 (m, 8H), 1.90 (m, 1H), 1.79-1.22 (m, 8H), 0.79 (d, J = 6.7 Hz, 3H), 0.75 (d, J = 6.7 Hz, 3H). MS (ESI + ) C 66 H 80 N 17 O 17 + [M+H] + Calculated value: 1382.5, measured value: 1382.81.

[0487] Preparation of 5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)-propanamide)thiophene-2-carboxylic acid (XR23) [ka]

[0488] 5-aminothiophene-2-carboxylate benzyl (XJ16) A suspension of benzyl 5-nitrothiophene-2-carboxylate (XJ15) (1.55 g, 5.89 mmol, prepared as described in International Publication No. 2007 / 018508) in MeOH (50 mL) was mixed with saturated aqueous NH4Cl (7 mL) and zinc powder (3.8 g). The suspension was stirred at room temperature for 2.5 hours and then filtered through Celite®. The solid was washed with MeOH, and the filtrate was concentrated. Ether was added, the organic phase was washed with brine, dried (Na2SO4), filtered, concentrated, and purified by flash chromatography (silica gel, DCM:Ether 1:0 to 4:1) to obtain XJ16 as a purple solid (0.90 g, 66%). 1 H NMR (400 MHz, CDCl3) ppm = 7.45 (d, J = 4.0 Hz, 1H), 7.40-7.29 (m, 5H), 6.03 (d, J = 4.0 Hz, 1H), 5.26 (s, 2H), 1.52 (br s, 2H). 13 C NMR (100 MHz, CDCl3) ppm = 162.5, 159.5, 136.4, 135.3, 128.5, 128.0, 117.1, 107.8, 66.1. MS (ESI + ) C 12 H 12 NO2S + [M+H] + Calculated value: 234.05, measured value: 234.06.

[0489] (S)-5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)thiophen-2-carboxylate benzyl(XJ17) To a suspension of Fmoc-Ala-OH (1.82 g, 5.84 mmol) in DCM (18 mL), oxalyl chloride (1.0 mL, 11.7 mmol) and 10 drops of DMF were added at 0°C. The mixture was stirred at room temperature for 1 hour and then concentrated. The unpurified acid chloride was dissolved in DCM (8 mL) and subsequently added to a solution of XJ16 (0.90 g, 3.90 mmol) in cooled (0°C) DCM (8 mL). Et3N (1.6 mL, 11.7 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, DCM: siRNA 1:0 to 1:1) to obtain XJ17 as a yellow foam (0.66 g, 32%). MS (ESI + ) C 30 H 27 N2O5S + [M+H] + Calculated value: 527.16, measured value: 527.36.

[0490] 5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)thiophene-2-carboxylate benzyl(XJ18) Amine XJ17 protected with Fmoc (0.66 g, 1.25 mmol) was dissolved in DMF (10 mL). Piperidine (0.62 mL, 6.2 mmol) was added at room temperature, and the mixture was stirred for 30 minutes. The reaction mixture was concentrated, and the solvent was evaporated with toluene / DCM to obtain the deprotected amine as a yellow solid. The material was redissolved in DCM (10 mL), and Boc-L-Val-OSu (0.55 g, 1.75 mmol) and DIPEA (0.54 mL, 3.1 mmol) in DCM (10 mL) were added at 0°C, and the mixture was then stirred at room temperature for 2.5 hours. After concentration, the crude product was purified by flash chromatography (silica gel, DCM:siRNA 1:0 to 4:1) to obtain amide XJ18 as a colorless syrup (0.48 g, 77%). 1H NMR (400 MHz, CDCl3) ppm = 10.76 (br s, 1H), 7.61 (d, J = 4.2 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.42-7.28 (m, 5H), 6.72 (d, J = 3.7 Hz, 1H), 5.59 (d, J = 6.7 Hz, 1H), 5.29 (s, 2H), 4.71 (q, J = 7.1 Hz, 1H), 4.02 (br t, J = 5.6 Hz, 1H), 2.15 (m, 1H), 1.45 (d, J = 7.1 Hz, 3H), 1.42 (s, 9H), 0.96 (dd, J = (17.3, 6.8 Hz, 6H). 13 C NMR (100 MHz, CDCl3) ppm = 172.1, 169.6, 162.7, 146.0, 136.0, 131.9, 128.4, 128.0, 127.9, 123.5, 112.4, 80.0, 66,1, 60.4, 48.8, 30.6, 28.2, 19.2, 17.7, 17.5. MS (ESI + ) C 25 H 34 N3O6S + [M+H] + Calculated value: 504.21, measured value: 504.31.

