Antibody drug conjugates comprising trabectedin and rubitedin derivatives

Abstract

The invention relates to novel trabectedin and rubitedin derivatives, corresponding antibody drug conjugates and a method for preparing the antibody drug conjugates. The present invention also relates to a pharmaceutical dosage form comprising a novel trabectedin or rubitedin derivative or a corresponding antibody drug conjugate. Furthermore, the present invention relates to novel trabectedin and rubitedin derivatives, corresponding antibody drug conjugates and corresponding pharmaceutical dosage forms for use as a medicament or for the treatment of specific cancer types.

Description

[0001] This patent application claims priority to and the benefits of international patent application PCT / SG2023 / 050138 filed on March 7, 2023.

[0002] The invention disclosed herein relates to novel trabectedin and lubectedin derivatives, corresponding antibody-drug conjugates, and methods for preparing the antibody-drug conjugates. The invention also relates to pharmaceutical dosage forms comprising the novel trabectedin or lubectedin derivatives or corresponding antibody-drug conjugates. In addition, the invention also relates to novel trabectedin and lubectedin derivatives, corresponding antibody-drug conjugates, and corresponding pharmaceutical dosage forms for use as medicaments or for treating specific cancer types. Background of the Invention

[0004] In recent years, cancer treatment has made progress with the development of drugs that can more effectively target and kill cancer cells. In this regard, researchers have taken advantage of cell surface receptors and antigens that are selectively expressed by target cells (e.g. cancer cells). This has led to the development of drugs based on (monoclonal) antibodies (mAbs), for example, antibodies that bind to tumor-specific or tumor-associated antigens. In order to achieve inhibition of tumor cells, cytotoxic compounds such as chemotherapy drugs, bacterial toxins and plant toxins, as well as radionuclides, can be chemically linked to target-specific antibodies as payloads via suitable linker chemistries. The resulting antibody drug conjugates (ADCs) are able to deliver cytotoxic compounds in a targeted manner, and therefore generally allow for more effective cancer treatment.

[0005] ADCs represent a challenging area of ​​development due to the inherent complexity of payloads, linker chemistries, and existing antibodies, and the continued need to develop alternative or improved ADCs. Therefore, careful consideration is required when selecting the various components.

[0006] ADC technology is believed to have potential advantages over other approaches, including improved efficacy compared to antibody drugs and a lower risk of side effects compared to traditional anticancer agents due to its ability to target cancer cells with minimal impact on healthy cells and release the drug only under specific conditions.

[0007] Nonetheless, there is still a need to develop alternative or improved cytotoxic compounds that can be used both as direct anticancer drugs and as payloads in corresponding ADCs.

[0008] In this regard, trabectedin and the structurally related rubitidin, which belong to the ecteinascidin family, have recently attracted the attention of cancer researchers:

[0009]

[0010] Trabectedin and rubectedin are DNA alkylating agents that can induce tumor cell apoptosis, reshape the tumor microenvironment, and inhibit angiogenesis. They exhibit potent cytotoxicity in many different cancer cell lines across multiple cancer types.

[0011] WO 2011 / 147828 A1 discloses the synthesis of derivatives of both trabectedin and lubectedin. Later, WO 2020 / 084115 A1 and WO 2021 / 214126 A1 disclosed lubectedin derivatives and corresponding ADCs. SUMMARY OF THE INVENTION

[0013] The present invention itself provides novel compounds derived from trabectedin and rubectedin and corresponding antibody drug conjugates, which are useful in the treatment of cancer.

[0014] While the parent compounds trabectedin and rubectedin have been extensively investigated for their medical uses in both traditional small molecule therapeutics and ADC approaches, less progress has been made with their derivatives to date.

[0015] In contrast, the trabectedin and rubectedin derivatives according to the present invention did show better in vitro potency than the parent drug, and they were successfully tested against several tumor cell lines in the form of conventional active ingredients and also in a target-specific manner using the corresponding ADCs.

[0016] The present invention generally relates to compounds of formula (I) or pharmaceutically acceptable salts, esters, solvates, tautomers or stereoisomers thereof:

[0017]

[0018] in

[0019] -R1 is a substituent having a negative inductive effect;

[0020] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0021] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n(C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl;

[0022] -R4 is selected from -H, -CH2OR R4 、-CH2NHR R4 and -CH2C(=O)OR R4 , where R R4 Is -H or -C 1-4 alkyl; and

[0023] -R5 is -H or -C 1-4 alkyl.

[0024] The present invention also relates to a compound of formula (VII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0025]

[0026] in

[0027] -R1 is a substituent having a negative inductive effect;

[0028] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0029] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0030] -R4 is -H or -C 1-4alkyl.

[0031] The present invention also relates to a compound of formula (XIII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0032]

[0033] in

[0034] -R1 is a substituent having a negative inductive effect;

[0035] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0036] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0037] -R4 is -H or -CH2NHR R4 , where R R4 Is -H or -C 1-4 alkyl.

[0038] "Negative inductive effect" refers to a local change in electron density caused by electron-withdrawing functional groups or atoms elsewhere in the molecule, resulting in the appearance of a permanent dipole in the σ (sigma) bond. In the meaning of the present invention, a substituent with a negative inductive effect is based on hydrogen, i.e., a substituent (including a single atom substituent) whose negative inductive effect is stronger than that of hydrogen. Examples of suitable substituents include carbonyl (-C (= O) R), carboxyl (-C (= O) OR), -OH, -I, -Br, -Cl, -NO2, -NH2, -COOH, -F and -C≡N. Preferred suitable substituents can include carbonyl, -OH, -NO2, -F and -C≡N, more preferably -OH, -F and -C≡N, and most preferably -OH and -C≡N.

[0039] Unless otherwise defined, the term "alkyl" as used herein refers to an unsubstituted saturated hydrocarbon group (branched or straight chain). Where indicated, the number of carbons refers to all carbons present in the group, including carbons in side chains. For example, the term "C 1-4 "Alkyl" is intended to include -CH3, -CH2CH3, -CH2CH2CH3 (n-propyl), -CH(CH3)2 (isopropyl), -CH2CH2CH2CH3 (n-butyl), -CH2CH2CH(CH3)2 (isobutyl), -CH(CH3)CH2CH3 (sec-butyl) and -CH(CH3)3 (tert-butyl). Wherever C is mentioned herein, 1-4 Alkyl, it should be understood that -CH3 is its preferred embodiment.

[0040] More specifically, according to a first aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0041]

[0042] in

[0043] -R1 is a substituent having a negative inductive effect;

[0044] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0045] -R3 is -H or -X(CH2)m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl;

[0046] -R4 is selected from -H, -CH2OR R4 、-CH2NHR R4 and -CH2C(=O)OR R4 , where R R4 Is -H or -C 1-4 alkyl; and

[0047] -R5 is -H or -C 1-4 alkyl;

[0048] Provided that compounds of formula (I) having the following combinations of R2, R3, R4 and R5 are not included:

[0049] -R2 is -OCH3, R3 is -OH, R4 is -H and R5 is -H.

[0050] According to other aspects of the present invention, there is provided a compound of formula (VII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0051]

[0052] in

[0053] -R1 is a substituent having a negative inductive effect;

[0054] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0055] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(RR3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0056] -R4 is -H or -C 1-4 alkyl;

[0057] Provided that compounds of formula (VII) having the following combinations of R2, R3 and R4 are not included:

[0058] -R2 is -H, R3 is -H and R4 is -H; and

[0059] -R2 is -OCH3, R3 is -H and R4 is -H.

[0060] According to other aspects of the present invention, there is provided a compound of formula (XIII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0061]

[0062] in

[0063] -R1 is a substituent having a negative inductive effect;

[0064] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0065] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0066] -R4 is -H or -CH2NHR R4 , where R R4 Is -H or -C 1-4 alkyl.

[0067] The compounds provided according to the present invention can be used as the payload of the corresponding antibody drug conjugates. Therefore, another aspect of the present invention relates to an antibody drug conjugate of formula (XIX):

[0068]

[0069] in

[0070] -Ab represents an antibody, an antigen-binding fragment, or an immunologically active portion thereof;

[0071] -L represents a linker; and

[0072] -D represents a compound selected from trabectedin, rubectedin, and a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, preferably a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof;

[0073] in

[0074] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0075] -z is an integer from 1 to 20.

[0076] Another aspect of the invention relates to a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to the previous aspect for use as a medicament or, according to yet another aspect, for use in the treatment of cancer, preferably ovarian cancer, gastric cancer or breast cancer.

[0077] Another aspect of the invention relates to an antibody drug conjugate as defined above for use as a medicament, or according to another aspect, for use in the treatment of cancer, preferably ovarian cancer, gastric cancer or breast cancer.

[0078] According to another aspect of the present invention, a method for preparing an antibody drug conjugate of formula (XIX) is provided:

[0079]

[0080] The method comprises coupling an antibody, an antigen-binding fragment or an immunologically active portion thereof (Ab) to a compound (D) via a linker (L), wherein

[0081] -D is a compound selected from trabectedin, rubectedin, and a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, preferably a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof;

[0082] -L is a linker disclosed below:

[0083] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0084] -z is an integer from 1 to 20.

[0085] Another aspect of the present invention relates to a pharmaceutical dosage form comprising a therapeutically effective amount of a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to the aforementioned aspects or a therapeutically effective amount of an antibody drug conjugate according to the aforementioned aspects.

[0086] Yet another aspect of the present invention relates to a method for treating cancer, wherein the method comprises administering to a subject a therapeutically effective amount of a compound according to the preceding aspects, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, a therapeutically effective amount of an antibody drug conjugate according to the preceding aspects, or a pharmaceutical dosage form according to the preceding aspects.

[0087] The compounds of the present invention have been found to exhibit improved efficacy against the proliferation of various tumor cell lines. These tumor cell lines include OV-90 (CRL-11732) associated with ovarian papillary serous adenocarcinoma, NCI-N87 (CRL-5822) associated with gastric cancer, and MDA-MB-231 associated with breast adenocarcinoma. As demonstrated, the 50% cytotoxic concentration (CC) of the compounds of the present invention was significantly higher than that of the parent compounds trabectedin and rubectedin, respectively. 50 This discovery makes these compounds potent active ingredients in the treatment of ovarian, breast and gastric cancers.

[0088] In addition, it was found that the compounds of the present invention are potent payloads when used in antibody drug conjugates. Specifically, these compounds were tested as payloads in combination with different chemical linkers and cetuzumab (anti-ROR1), gosartan (anti-TROP2) and trastuzumab (anti-HER2). Other antibodies tested included paltratuzumab (anti-HER3), mituximab (anti-FRα) and cetuximab (anti-EGFR). It is worth noting that there was a certain correlation between the cytotoxicity of the compounds themselves and the effects of the corresponding antibody drug conjugates on the cell lines NCI-N87 and MDA-MB-231 (also in CC 50 In some cases, it is even believed that the cytotoxicity of the antibody drug conjugate is superior to the cytotoxic effects of the payload and antibody alone. This makes the antibody drug conjugate of the present invention a potent candidate molecule for the treatment of various cancer types, particularly cancers associated with the expression of ROR1, TROP2 or HER2 (sometimes also referred to as HER2 / new). In terms of cancer types, the antibody drug conjugate of the present invention can be used for the targeted treatment of ovarian cancer, breast cancer and gastric cancer.

[0089] Detailed description of implementation plan

[0090] Advantageous embodiments will be discussed in more detail below. It should be understood that these details apply to all aspects of the invention.

[0091] As described above, one aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0092]

[0093] in

[0094] -R1 is a substituent having a negative inductive effect;

[0095] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0096] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n(C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl;

[0097] -R4 is selected from -H, -CH2OR R4 、-CH2NHR R4 and -CH2C(=O)OR R4 , where R R4 Is -H or -C 1-4 alkyl; and

[0098] -R5 is -H or -C 1-4 alkyl;

[0099] Provided that compounds of formula (I) having the following combinations of R2, R3, R4 and R5 are not included:

[0100] -R2 is -OCH3, R3 is -OH, R4 is -H and R5 is -H.

[0101] According to one embodiment, R1 in formula (I) is -OH or -C≡N. According to a preferred embodiment, R1 is -C≡N. It has been found that, in some cases, if R1 is -C≡N, the potency is more significantly enhanced relative to the parent compound trabectedin than if R1 is -OH.

[0102] According to a further embodiment, the present invention relates to a compound of formula (I) as shown above or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0103] -R2 is -H, -OR R2 、-OCH2CH2OR R2 、-OCH2C(CH3)2OR R2 、-OCH2CH2NHR R2 、-OCH2C(=O)OR R2 、-NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2CH2NH2, -OCH2C(=O)OH, -NH2 or -NHC(=O)CH2OH; and / or

[0104] -R3 is -H, -OR R3 、-NHR R3 or -NHC(=O)CH2OR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H, -OH, -NH2 or -NHC(=O)CH2OH.

[0105] According to a further embodiment, the present invention relates to a compound of formula (I) as shown above or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0106] -R4 is -H, -CH2OR R4 or -CH2NHR R4 , where R R4 Is -H or -C 1-4 Alkyl, preferably R4 is -H, -CH2OH or -CH2NH2; and / or

[0107] -R5 is -H or -C 1-4 Alkyl, preferably R5 is -H or -CH3.

[0108] According to a further embodiment, the present invention relates to a compound of formula (I) as shown above or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0109] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0110] -R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2CH2NH2, -OCH2C(=O)OH, -NH2 or -NHC(=O)CH2OH, preferably R2 is -OCH2CH2OH;

[0111] -R3 is -H, -OH, -NH2 or -NHC(=O)CH2OH, preferably R3 is -NH2;

[0112] -R4 is -H, -CH2OH or -CH2NH2, preferably R4 is -CH2NH2; and

[0113] -R5 is -H or -CH3, preferably R5 is -CH3.

[0114] For example, formula (I) can be one of the following:

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Additionally or alternatively, formula (I) may be, for example, one of the following:

[0121]

[0122] In another embodiment of the present invention:

[0123] -R2 is -H, -OR R2 、-OCH2CH2OR R2 、-OCH2C(=O)OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(=O)OH or -NH2; and / or

[0124] -R3 is -H, -OR R3 、-OCH2CH2NHR R3 、-NHR R3 、-NHC(=O)CH2OR R3 or -NHC(=O)CH2NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H, -OH, OCH2CH2NH2, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2.

[0125] In yet another embodiment of the present invention:

[0126] -R4 is -H, -CH2OR R4 or -CH2NHR R4 , where R R4 Is -H or -C 1-4 Alkyl, preferably R4 is -H; and / or -R5 is -H or -C 1-4 Alkyl, preferably R5 is -H.

[0127] In a preferred embodiment of the present invention:

[0128] -R1 is -OH or -C≡N;

[0129] -R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(=O)OH or -NH2;

[0130] -R3 is -H, -OH, -OCH2CH2NH2, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2;

[0131] -R4 is -H; and

[0132] -R5 is -H.