[0491] 5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)thiophene-2-carboxylic acid (XR23) To a solution of amide XJ18 (0.48 g, 0.95 mmol) in siRNA (10 mL), palladium (0.3 g, 10% on activated carbon) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 days. The reaction mixture was purged with N2 and filtered through Celite®. The residue was washed with siRNA, and the filtrate was concentrated to obtain XR23 as a white solid (0.28 g, 72%). 1H NMR (400 MHz, DMSO-d6) ppm = 12.59 (br s, 1H), 11.62 (s, 1H), 8.24 (d, J = 6.6 Hz, 1H), 7.51 (d, J = 4.0 Hz, 1H), 6.70 (s, 1H), 6.69-6.68 (m, 1H), 4.45-4.38 (m, 1H), 3.84 (br t, J = 7.75 Hz, 1H), 1.99-1.91 (m, 1H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz (3H). 13 C NMR (100 MHz, CDCl3) ppm = 171.8, 170.5, 164.0, 156.0, 146.1, 131.9, 124.2, 112.3, 78.5, 59.8, 48.8, 30.9, 28.6, 19.6, 18.5, 18.1. MS (ESI + ) C 18 H 28 N3O6S + [M+H] + Calculated value: 414.16, measured value: 414.29.

[0492] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)thiophene-2-carboxamide)pentanoic acid (XR16) manufacturing [ka]

[0493] (S)-5-(5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanamide)thiophen-2-carboxamide)-2-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)methyl pentanoate (XR15) Amine XT48 (50 mg, 0.1 mmol) was reacted with XR23 (41.0 mg, 0.099 mmol), HATU (45.3 mg, 0.119 mmol), and DIPEA (0.104 mL, 0.595 mmol) in DMF (1.0 mL) according to general procedure XXA. The crude product was purified by flash chromatography (silica gel, DCM:MeOH, 1:0 to 4:1) to obtain impure XR15 (105 mg), which was used without further purification. MS (ESI + ) C 39 H 51 N 12 O9S + [M+H] + Calculated value: 863.36, measured value: 863.83.

[0494] (S)-2-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)thiophene-2-carboxamide)pentanoic acid (XR16) A solution of amide XR15 (43 mg, 0.050 mmol) in THF / water (1:1, 1 mL) was treated with LiOH (6 mg, 0.25 mmol) for 90 minutes at room temperature. AcOH (0.03 mL, 0.5 mmol) and toluene (5 mL) were added, and the mixture was concentrated. The crude product was dissolved in DCM (2 mL), and TFA (1 mL) was added at 0°C. After 40 minutes, the mixture was concentrated, the solvent was evaporated with DCM, and the mixture was dried under reduced pressure. The deprotected intermediate was dissolved in DMF (0.5 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (18.43 mg, 0.060 mmol) was added at room temperature, followed by DIPEA (0.017 mL, 0.100 mmol). Further addition of DIPEA (>5 equivalents) was made the reaction mixture sufficiently basic, and the remaining acid was made basic. The mixture was stirred at room temperature for 3 hours and then concentrated. XR16 was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient) to obtain a yellow solid (11.6 mg, 26%). 1 H NMR (400 MHz, DMSO-d6) ppm = 12.95 (br s, 1H), 12.47 (m, 1H), 11.3 (s, 1H), 9.12 (br s, 1H), 8.82 (s, 1H), 8.29 (d, J = 6.7 Hz, 1H), 8.26 (t, J = 5.6 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 4.1 Hz, 1H), 7.00 (s, 2H), 6.88 (m, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 4.1 Hz, 1H), 4.60 (d, J = 4.8 Hz, 2H), 4.36 (m, 2H), 4.15 (m, 1H), 3.21 (m, 3H), 2.23-1.89 (m, 4H), 1.89-1.68 (m, 2H), 1.65-1.42 (m, 6H), 1.31 (d, J = 7.2 Hz, 3H), 1.26-1.13 (m, 4H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H). MS (ESI + C 42 H 52 N 13 O9S + [M+H] + The calculated value is 914.37 and the measured value is 914.49.