[0133] In a more preferred embodiment of the present invention, formula (I) is one of the following:

[0134]

[0135]

[0136]

[0137]

[0138] Some compounds of the present invention exhibit particularly high activity against individual cell lines, such as OV-90, which indicates that they have excellent therapeutic effects on ovarian cancer, especially ovarian papillary serous carcinoma. Therefore, in another embodiment, the present invention relates to a compound of formula (I) as shown above or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0139] -R1 is -C≡N;

[0140] -R2 is -H, -OCH3 or -OCH2CH2OH;

[0141] -R3 is -OH, -OCH2CH2NH2, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2;

[0142] -R4 is -H; and

[0143] -R5 is -H.

[0144] For example, in another more preferred embodiment of the present invention, formula (I) is one of the following formulas:

[0145]

[0146]

[0147]

[0148] Other compounds of the present invention exhibit particularly high activity against individual cell lines, such as MDA-MB-231, indicating that they have excellent therapeutic effects on breast cancer, particularly breast adenocarcinoma. Therefore, in this embodiment, the present invention relates to a compound of formula (I) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0149] -R1 is -C≡N;

[0150] -R2 is -H, -OCH3 or -OCH2CH2OH;

[0151] -R3 is -OH, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2;

[0152] -R4 is -H; and

[0153] -R5 is -H.

[0154] For example, in a more preferred embodiment of the present invention, formula (I) is one of the following:

[0155]

[0156]

[0157] As described above, another aspect of the present invention relates to a compound of formula (VII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0158]

[0159] in

[0160] -R1 is a substituent having a negative inductive effect;

[0161] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0162] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3)2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0163] -R4 is -H or -C 1-4 alkyl;

[0164] Provided that compounds of formula (VII) having the following combinations of R2, R3 and R4 are not included:

[0165] -R2 is -H, R3 is -H and R4 is -H; and

[0166] -R2 is -OCH3, R3 is -H and R4 is -H.

[0167] According to one embodiment, R1 in formula (VII) is -OH or -C≡N. According to a preferred embodiment, R1 is -C≡N. It has been found that, in some cases, if R1 is -C≡N, the potency is more significantly enhanced relative to the parent compound, rubitidine, compared to when R1 is -OH.

[0168] According to a further embodiment, the present invention relates to a compound of formula (VII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0169] -R2 is -H, -OR R2 、-NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -NH2 or -NHC(=O)CH2OH; and / or

[0170] -R3 is -H, -NHR R3 or -NHC(=O)CH2OR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H, -NH2 or -NHC(=O)CH2OH.

[0171] According to a further embodiment, the present invention relates to a compound of formula (VII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0172] -R4 is -H or -CH3.

[0173] According to a further embodiment, the present invention relates to a compound of formula (VII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0174] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0175] -R2 is -H, -OCH3, -NH2 or -NHC(=O)CH2OH, preferably R2 is -NH2; -R3 is -H, -NH2 or -NHC(=O)CH2OH, preferably R3 is -NH2; and -R4 is -H or -CH3, preferably R4 is -CH3.

[0176] For example, formula (VII) can be one of the following:

[0177]

[0178]

[0179]

[0180] Additionally or alternatively, formula (VII) may be, for example, one of the following:

[0181]

[0182]

[0183] In another embodiment of the present invention:

[0184] -R2 is -H, -NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -NH2 or -NHC(=O)CH2OH; and / or

[0185] -R3 is -H or -NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H or -NH2.

[0186] In yet another embodiment of the present invention:

[0187] -R4 is -H or -CH3.

[0188] In a preferred embodiment of the present invention:

[0189] -R1 is -OH or -C≡N;

[0190] -R2 is -H, -NH2 or -NHC(=O)CH2OH;

[0191] -R3 is -H or -NH2; and

[0192] -R4 is -H or -CH3.

[0193] In a more preferred embodiment of the present invention, formula (VII) is one of the following:

[0194]

[0195]

[0196] As described above, some compounds of the present invention exhibit particularly high activity against individual cell lines, such as OV-90, which indicates that they have excellent therapeutic effects on ovarian cancer, particularly ovarian papillary serous carcinoma. Therefore, in another embodiment, the present invention relates to a compound of formula (VII) as shown above or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0197] -R1 is -C≡N;

[0198] -R2 is -NH2;

[0199] -R3 is -H; and

[0200] -R4 is -CH3.

[0201] Thus, in this embodiment, Formula (VII) has the following formula:

[0202]

[0203] Other compounds of the present invention exhibit particularly high activity against individual cell lines, such as MDA-MB-231, indicating that they have excellent therapeutic effects on breast cancer, particularly breast adenocarcinoma. Therefore, in this embodiment, the present invention relates to a compound of formula (VII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0204] -R1 is -C≡N;

[0205] -R2 is -H or NH2;

[0206] -R3 is -H or -NH2; and

[0207] -R4 is -H.

[0208] For example, in a more preferred embodiment of the present invention, formula (I) is one of the following:

[0209]

[0210]

[0211] As described above, another aspect of the present invention relates to a compound of formula (XIII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0212]

[0213] in

[0214] -R1 is a substituent having a negative inductive effect;

[0215] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0216] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0217] -R4 is -H or -CH2NHR R4 , where R R4 Is -H or -C 1-4 alkyl.

[0218] According to one embodiment, R1 in formula (XIII) is -OH or -C≡N. According to a preferred embodiment, R1 is -C≡N. It has been found that, in some cases, if R1 is -C≡N, the potency is more significantly enhanced relative to the parent compound, rubitidine, compared to when R1 is -OH.

[0219] According to another embodiment, the present invention relates to a compound of formula (XIII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0220] -R2 is -OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -OCH3 or -NH2; and / or

[0221] -R3 is -H.

[0222] According to another embodiment, the present invention relates to a compound of formula (XIII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0223] -R4 is -H or -CH2NH2.

[0224] According to another embodiment, the present invention relates to a compound of formula (XIII) as shown above, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0225] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0226] -R2 is -OCH3 or -NH2, preferably R2 is -NH2;

[0227] -R3 is -H; and

[0228] -R4 is -H or -CH2NH2, preferably R4 is -CH2NH2.

[0229] For example, formula (XIII) can be one of the following:

[0230]

[0231]

[0232] Additionally or alternatively, formula (XIII) may be, for example, the following:

[0233]

[0234] In another embodiment of the present invention:

[0235] -R2 is -OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4Alkyl, preferably R2 is -OCH3 or -NH2; and / or -R3 is -H.

[0236] In yet another embodiment of the present invention:

[0237] -R4 is -H.

[0238] In a preferred embodiment of the present invention:

[0239] -R1 is -OH or -C≡N;

[0240] -R2 is -OCH3 or -NH2;

[0241] -R3 is -H; and

[0242] -R4 is -H.

[0243] In a more preferred embodiment of the present invention, formula (XIII) is one of the following:

[0244]

[0245]

[0246] In all aspects and corresponding embodiments described above, preferably R R2 、R R3 and R R4 independently can be -H or -CH3.

[0247] Likewise, as described above, another aspect of the present invention relates to an antibody drug conjugate of formula (XIX):

[0248]

[0249] in

[0250] -Ab represents an antibody, an antigen-binding fragment, or an immunologically active portion thereof;

[0251] -L represents a linker; and

[0252] -D represents a compound selected from trabectedin, rubectedin and the compounds according to the aforementioned aspects and embodiments, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, preferably a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to the aforementioned aspects and embodiments;

[0253] And among them

[0254] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0255] -z is an integer from 1 to 20.

[0256] As defined herein, in the antibody drug conjugates of the present invention, compound D is covalently linked to L via a nitrogen or oxygen present in the compound. Those skilled in the art will understand that, formally, this means that the linker L replaces a proton (H) present in the corresponding compound of the present invention. For example, if the compound has the formula:

[0257]

[0258] The antibody drug conjugate can then be described by the structure of the following formula (XIX), wherein the compound is linked to the linker L via the nitrogen present in R2:

[0259]

[0260] Unless expressly stated otherwise, it will be understood by those skilled in the art that D may be covalently linked to the linker L through any other nitrogen or oxygen atom present in the compound. However, in a preferred embodiment of the present invention, D is covalently linked to L through a nitrogen or oxygen present in any one of R2, R3 or R4, preferably any one of R2 or R3 (for both the trabectedin and lubectedin derivatives disclosed herein).

[0261] Antibody drug conjugates are characterized by a specific so-called drug-antibody ratio (also referred to as DAR). For a single molecule as shown above, the drug-antibody ratio is an integer, denoted herein as "z". Therefore, in one embodiment of the invention, z is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, more preferably 2 or 4. However, due to the manufacturing process, the measured drug-antibody ratio of a given sample (consisting of a large number of antibody drug conjugate molecules) is generally not an integer, but an average. Therefore, in another embodiment, the antibody drug conjugate is characterized by a specific average drug-antibody ratio, which can be less than 1.5, or in the range of at least 1.5 to less than 2.5 or at least 2.5 to less than 3.5.

[0262] In the antibody drug conjugate of formula (XIX) of the present invention, Ab represents an antibody, an antigen-binding fragment or an immunologically active portion thereof. In a preferred embodiment of the present invention, Ab represents an antibody.

[0263] Generally, the present invention is not limited to specific antibodies. However, taking into account the structure of the compounds of the present invention and the inhibitory effect on specific cancer cell lines caused by the structure of each compound, some combinations may be beneficial. In terms of their targets, it has been found that anti-ROR1 antibodies, anti-TROP2 antibodies, and anti-HER2 antibodies may be beneficial. Other available antibodies include anti-HER3 antibodies, anti-FRα antibodies, and anti-EGFR antibodies. Therefore, in another embodiment of the present invention, antibody, its antigen-binding fragment or immunologically active portion (Ab) is selected from anti-HER2 antibodies, anti-ROR1 antibodies, anti-TROP2 antibodies, anti-HER3 antibodies, anti-FRα antibodies, and anti-EGFR antibodies. In a preferred embodiment, antibody, its antigen-binding fragment or immunologically active portion (Ab) is selected from anti-HER2 antibodies, anti-ROR1 antibodies, anti-TROP2 antibodies, anti-HER3 antibodies, and anti-EGFR antibodies, more preferably anti-HER2 antibodies, anti-TROP2 antibodies, and anti-HER3 antibodies, even more preferably anti-HER2 antibodies and anti-TROP2 antibodies, and most preferably anti-HER2 antibodies. In alternative embodiments, the antibody, its antigen-binding fragment or immunologically active portion (Ab) is selected from an anti-ROR1 antibody or an anti-EGFR antibody, preferably an anti-ROR1 antibody. Therefore, specific antibodies that can be used for the purposes of the present invention include Cirmtuzumab (as an example of an anti-ROR1 antibody), Sacituzumab (as an example of an anti-TROP2 antibody) and Trastuzumab (as an example of an anti-HER2 antibody). Other available antibodies include Patritumab (as an example of an anti-HER3 antibody), Mirvetuximab (as an example of an anti-FRα antibody) and Cetuximab (as an example of an anti-EGFR antibody). Therefore, in a more specific embodiment of the present invention, the antibody, its antigen-binding fragment or immunologically active portion (Ab) is selected from Cirmtuzumab, Gosacituzumab, Trastuzumab, Patritumab, Mirvetuximab and Cetuximab. In a preferred embodiment, the antibody, its antigen-binding fragment or immunologically active portion (Ab) is selected from citrullumab, goxatuzumab, trastuzumab, pertrastuzumab and cetuximab, more preferably goxatuzumab, trastuzumab and pertrastuzumab, still more preferably goxatuzumab and trastuzumab, and most preferably trastuzumab. In an alternative embodiment, the antibody, its antigen-binding fragment or immunologically active portion (Ab) is citrullumab or cetuximab, preferably citrullumab.

[0264] In another embodiment of the present invention, the anti-ROR1 antibody has an antigen-binding fragment (Fb) comprising a heavy chain and a light chain. ab), wherein the sequence of the heavy chain is preferably substantially identical to SEQ ID NO. 3 disclosed in US2022 / 0133901 A1, and the sequence of the light chain is substantially identical to SEQ ID NO. 4 disclosed in US2022 / 0133901 A1.

[0265] For example, in another embodiment, an antigen-binding fragment (F ab ) has the following sequence (SEQ ID NO.1):

[0266] QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0267] And the light chain has the following sequence (SEQ ID NO.2):

[0268] DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0269] In another embodiment of the present invention, the anti-TROP2 antibody has an antigen-binding fragment (F) comprising a heavy chain and a light chain. ab ), wherein the sequence of the heavy chain is preferably substantially identical to chain ID INN 10418_H, and the sequence of the light chain is substantially identical to chain ID INN10418_L, as disclosed in the International Immunogenetics Information System (IMGT) mAb database as INN number 10418 (IMGT publication date: March 20, 2017).

[0270] For example, in another embodiment, an antigen-binding fragment (F ab ) has the following sequence (SEQ ID NO.3):

[0271] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0272] And the light chain has the following sequence (SEQ ID NO.4):

[0273] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0274] In another embodiment of the present invention, the anti-HER2 antibody has an antigen-binding fragment (Fb) comprising a heavy chain and a light chain. ab ), wherein the sequence of the heavy chain is preferably substantially identical to the anti-HER2 heavy chain disclosed in DrugBank with accession number DB00072, and the sequence of the light chain is substantially identical to the anti-HER2 light chain disclosed in DrugBank with accession number DB00072.

[0275] For example, in another embodiment, an antigen-binding fragment (F ab ) has the following sequence (SEQ ID NO.5):

[0276] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0277] And the light chain has the following sequence (SEQ ID NO.6):

[0278] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0279] In another embodiment of the present invention, the anti-HER3 antibody has an antigen-binding fragment (Fb) comprising a heavy chain and a light chain. ab ), wherein the sequence of the heavy chain is preferably substantially identical to chain ID INN 9549_H, and the sequence of the light chain is substantially identical to chain ID INN9549_L, as disclosed in the International Immunogenetics Information System (IMGT) mAb database under INN number 9549.

[0280] For example, in another embodiment, an antigen-binding fragment (F ab ) has the following sequence (SEQ ID NO.7):

[0281] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0282] And the light chain has the following sequence (SEQ ID NO.8):

[0283] DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0284] In another embodiment of the present invention, the anti-FRα antibody has an antigen-binding fragment (F) comprising a heavy chain and a light chain. ab ), wherein the sequence of the heavy chain is preferably substantially identical to chain ID INN 10187_H, and the sequence of the light chain is substantially identical to chain ID INN10187_L, as disclosed in the International Immunogenetics Information System (IMGT) mAb database as INN number 10187 (IMGT publication date: July 26, 2016).

[0285] For example, in another embodiment, an antigen-binding fragment of an anti-FRα antibody (Fab ) has the following sequence (SEQ ID NO.9):

[0286] QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYDGSRAMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0287] And the light chain has the following sequence (SEQ ID NO.10):

[0288] DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0289] In another embodiment of the present invention, the anti-EGFR antibody has an antigen-binding fragment (F) comprising a heavy chain and a light chain. ab ), wherein the sequence of the heavy chain is preferably substantially identical to the anti-EGFR heavy chain disclosed in DrugBank with accession number DB00002, and the sequence of the light chain is substantially identical to the anti-EGFR light chain disclosed in DrugBank with accession number DB00002.