[0495] (R)-2-(4-(((2,5-diaminopteridine-6-yl)methyl)amino)benzamide)-5-(5-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methyl-butanamide)propanamide)thiophene-2-carboxamide)methyl pentanoate (XJ21)

change

[0496] XR15 (83 mg, 0.096 mmol) was dissolved in MeOH (1.5 mL), and hydrochloric acid (6N, 0.9 mL, 5.51 mmol) was added. The mixture was stirred at 50°C for 1 hour, then cooled to room temperature, diluted with MeOH, and the solvent was evaporated with toluene (2×) and dried under reduced pressure. The product was dissolved in DMF (1 mL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.029 g, 0.095 mmol) and DIPE (0.1 mL, 0.57 mmol) were added sequentially at 0°C. The resulting mixture was stirred at room temperature for 2 hours and then concentrated. The crude solid was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient 20% to 50%). The product fraction was collected, MeCN was removed using a rotary evaporator, and the aqueous solution was freeze-dried to obtain XJ21 as a yellow foamy substance (25 mg, 28%). 1H NMR (400 MHz, DMSO-d6) ppm = 11.32 (s, 1H), 9.30 (bs, 1H), 9.26 (bs, 1H), 8.84 (s, 1H), 8.32 (dd, J = 15.5, 7.0 Hz, 2H), 8.27-8.24 (m, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.61 (br d, J = 8.6 Hz, 1H), 7.49 (d, J = 4.1 Hz, 1H), 7.0 (s, 2H), 6.91 (br s, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.66 (d, J = 4 Hz, 1H), 4.62 (br s, 2H), 4.43-4.34 (m, 2H), 4.15 (t, J = 7.7 Hz, 1H), 3.62 (s, 3H), 3.24-3.19 (m, 2H), 2.22-2.07 (m, 2H), 1.99-1.90 (m, 1H), 1.86-1.76 (m, 2H), 1.65-1.42 (m, 7H), 1.31 (d, J = 7.1 Hz, 3H), 1.22-1.14 (m, 2H), 1.04 (d, J = 6.1 Hz, 2H), 0.86 (br d, J = 6.7 Hz, 3H), 0.82 (br d, J = 6.7 Hz, 3H). MS (ESI + C 43 H 54 N 13 O9S + [M+H] + The calculated value is 928.38 and the measured value is 928.60.

[0497] Production of methyl 2-ethynyl-5-nitrobenzoate (XR18)

change

[0498] 5-ニトロ-2-((トリメチルシリル)エチニル)benzoic acid メチル(XR17) Ethinyltrimethylsilane (0.71 mL, 5.2 mmol), methyl 2-iodo-5-nitrobenzoate (1.06 g, 3.44 mmol), copper(I) iodide (33 mg, 0.17 mmol), and DMF (15 mL) were placed in a microwave vial, and the solution was purged with N2. Pd(PPh3)2Cl2 (48 mg, 0.069 mmol) and Et3N (0.48 mL, 3.4 mmol) were added, and the mixture was purged again with N2. The vial was stoppered and heated in a microwave at 80°C for 3.5 hours. The mixture was then concentrated, and the crude product was purified by flash chromatography (silica gel, heptane:siRNA, 1:0 to 0:1) to obtain TMS-alkyne XR17 (620 mg, 65%). 1 H NMR (400 MHz, CDCl3) ppm = 8.77 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 3.98 (s, 3H), 0.31 (s, 9H). 13 C NMR (100 MHz, CDCl3) ppm = 164.7, 146.6, 135.5, 133.6, 129.7, 125.8, 125.6, 106.9, 101.3, 52.6, -0.4.