[0290] For example, in another embodiment, an antigen-binding fragment (F ab ) has the following sequence (SEQ ID NO.11):

[0291] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0292] And the light chain has the following sequence (SEQ ID NO.12):

[0293] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0294] The term "substantially identical" can refer to sequences of identical sequence units (e.g., amino acid residues) of a specific percentage when compared and aligned for maximum correspondence within a comparison window (preferably within the entire sequence). Thus, in additional embodiments, it will be understood that substantially identical sequences are at least 80% identical, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99%. In alternative embodiments, the sequences are identical (100% sequence identity) within the entire sequence.

[0295] Percent sequence identity (%) relative to a reference protein sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Alignment for determining percent amino acid sequence identity can be accomplished in a variety of ways known to those skilled in the art. As used herein, percent sequence identity refers to identity as determined by BLAST using the so-called blastp algorithm (protein-protein BLAST).

[0296] Typically, D in Formula (XIX) is a compound selected from trabectedin, rubectedin, and a compound of Formula (I), (VII), or (XIII) disclosed herein, or a pharmaceutically acceptable salt, ester, solvate, tautomer, or stereoisomer thereof. In a preferred embodiment, D is a compound of Formula (I), (VII), or (XIII) disclosed herein, or a pharmaceutically acceptable salt, ester, solvate, tautomer, or stereoisomer thereof.

[0297] Useful combinations of an antibody, antigen-binding fragment or immunologically active portion thereof (Ab) and a compound of formula (XIX), a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof used as a payload (D) include any combination of the individual embodiments described above for Ab and D, respectively.

[0298] Some particularly useful embodiments are described below. For example, in one embodiment, D is:

[0299] -Compounds of formula (I), wherein R1 is -OH or -C≡N, and R2 is selected from -H, -OCR R2 and -CH2CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is selected from -OR R3 、-NHR R3 and -NHC(=O)CH2NHR R3 , where R R3 Is -H or -C 1-4Alkyl, R4 is -H or -C 1-4 Alkyl, and R5 is -H or -C 1-4 Alkyl, preferably R1 is -OH or -C≡N, R2 is selected from -H, -OCH3 and -CH2CH2OH, R3 is selected from -OH, -NH2 and -NHC(=O)CH2NH2, R4 is -H, and R5 is -H; or

[0300] - A compound of formula (VII) wherein R1 is -OH or -C≡N, and R2 is -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -H or -C 1-4 Alkyl, and R4 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -NH2, R3 is -H, and R4 is -H;

[0301] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0302] Specifically, in a preferred embodiment, D is one of the following formulae:

[0303]

[0304]

[0305] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0306] In another embodiment of the present invention, D is:

[0307] - a compound of formula (I), wherein R1 is -C≡N, R2 is selected from -H, -OCR R2 and -CH2CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is selected from -OR R3 、-NHR R3 and -NHC(=O)CH2NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, R4 is -H or -C 1-4 Alkyl, and R5 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is selected from -H, -OCH3 and -CH2CH2OH, R3 is selected from -OH, -NH2 and -NHC(=O)CH2NH2, R4 is -H, and R5 is -H; or

[0308] - A compound of formula (VII) wherein R1 is -OH or -C≡N, and R2 is -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -H or -C 1-4 Alkyl, and R4 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -NH2, R3 is -H, and R4 is -H;

[0309] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0310] Specifically, in a preferred embodiment, D is one of the following formulae:

[0311]

[0312]

[0313] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0314] In yet another embodiment of the present invention, D is:

[0315] - a compound of formula (I), wherein R1 is -C≡N, R2 is selected from -H, -OCR R2 and -CH2CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -OR R3 or -NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, R4 is -H or -C 1-4 Alkyl, and R5 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is selected from -H, -OCH3 and -CH2CH2OH, R3 is -OH or -NH2, R4 is -H, and R5 is -H; or

[0316] - A compound of formula (VII) wherein R1 is -OH or -C≡N, and R2 is -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -H or -C 1-4 Alkyl, and R4 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -NH2, R3 is -H, and R4 is -H;

[0317] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0318] Specifically, in a preferred embodiment, D is one of the following formulae:

[0319]

[0320]

[0321] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0322] In yet another embodiment of the present invention, D is:

[0323] - a compound of formula (I) wherein R1 is -C≡N, R2 is -H or -CH2CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -OR R3 or -NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, R4 is -H or -C 1-4 Alkyl, and R5 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -H or -OCH3, R3 is -OH or -NH2, R4 is -H, and R5 is -H; or

[0324] - A compound of formula (VII) wherein R1 is -OH or -C≡N, and R2 is -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -H or -C 1-4 Alkyl, and R4 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -NH2, R3 is -H, and R4 is -H;

[0325] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0326] Specifically, in a preferred embodiment, D is one of the following formulae, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0327]

[0328] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0329] In yet another embodiment of the present invention, D is:

[0330] - a compound of formula (I) wherein R1 is -C≡N, R2 is -H or -CH2CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, R3 is -OR R3 or -NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, R4 is -H or -C 1-4 Alkyl, and R5 is -H or -C 1-4 Alkyl, preferably R1 is -C≡N, R2 is -H or -OCH3, R3 is -OH or -NH2, R4 is -H, and R5 is -H;

[0331] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0332] Specifically, in a preferred embodiment, D is one of the following formulae:

[0333]

[0334]

[0335] or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer of any of the foregoing.

[0336] The structure of the linker L present in formula (XIX) describing the antibody drug conjugate of the present invention is not believed to be particularly critical, but it can be defined as follows:

[0337]

[0338] in

[0339] - T and U are independently spacer units, each of which has 1 to 24 chain carbon atoms, wherein t and u are independently 0 or 1, preferably t+u≥1;

[0340] - each A is independently an amino acid unit, wherein a is an integer from 0 to 12;

[0341] -H is a hydrophilic unit, wherein h is 0 or 1; and

[0342] -Y is a coupling group covalently linked to Ab;

[0343] It is preferred that the coupling group Y is covalently linked to the sulfur atom present in Ab.

[0344] Therefore, the antibody drug conjugate of the present invention defined by formula (XIX) can be described as follows:

[0345]

[0346] For example, if the compound has the formula:

[0347]

[0348] The antibody drug conjugate can then be described by the structure of the following formula (XIX), wherein the compound is linked to the linker L via the nitrogen present in R2:

[0349]

[0350] In a preferred embodiment of the present invention, the coupling group Y of the linker L is covalently linked to a sulfur atom present in Ab, which means that a sulfur atom is present in the antibody, antigen-binding fragment or immunologically active portion thereof. Thus, the antibody drug conjugate represented by formula (XIX) may have the following structure:

[0351]

[0352] Typically, T and U are spacer units that can be independently selected from spacer units having 1 to 24 chain carbon atoms. The subscripts t and u are independently 0 or 1, which means that both units may not exist, or both units may exist. Preferably, the linker comprises at least one of T and U, i.e., t+u≥1. Chain carbon is the carbon present in the chain connecting T or U to the respective adjacent units or compound D, including the carbon of the ring that constitutes part of the chain. If a substituent or side chain is present, the carbon of the substituent or side chain is not included.

[0353] Those skilled in the art are very familiar with spacers that can be used to connect to chemical linkers (including in antibody drug conjugates). Available spacers include branched or linear polyalkylene chains and polyethylene glycol spacers, which have up to 24 chain carbon atoms. However, spacers may also exhibit more complex structures and may include functional groups such as carbonyls. In addition, spacers that can be used for the purposes of the present invention may include cyclic structures, such as aromatic and non-aromatic hydrocarbons or heterocycles (including sugars).

[0354] In one embodiment of the present invention, the spacer unit U is one of the following formulae:

[0355]

[0356]

[0357] When present, n is preferably an integer from 5 to 10, more preferably an integer from 6 to 8, and most preferably 7.

[0358] According to yet another embodiment of the present invention, the spacer unit T is one of the following formulae:

[0359]

[0360] wherein n is preferably an integer from 1 to 8, more preferably an integer from 1 to 6, and most preferably 2 or 5;

[0361] or

[0362]

[0363] wherein m is preferably 1, and n is an integer of 2 to 8, more preferably an integer of 4 to 7, and most preferably 5 or 6.

[0364] The linker L may contain a hydrophilic unit H. Those skilled in the art are generally familiar with chemical structures that can introduce hydrophilicity, thereby increasing the tendency of a given compound or antibody drug conjugate related to the present invention to be solvated in an aqueous environment. Typically, the hydrophilic unit H is rich in oxygen. According to one embodiment of the present invention, the hydrophilic unit H is derived from a PEGylated amino acid, and preferably H is one of the following formulas:

[0365]

[0366] wherein n is more preferably an integer of 5 to 10, still more preferably an integer of 6 to 8, and most preferably 7; or

[0367]

[0368] wherein n is more preferably an integer from 5 to 10, still more preferably an integer from 6 to 8, and most preferably 7. Additionally or alternatively, the hydrophilic unit H may be of the formula:

[0369]

[0370] wherein n is preferably an integer of 2 to 8, more preferably an integer of 4 to 7, and most preferably 5 or 6.

[0371] The coupling group Y present in the antibody drug conjugate can be derived from any group that can bind to an antibody, an antigen-binding fragment thereof, or an immunologically active portion (Ab) in an unreacted form. Since it is preferred that the coupling group Y is covalently linked to the sulfur atom of the antibody, its antigen-binding fragment, or the immunologically active portion, Y is preferably derived from a functional group containing a double bond. In this sense, a particularly suitable functional group is an α, β-unsaturated carbonyl group. Therefore, in one embodiment, Y is a coupling group derived from maleimide, maleamic acid ester, or propenamide, preferably the coupling group Y is derived from maleimide. In a more specific and more preferred embodiment, Y is such that L is one of the following formulae:

[0372]

[0373] wherein R6, if present, is -H or -C 1-4 Alkyl, preferably -H or -CH3.

[0374] The linker L may optionally contain one or more amino acid units A, so L may comprise a structure which may be represented by (A) 0-12 The structural motif of a wherein a is an integer from 0 to 12. In further embodiments, a is an integer from 1 to 10, more preferably an integer from 1 to 5, still more preferably an integer from 1 to 4, and most preferably 2 or 4.

[0375] Each individual unit designated A has the following structure, which should include all protein-forming amino acids, as well as other amino acids where R7 is p-hydroxybenzyl or -(CH2)3NHC(C=O)NH2 (citrulline):

[0376]

[0377] In a preferred embodiment of the present invention, R7 is independently selected from -H (glycine), -CH3 (alanine), -C(H)(CH3)2 (valine), benzyl (phenylalanine), p-hydroxybenzyl and -(CH2)3NHC(C=O)NH2 (citrulline); more preferably, R7 is independently selected from -H (glycine), -C(H)(CH3)2 (valine), benzyl (phenylalanine) and -(CH2)3NHC(C=O)NH2 (citrulline).

[0378] In some cases, specific combinations of amino acids are preferred. In particularly preferred embodiments, this includes the combination known as "GGFG" (Glycine-Glycine-Phenylalanine-Glycine), and the combination known to those skilled in the art as "Val-Cit" (Valine-Citrulline). Thus, in these embodiments, the linker L can be one of the following formulae:

[0379]

[0380] Additionally or alternatively, advantageous combinations include those known as "GGGG-Val-Cit" and "Val-Ala". Thus, the linker L may be one of the following formulae:

[0381]

[0382] In further embodiments of the present invention, H and h, A and a, and U and u are such that L is one of the following formulae:

[0383]

[0384]

[0385]

[0386] In a further embodiment of the invention, L is such that formula (XIX) is one of the following:

[0387]

[0388]

[0389]

[0390]

[0391] Yet another aspect of the present invention relates to a compound, pharmaceutically acceptable salt, ester, solvate, tautomer, or stereoisomer as disclosed herein for use as a medicament, with a more specific aspect relating to use in the treatment of cancer, preferably ovarian cancer, gastric cancer, or breast cancer. In a more preferred embodiment, the cancer types include ovarian cancer and breast cancer. Particularly preferred embodiments relate to a compound, pharmaceutically acceptable salt, ester, solvate, tautomer, or stereoisomer as disclosed herein for use in the treatment of papillary serous ovarian carcinoma or breast adenocarcinoma.

[0392] As demonstrated by way of example, the compounds of the present invention exhibit improved efficacy. This discovery makes these compounds potent active ingredients in conventional chemotherapy for ovarian, breast and gastric cancers, particularly ovarian and breast cancers.

[0393] Yet another aspect of the present invention relates to the antibody drug conjugates disclosed herein for use as a medicament, with a more specific aspect relating to use in the treatment of cancer, preferably ovarian cancer, gastric cancer, or breast cancer. Furthermore, in a more preferred embodiment, the cancer types include ovarian cancer and breast cancer. In an alternative embodiment, the cancer types include gastric cancer and breast cancer. Particularly preferred aspects relate to the antibody drug conjugates disclosed herein for use in the treatment of gastric cancer or breast adenocarcinoma.

[0394] A preferred embodiment of the present invention relates to the antibody drug conjugates disclosed herein for use as a medicament, wherein

[0395] - the antibody is an anti-ROR1 antibody, preferably cimetuzumab, and / or, wherein the present invention relates to an antibody drug conjugate for use in treating ROR1-positive ovarian cancer;

[0396] - the antibody is an anti-TROP2 antibody, preferably gosartuzumab, and / or, wherein the present invention relates to an antibody drug conjugate for use in treating TROP2-positive gastric cancer;

[0397] - the antibody is an anti-HER2 antibody, preferably trastuzumab, and / or, wherein the present invention relates to an antibody drug conjugate for use in the treatment of HER2-positive breast cancer;

[0398] - the antibody is an anti-HER3 antibody, preferably Pertrastuzumab, and / or, wherein the present invention relates to an antibody drug conjugate for use in treating HER3-positive gastric cancer;

[0399] - the antibody is an anti-FRα antibody, preferably mituximab, and / or, wherein the present invention relates to an antibody drug conjugate for use in the treatment of FRa-positive ovarian cancer;

[0400] - the antibody is an anti-EGFR antibody, preferably cetuximab, and / or, wherein the present invention relates to an antibody drug conjugate for use in the treatment of EGFR-positive breast cancer.

[0401] In the aforementioned embodiments, the antibodies may have the structures disclosed above. According to the present invention, anti-ROR1 antibodies may be more preferred, in particular cimetuzumab.

[0402] According to another aspect of the present invention, a method for preparing an antibody drug conjugate of formula (XIX) is provided:

[0403]

[0404] The method comprises coupling an antibody, an antigen-binding fragment or an immunologically active portion thereof (Ab) to a compound (D) via a linker (L), wherein

[0405] -D is a compound selected from trabectedin, rubectedin, and a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, preferably a compound of formula (I), (VII) or (XIII) disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof;

[0406] -L is a linker as disclosed above;

[0407] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0408] -z is an integer from 1 to 20.

[0409] In a representative method, compound D is synthesized and first reacted with a linker to obtain a linker-payload building unit. Based on the definitions provided above, the linker-payload building unit of the present invention can be represented as follows:

[0410] Y*-(T) t —(H) h -(A) a -(U) u -D

[0411] wherein Y* represents the unreacted form of the coupling group Y capable of reacting with Ab, for example, with a thiol group present in the antibody. Thus, Y* can be a functional group containing a carbon-carbon double bond, such as maleimide, maleamic acid ester or malonamide.