[0499] Methyl 2-ethynyl-5-nitrobenzoate (XR18) Alkyne XR17 (822 mg, 2.96 mmol) and K2CO3 (41.0 mg, 0.296 mmol) were dissolved in MeOH (15 mL) at room temperature, and the mixture was stirred for 30 minutes. Ether (50 mL) was added, and the mixture was washed with saturated NaHCO3 aqueous solution (2 ×) and brine. The organic phase was dried over MgSO4, filtered, and concentrated to obtain XR18 (quantified). 1H NMR (400 MHz, CDCl3) ppm = 8.81 (d, J = 2.4 Hz, 1H), 8.32 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 4.00 (s, 3H), 3.72 (s, 1H). 13 C NMR (100 MHz, CDCl3) ppm = 164.3, 147.0, 136.0, 133.8, 129.0, 126.1, 125.5, 87.9, 80.4, 52.8.

[0500] Manufacturing of (S)-5-azido-2-(((benzyloxy)carbonyl)amino)pentanoic acid (XR19) [ka]

[0501] To a solution of NaN3 (10 g, 154 mmol) in ice-cold water (25 mL) / DCM (44 mL), trifluic anhydride (5.2 mL, 30.8 mmol) was added dropwise. The resulting mixture was stirred at 0°C for 2 hours. The organic phase was separated, and the aqueous phase was extracted with DCM (2 × 20 mL). The combined organic phase was washed with Na2CO3 aqueous solution (1 M) and added to a solution of Z-Orn-OH (4.10 g, 15.4 mmol), K2CO3 (3.40 g, 24.6 mmol), and CuSO4·5H2O (0.077 g, 0.308 mmol) in a water / MeOH (1:2, 150 mL) mixture at room temperature. After stirring at room temperature for 18 hours, the organic solvent was evaporated, the aqueous slurry was diluted with water (150 mL), and the pH was adjusted to 2 with concentrated hydrochloric acid. The aqueous phase was extracted three times with phenylethylamine, the combined organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain crude XR19 (4.5 g), which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl3) ppm = 7.36 (m, 5H), 5.34 (d, J = 8.3 Hz, 1H), 5.12 (s, 2H), 4.43 (m, 1H), 3.34 (m, 2H), 1.99 (m, 1H), 1.84-1.61 (m, 3H). MS (ESI + ) C 13 H 17 N4O4 + [M+H] + Calculated value: 293.12, measured value: 293.39.

[0502] Preparation of (S)-5-amino-2-(1-(4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)-1H-1,2,3-triazole-4-yl)benzoic acid (XR22) [ka]