[0412] In a preferred embodiment, the method of the present invention comprises the step of covalently linking D to L via any one of R2, R3 or R4, preferably the nitrogen or oxygen present in any one of R2 or R3.

[0413] In order to achieve the binding of the linker-payload building block to the antibody, the antibody, its antigen-binding fragment or immunologically active portion (Ab) is preferably first reacted with a reducing agent to reduce one or more disulfide groups contained therein, depending on the amount of reducing agent used and the desired drug-antibody ratio. Tris(2-carboxyethyl)phosphine (TCEP) can be used as a preferred reducing agent. Only after reduction, an appropriate amount of the linker-payload building block reacts with the (partially) reduced antibody, its antigen-binding fragment or immunologically active portion to provide an antibody drug conjugate of formula (XIX) of the present invention having the desired drug-antibody ratio.

[0414] In one embodiment of the method of the present invention, z is an integer of 1 to 10, preferably an integer of 1 to 6, more preferably an integer of 2 to 4, and still more preferably 2 or 4.

[0415] Antibodies used in the methods of the present invention generally include anti-HER2 antibodies, anti-ROR1 antibodies, anti-TROP2 antibodies, anti-HER3 antibodies, anti-FRα antibodies, and anti-EGFR antibodies. Particularly useful antibodies are those described as preferred above. These antibodies can also have the structure disclosed above. For example, the antibody, its antigen-binding fragment, or immunologically active portion (Ab) can be an anti-HER2 antibody, such as trastuzumab.

[0416] The present invention also relates to corresponding pharmaceutical dosage forms, which contain a therapeutically effective amount of a compound disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, or a therapeutically effective amount of an antibody drug conjugate disclosed herein.

[0417] Also disclosed herein are methods for treating cancer, wherein the method comprises administering to an individual a therapeutically effective amount of a compound disclosed herein, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, or a therapeutically effective amount of an antibody drug conjugate disclosed herein, or a pharmaceutical dosage form disclosed above. Preferred cancer types include ovarian cancer, gastric cancer or breast cancer, wherein ovarian cancer and breast cancer may be preferred in terms of efficacy.

[0418] The following numbered embodiments further define the invention disclosed herein:

[0419] 1. A compound of formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0420]

[0421] in

[0422] -R1 is a substituent having a negative inductive effect;

[0423] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0424] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 RR3 , wherein m and n are independently integers from 0 to 10, wherein X is O or NH, and R R3 are independently -H or -C 1-4 alkyl;

[0425] -R4 is selected from -H, -CH2OR R4 、-CH2NHR R4 and -CH2C(=O)OR R4 , where R R4 Is -H or -C 1-4 alkyl; and

[0426] -R5 is -H or -C 1-4 alkyl;

[0427] Provided that compounds of formula (I) having the following combinations of R2, R3, R4 and R5 are not included:

[0428] -R2 is -OCH3, R3 is -OH, R4 is -H and R5 is -H.

[0429] 2. The compound according to embodiment 1, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein R1 is -OH or -C≡N, preferably -C≡N.

[0430] 3. A compound according to any one of embodiments 1 or 2, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0431] -R2 is -H, -OR R2 、-OCH2CH2OR R2 、-OCH2C(CH3)2OR R2 、-OCH2CH2NHR R2 、-OCH2C(=O)OR R2 、-NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2CH2NH2, -OCH2C(=O)OH, -NH2 or -NHC(=O)CH2OH; and / or

[0432] -R3 is -H, -OR R3 、-NHR R3 or -NHC(=O)CH2OR R3 , where R R3 Is -H or -C1-4 Alkyl, preferably R3 is -H, -OH, -NH2 or -NHC(=O)CH2OH.

[0433] 4. A compound according to any one of embodiments 1 to 3, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0434] -R4 is -H, -CH2OR R4 or -CH2NHR R4 , where R R4 Is -H or -C 1-4 Alkyl, preferably R4 is -H, -CH2OH or -CH2NH2; and / or

[0435] -R5 is -H or -C 1-4 Alkyl, preferably R5 is -H or -CH3.

[0436] 5. A compound according to any one of embodiments 1 to 4, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0437] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0438] -R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(CH3)2OH, -OCH2CH2NH2, -OCH2C(=O)OH, -NH2 or -NHC(=O)CH2OH, preferably R2 is -OCH2CH2OH;

[0439] -R3 is -H, -OH, -NH2 or -NHC(=O)CH2OH, preferably R3 is -NH2;

[0440] -R4 is -H, -CH2OH or -CH2NH2, preferably R4 is -CH2NH2; and

[0441] -R5 is -H or -CH3, preferably R5 is -CH3.

[0442] 6. The compound according to embodiment 1 or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein formula (I) is:

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] 7. A compound of formula (VII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0449]

[0450] in

[0451] -R1 is a substituent having a negative inductive effect;

[0452] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0453] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0454] -R4 is -H or -C 1-4 alkyl;

[0455] Provided that compounds of formula (VII) having the following combinations of R2, R3 and R4 are not included:

[0456] -R2 is -H, R3 is -H and R4 is -H; and

[0457] -R2 is -OCH3, R3 is -H and R4 is -H.

[0458] 8. The compound according to embodiment 7, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein R1 is -OH or -C≡N, preferably -C≡N.

[0459] 9. A compound according to any one of embodiments 7 or 8, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0460] -R2 is -H, -OR R2 、-NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -NH2 or -NHC(=O)CH2OH; and / or

[0461] -R3 is -H, -NHR R3 or -NHC(=O)CH2OR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H, -NH2 or -NHC(=O)CH2OH.

[0462] 10. A compound according to any one of embodiments 7 to 9, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0463] -R4 is -H or -CH3.

[0464] 11. A compound according to any one of embodiments 7 to 10, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0465] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0466] -R2 is -H, -OCH3, -NH2 or -NHC(=O)CH2OH, preferably R2 is -NH2;

[0467] -R3 is -H, -NH2 or -NHC(=O)CH2OH, preferably R3 is -NH2; and

[0468] -R4 is -H or -CH3, preferably R4 is -CH3.

[0469] 12. The compound according to embodiment 7, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein formula (VII) is:

[0470]

[0471]

[0472]

[0473] 13. A compound of formula (XIII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof:

[0474]

[0475] in

[0476] -R1 is a substituent having a negative inductive effect;

[0477] -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is O or NH, and R R2 are independently -H or -C 1-4 alkyl;

[0478] -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is O or NH, and R R3 are independently -H or -C 1-4 alkyl; and

[0479] -R4 is -H or -CH2NHR R4 , where R R4 Is -H or -C 1-4 alkyl.

[0480] 14. The compound according to embodiment 13, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein R1 is -OH or -C≡N, preferably -C≡N.

[0481] 15. The compound according to any one of embodiments 13 or 14, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0482] -R2 is -OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -OCH3 or -NH2; and / or

[0483] -R3 is -H.

[0484] 16. A compound according to any one of embodiments 13 to 15, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0485] -R4 is -H or -CH2NH2.

[0486] 17. A compound according to any one of embodiments 13 to 16, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein

[0487] -R1 is -OH or -C≡N, preferably R1 is -C≡N;

[0488] -R2 is -OCH3 or -NH2, preferably R2 is -NH2;

[0489] -R3 is -H; and

[0490] -R4 is -H or -CH2NH2, preferably R4 is -CH2NH2.