[0503] (S)-2-(1-(4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentyl)-1H-1,2,3-triazole-4-yl)-5-methyl nitrobenzoate (XR20) To a solution of azide XR19 (533 mg, 0.912 mmol) and alkyne XR18 (187 mg, 0.912 mmol) in THF (7 mL), CuSO4·5H2O (175 mg, 0.702 mmol) in water (1.5 mL) was added at room temperature. After purging the solution with N2 for 5 minutes, sodium ascorbate (271 mg, 1.39 mmol) in water (1.5 mL) was added. The yellow solution was stirred for 5 minutes, at which point ULC-MS indicated the completion of the reaction. SiO (20 mL) and water / brine (1:1, 10 mL) were added to separate the layers, and the aqueous phase was extracted with SiO (2 ×). The combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. The triazole was purified by flash chromatography (silica gel, heptane:SiO, 1:0 to 0:1). The isolated product was dissolved in DMF (10 mL), and Cs2CO3 (163 mg, 0.500 mmol) and MeI (60 μL, 0.964 mmol) were added at room temperature. The reaction mixture was stirred for 16 hours. After concentration, the crude product was purified by flash chromatography (silica gel, heptane:ethyl, 1:0 to 0:1) to obtain ester XR20 (380 mg, 81%, 2 steps). 1H NMR (400 MHz, CDCl3) ppm = 8.76 (d, J = 2.4 Hz, 1H), 8.39 (dd, J = 8.7 Hz, 2.4 Hz, 1H), 8.19 (d, J = 8.7 Hz, 1H), 8.06 (s, 1H), 7.34 (m, 5H), 5.43 (d, J = 7.9 Hz, 1H), 5.12 (s, 2H), 4.47 (m, 3H), 3.91 (s, 3H), 3.75 (s, 3H), 2.11-1.91 (m, 2H), 1.71 (m, 2H). MS (ESI + ) C 24 H 26 N5O8 + [M+H] + Calculated value: 512.18, measured value: 512.33.

[0504] (S)-2-(1-(4-(4-(N-((2,4-diaminopteridine-6-yl)methyl)formamide)benzamide)-5-methoxy-5-oxopentyl)-1H-1,2,3-triazole-4-yl)-5-methyl nitrobenzoate (XR21) 5 mL of HBr (33%) in acetic acid was added at 0°C to a solution of ester XR20 (300 mg, 0.587 mmol), and the reaction mixture was stirred at 0°C for 75 minutes. The mixture was concentrated, and the solvent was evaporated with toluene (2×), chloroform, and ether. MS (ESI + ) C 16 H 20 N5O6 + [M+H] + Calculated value: 378.14, measured value: 378.19.

[0505] The crude product was reacted with XT7 (166 mg, 0.489 mmol), HATU (223 mg, 0.59 mmol), and DIPEA (0.513 mL, 2.93 mmol) in DMF (5 mL) according to the general procedure XXA. The mixture was purified by flash chromatography (silica gel, DCM:MeOH, 1:0 to 4:1) to obtain amide XR21 (240 mg, 70%). MS (ESI + ) C 31 H 31 N 12 O8 + [M+H] + Calculated value: 699.24, measured value: 699.62.

[0506] (S)-5-amino-2-(1-(4-carboxy-4-(4-(((2,4-diaminopteridine-6-yl)methyl)amino)benzamide)butyl)-1H-1,2,3-triazole-4-yl)benzoic acid (XR22) To a solution of amide XR21 (280 mg, 0.423 mmol) in DMF (3 mL), saturated NH4Cl aqueous solution (600 μL) and zinc powder (830 mg, 12.7 mmol) were added. After the UPLC indicated completion of the reaction (approximately 3 hours), the reaction mixture was diluted with DMF (6 mL) and filtered through Celite®. The filtrate was stirred overnight in air, then concentrated, suspended in MeOH (6 mL), stirred for 30 minutes, and filtered. The solid was collected, washed with ether, air-dried, and used in the next step without further purification. The unpurified intermediate (109 mg) was dissolved in THF / water (1:1 2 mL). LiOH (36 mg, 0.85 mmol) was added, and the mixture was stirred for 6 hours. The reaction mixture was acidified with AcOH (0.100 mL, 1.7 mmol), concentrated, and the solvent was evaporated with toluene (2 ×). A portion of the crude product was purified by preparative RP-HPLC (water × 0.1% TFA / MeCN × 0.1% TFA, gradient) to obtain XR22 (11 mg). 1H NMR (400 MHz, DMSO-d6) ppm = 12.57 (br s, 2H), 9.32 (d, J = 14.1 Hz, 2H), 8.84 (s, 1H), 8.61 (br s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.05 (s, 1H), 7.90 (br s, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.74 (m, 3H), 4.62 (s, 2H), 4.38 (m, 3H), 2.00-1.71 (m, 4H). MS (ESI + C 28 H 29 N 12 O5 + [M+H] + The calculated value is 613.24 and the measured value is 613.37.

[0507] Uses of compounds がん cell line Human tumor cell lines SK-BR-3, SW-620, A-549, BT-474, AU-565, and SK-OV-3 were obtained from the American Type Culture Collection (Rockville, MD, USA). Jurkat NucLight Red cells were obtained from Essen BioScience Inc. (Ann Arbor, MI, USA). SK-BR-3 and SK-OV-3 cells were cultured in McCoys 5A medium (Lonza; Walkersville, MD, USA) supplemented with 10% v / w fetal bovine serum (FBS), heat-inactivated serum (HI) (Gibco-Life Technologies; Carlsbad, CA), and 80 U / mL Pen / Strep (Gibco-Life Technologies) in a humidified incubator at 37°C and 5% CO2. SW-620 cells were cultured similarly in RPMI1640 medium (Lonza) containing 10% v / w FBS HI and 80 U / mL Pen / Strep. A-549 cells were cultured similarly in F-12K Nutrient Mixture (1×) (Gibco-Life Technologies) supplemented with 80 U / mL Pen / Strep and 5% v / w certified (Q) FBS (Gibco-Life Technologies). BT-474 cells and AU-565 cells were cultured similarly in RPMI1640 medium (Lonza) containing 10% v / w certified (Q) FBS (Gibco-Life Technologies) and 80 U / mL Pen / Strep. Jurkat NucLight Red cells were similarly cultured in RPMI1640 medium (Lonza) containing 10% v / w FBS HI, 80 U / mL Pen / Strep, and 0.5 μg / mL puromycin (Gibco-Life Technologies).

[0508] In vitro cell survival assay Cells in complete growth medium were placed in 96-well plates (90 μL / well) and incubated at 37°C, 5% CO2, and the following cell densities: SK-BR-3; 6500 cells per well (same for subsequent densities), SW-620; 4000, A-549; 2500, BT-474; 10000, AU-565; 5000, Jurkat NucLight Red; 3000, SK-OV-3; 4000. After incubation overnight, ten log10 dilutions of the free drug were performed using DMSO (Sigma-Aldrich). These free drug dose-response curves were further diluted tenfold with complete growth medium. For the ADC of the present invention, ten log10 dilutions were performed using complete growth medium. 10 μL of the composition containing the free drug or the ADC of the present invention was added to the assay plate (final DMSO content of the free drug was 1%). Six days later, cell viability was evaluated using a luminescence assay kit (CellTiter-Glo® (CTG), Promega Corporation) according to the manufacturer's instructions. Cell viability was calculated by dividing the measured luminescence of each free drug or ADC at different concentrations by the average value of untreated cells (1% DMSO (free drug) or 100% complete growth medium (ADC)) and multiplying by 100.

[0509] The curves were fitted using curve fitting software (GraphPad Prism, version 8.4.0 for Windows®, GraphPad, San Diego, CA or Electronic Laboratory Notebook (ELN) add-in BioAssay, version 12.1.8.11, Perkin Elmer, Waltham, MA) with a nonlinear regression using a variable-gradient (4-variable) sigmoid dose-response equation. Relative IC 50 The value was calculated as the midpoint of the lower and upper limits of the curve when using a four-variable logistic fit. The data was used to obtain the mean IC from at least one of the two experiments. 50 It was reported as a value.

[0510] Experiments on free folate antagonists result [Table 1]

[0511] Comparative compound 1 [ka]

[0512] Comparative compound 1 was synthesized according to the procedure described in Rosowsky et al, J. Med. Chem. 1998, 41, 5310-5319.

[0513] Compounds XJ4, XX5, XX7, XX12, and XT35, which lack a COOH group on the phenyl ring, exhibit lower activity than talotrexin and other folate antagonist compounds that possess a COOH group. Substitution of COOH with SO3H or tetrazole had little effect on folate antagonist activity, but substitution of COOH with CN appeared to reduce activity in SK-BR-3 cells. For amide bond bioequivalents, substitution of the amide bond (Q) with triazole had little effect on folate antagonist activity. Introducing NH2 or OH at the para position of the phenyl ring yields compounds with folate antagonist activity comparable to talotrexin. The folate antagonist activity of compounds with NH2 at the meta position is approximately 10 times lower compared to the corresponding para compounds and (NH2-less) talotrexin.

[0514] Experiments on antibody-drug conjugates Production of DAR2 site-specific complex To a solution of HC41C-modified trastuzumab (10 mg / mL in 100 mM histidine, pH 5), 2-(diphenylphosphino)benzenesulfonic acid (diPPBS) (16-32 molar equivalents per molar equivalent of modified antibody, 10 mM in water (MilliQ®)) was ...

Claims

1. Equation (Ia) 【Chemistry 1】 [In the formula, R １ O, NH ２ or OH; R ２ and R ２’ These are independently N, CH, or CMe; R ３ is NH, N(C １－５ alkyl), CH ２ , CH(C １－５ alkyl), CH(C ２－４ alkenyl), CH(C ２－４ alkynyl) or CH(C １－４ alkoxy); R ４ H, halogen, -COOH, OH, NH ２ , -CONH ２ , -CONHR, -CONHR ２ , C １－４ Alkyl, C １－４ Alkoxy, benzyloxy, tetrazole, -SO ３ H, -OSO ３ H, -PO ３ H ２ , -OPO ３ H ２ -CN or azid (where R is H and C) １－５ (Selected from alkyl) or R ４ teeth, 【Chemistry 2】 (Here, R ａ ' is H, CH ２ F, CHF ２ CF ３ and C １－６ Selected from alkyl groups, each R ａ These are independently H, F, and CH ２ F, CHF ２ CF ３ and C １－６ Selected from alkyl, and possibly two R ａ These are carboxylic acid biological equivalents selected from the group consisting of (which can combine to form a ring); Q is a single bond, -N(R) ７ )-(C=O)-, -(C=O)-N(R ７ ) -, -CH ２ N(R) ７ )-,-N(R ７ )CH ２ -, -N(R ７ ) SO ２ - or - SO ２ N(R) ７ ) - (Here, R ７ H, C １－４ Alkyl, C １－４ Alkenyl or C １－４ Alkinyl (preferably H), or Q is 【Transformation 3】 (Here, R ｂ H and C １－５ Selected from alkyl, T1, T1' and T1'' are independently selected from CH and N, and W1, W1' and W1'' are independently C, CH, S, N, NH, N(C) １－５ An amide bond biological equivalent selected from the group consisting of alkyl and O; X is O, NH, S, C １－５ Alkylene, C １－５ Alkenylene and C １－５ A linking group selected from alkynylenes; L is a linker containing a reactive part (RM), where RM is 【Chemistry 4】 (Here, X1 is selected from -Cl, -Br, -I, -F, -OH, -O-N-succinimide, -O-(4-nitrophenyl), -O-pentafluorophenyl, -O-tetrafluorophenyl, -O-C(O)-R9, and -O-C(O)-OR9, or C(O)-X1 is an active ester; X2 is selected from -Cl, -Br, -I, -O-mesyl, -O-trifuryl, and -O-tosyl; R9 may be substituted in some cases and selected from branched or unbranched C1-10 alkyl, C1-10 heteroalkyl, C3-10 cycloalkyl, C1-10 heterocycloalkyl, C5-10 aryl, or C1-10 heteroaryl. And; 【Transformation 5】 This means that the bond may be a single bond, or it may be a double bond that is not cumulative and, in some cases, not localized. A linker-drug compound represented by the term.

2. The linker-drug compound according to claim 1, wherein R1 is NH2 and R2 and R2' are N.

3. The linker-drug compound according to claim 1, wherein R4 is -COOH or tetrazole.

4. The linker-drug compound according to claim 2, wherein Q is -NH-(C=O)- and R4 is -COOH, tetrazole, or SO3H, or if R4 is -COOH, Q is triazole.

5. The linker-drug compound according to claim 2, represented by formula (Ic). 【Transformation 6】

6. RM is 【Transformation 7】 The linker-drug compound according to claim 1 or 2.

7. L 【Transformation 8】 [Here, m is an integer from 1 to 10, preferably 5; AA is an amino acid, preferably a neutral amino acid; p is 0, 1, 2, 3, or 4; q is an integer between 1 and 12, preferably 2; ES is a single bond or 【Chemistry 9】 An extension spacer selected from; RL is a single bond or 【Chemistry 10】 (Here, t is an integer from 1 to 10, and R １０ In some cases, C is substituted. １－４ It is an alkoxy, R １１ H, and possibly substituted C １－６ Alkyl, and possibly substituted C ６－１４ Aryl, or possibly substituted C-bond C ３－８ (It is a heteroaryl) [A detachable spacer selected from the following] And; Preferably L is 【Chemistry 11】 The linker-drug compound according to claim 6.

8. The linker-drug compound 【Chemistry 12】 The linker-drug compound according to claim 6.

9. The linker-drug compound 【Chemistry 13】 The linker-drug compound according to any one of claims 1 to 4.

10. Formula (III) Ab-(L-D) ｙ (III) [In the formula, Ab is an antibody or its antigen-binding fragment; L-D is a linker-drug compound according to any one of claims 1 to 9; y represents the average drug-antibody ratio between 1 and 16; The linker-drug compound is preferably bound to the antibody or its antigen-binding fragment via a cysteine ​​residue of the antibody or its antigen-binding fragment. An antibody-drug conjugate represented by [the specified symbol].

11. The antibody-drug conjugate 【Chemistry 14-1】 【Chemistry 14-2】 (Here, Ab is an antibody or its antigen-binding fragment; (y represents the average drug-antibody ratio of 1 to 16, preferably 1 to 10.) The antibody-drug conjugate according to claim 10.

12. The antibody or antigen-binding fragment is Annexin Al, B7H3, B7H4, BCMA, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242, CAIX, CCR2, CCR5, CD2, CD19, CD20, CD22, CD24, CD30, CD33, CD37, CD38, CD40, CD44, CD47, CD56, CD70, CD71, CD73, CD74, C D79, CD115, CD123, CD138, CD203c, CD303, CD333, CDCP1, CEA, CEACAM, claudin 4, claudin 7, CLCA-1, CLL1, c-ME T, Cripto, DLL3, EGFL, EGFR, EPCAM, EphA2, EPhB3, ETBR, FAP, FcRL5, FGFR3, FOLR1, FRβ, GCC, GD2, GITR, GLOBO H, GPA33, GPC3, GPNMB, HER2, p95HER2, HER3, HMW-MAA, Integrin α, IGF1R, TM4SF1, Lewis A-like carbohydrate, Lewis X, Lewis Y, LGR5, LIV1, Mesothelin, MN, MUC1, MUC16, NaPi2b, Nectin-4, Notch 3, PD-1, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4, TF, TF-Ag, Tag72, TNFα, TNFR, TROP2, uPAR, VEGFR and VLA The antibody-drug conjugate according to claim 10 or 11, which binds to a target antigen selected from the group consisting of the following.

13. A pharmaceutical composition comprising a linker-drug compound according to any one of claims 1 to 9 or an antibody-drug conjugate according to any one of claims 10 to 12, and one or more pharmaceutically acceptable additives.

14. A linker-drug compound according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, for use as a pharmaceutical.

15. A linker-drug compound according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, for use in the treatment of solid tumors or hematological cancers.

16. A linker-drug compound according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, for use in the treatment of autoimmune diseases, particularly rheumatoid arthritis.

17. A linker-drug compound according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, for use in treating infections caused by bacteria, viruses, parasites, or other organisms.

18. A linker-drug compound according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 12, or a pharmaceutical composition according to claim 13, for use in combination therapy with one or more other therapeutic agents.

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