[0491] 18. The compound according to embodiment 13, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein formula (XIII) is:

[0492]

[0493]

[0494] 19. Antibody drug conjugate of formula (XIX):

[0495]

[0496] in

[0497] -Ab represents an antibody, an antigen-binding fragment, or an immunologically active portion thereof;

[0498] -L represents a linker; and

[0499] -D represents a compound selected from trabectedin, rubectedin and a compound according to any one of Embodiments 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, preferably a compound according to any one of Embodiments 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer;

[0500] in

[0501] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0502] -z is an integer from 1 to 20.

[0503] 20. The antibody drug conjugate according to embodiment 19, wherein z is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, and still more preferably 2 or 4.

[0504] 21. The antibody drug conjugate according to any one of embodiments 19 or 20, wherein D is covalently linked to L through any one of R2, R3 or R4, preferably a nitrogen or oxygen present in any one of R2 or R3.

[0505] 22. The antibody drug conjugate according to any one of embodiments 19 to 21, wherein the antibody is:

[0506] -Anti-ROR1 antibody, preferably cimetuzumab;

[0507] - an anti-TROP2 antibody, preferably gosatuzumab; or

[0508] -Anti-HER2 antibody, preferably trastuzumab.

[0509] 23. The antibody drug conjugate according to any one of embodiments 19 to 22, wherein L has the following structure:

[0510]

[0511] in

[0512] - T and U are independently spacer units, each of which has 1 to 24 chain carbon atoms, wherein t and u are independently 0 or 1, preferably t+u≥1;

[0513] - each A is independently an amino acid unit, wherein a is an integer from 0 to 12;

[0514] -H is a hydrophilic unit, wherein h is 0 or 1; and

[0515] -Y is a coupling group covalently linked to Ab;

[0516] It is preferred that the coupling group Y is covalently linked to the sulfur atom present in Ab.

[0517] 24. The antibody drug conjugate according to embodiment 23, wherein Y is such that L is one of the following formulae:

[0518]

[0519] wherein R6, if present, is -H or -C 1-4 Alkyl, preferably -H or -CH3.

[0520] 25. The antibody drug conjugate according to any one of embodiments 23 or 24, wherein

[0521] -U is one of the following:

[0522]

[0523] wherein n is preferably an integer from 5 to 10, more preferably an integer from 6 to 8, and most preferably 7;

[0524] and / or

[0525] -T is one of the following:

[0526]

[0527] wherein n is preferably an integer from 1 to 8, more preferably an integer from 1 to 6, and most preferably 2 or 5;

[0528] or

[0529]

[0530] wherein preferably m is 1 and n is an integer from 2 to 8, more preferably an integer from 4 to 7, and most preferably 5 or 6; and / or

[0531] -H is derived from a PEGylated amino acid, preferably H is one of the following formulae:

[0532]

[0533] wherein n is more preferably an integer from 5 to 10, still more preferably an integer from 6 to 8, and most preferably 7;

[0534] or

[0535]

[0536] Among them, n is more preferably an integer of 5 to 10, still more preferably an integer of 6 to 8, and most preferably 7.

[0537] 26. The antibody drug conjugate according to any one of embodiments 23 to 25, wherein each A has the following structure:

[0538]

[0539] wherein R7 is independently selected from -H, -CH3, -C(H)(CH3)2, benzyl, p-hydroxybenzyl and -(CH2)3NHC(C=O)NH2; preferably R7 is independently selected from -H, -C(H)(CH3)2 and benzyl; and / or

[0540] a is an integer of 1 to 10, more preferably an integer of 1 to 5, still more preferably an integer of 1 to 4, and most preferably 2 or 4.

[0541] 27. The antibody drug conjugate according to embodiment 23, wherein H and h, A and a, and U and u are such that L is one of the following formulae:

[0542]

[0543]

[0544] 28. The antibody drug conjugate according to any one of embodiments 19 to 22, wherein L is such that formula (XIX) is one of the following:

[0545]

[0546]

[0547]

[0548] 29. A compound according to any one of embodiments 1 to 18, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, for use as a medicament.

[0549] 30. A compound according to embodiment 29 or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof for use in treating cancer, preferably ovarian cancer, gastric cancer or breast cancer, more preferably ovarian cancer or breast cancer.

[0550] 31. The antibody drug conjugate according to any one of embodiments 19 to 28 for use as a medicament.

[0551] 32. The antibody drug conjugate according to embodiment 31, for use in treating cancer, preferably ovarian cancer, gastric cancer or breast cancer, more preferably ovarian cancer or breast cancer.

[0552] 33. Method for preparing an antibody drug conjugate of formula (XIX):

[0553]

[0554] The method comprises coupling an antibody, an antigen-binding fragment or an immunologically active portion thereof (Ab) to a compound (D) via a linker (L), wherein

[0555] -D is a compound selected from trabectedin, rubectedin and a compound according to any one of embodiments 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, preferably a compound according to any one of embodiments 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer;

[0556] -L is a linker according to any one of embodiments 19 or 23 to 28;

[0557] - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and

[0558] -z is an integer from 1 to 20.

[0559] 34. The method according to embodiment 33, wherein z is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, and still more preferably 2 or 4.

[0560] 35. The method according to any one of embodiments 33 or 34, wherein the antibody is:

[0561] -Anti-ROR1 antibody, preferably cimetuzumab;

[0562] - an anti-TROP2 antibody, preferably gosatuzumab; or

[0563] -Anti-HER2 antibody, preferably trastuzumab.

[0564] 36. A pharmaceutical dosage form comprising a therapeutically effective amount of a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of embodiments 1 to 18, or a therapeutically effective amount of an antibody drug conjugate according to any one of embodiments 19 to 28.

[0565] 37. A method of treating cancer, wherein the method comprises administering to a subject a therapeutically effective amount of a compound according to any one of embodiments 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, a therapeutically effective amount of an antibody drug conjugate according to any one of embodiments 19 to 28, or a pharmaceutical dosage form according to embodiment 36.

[0566] 38. The method of embodiment 37, wherein the cancer is ovarian cancer, gastric cancer or breast cancer, preferably ovarian cancer or breast cancer.

[0567] The scope and significance of the present invention may be better understood based on the following examples which are intended to illustrate embodiments of the present invention. However, they should not be construed as limiting the scope of the claims in any way. Example

[0568] The following abbreviations will be used in this section, particularly in the schemes describing the preparation of the compounds and antibody drug conjugates of the invention:

[0569] ADC, antibody-drug conjugate

[0570] ATCC, American Type Culture Collection

[0571] Cbz, carboxybenzyl

[0572] CDI, 1,1'-carbonyldiimidazole

[0573] Citrulline

[0574] DAR, drug-antibody ratio

[0575] DCM, dichloromethane

[0576] DIPCDI, N,N'-diisopropylcarbodiimide

[0577] DIPEA, N,N-diisopropylethylamine

[0578] DMEM, Dulbecco's modified Eagle's medium

[0579] DMF, dimethylformamide

[0580] DMSO, dimethyl sulfoxide

[0581] DR, dose response

[0582] DTT, dithiothreitol

[0583] EDCI, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride

[0584] EDTA, ethylenediaminetetraacetic acid

[0585] EtOAc, ethyl acetate

[0586] FCS, fetal calf serum

[0587] Fmoc, 9-fluorenylmethoxycarbonyl

[0588] Gly, glycine

[0589] Hexane

[0590] HOBt, 1-hydroxybenzotriazole

[0591] HPLC, high performance liquid chromatography

[0592] ITS, insulin-transferrin-selenium medium supplement

[0593] MC, 6-maleimidocaproyl

[0594] MeOH, methanol

[0595] NAC, N-acetylcysteine

[0596] NMP, N-methyl-2-pyrrolidone

[0597] PAB, 4-aminobenzyl alcohol

[0598] PABC, p-aminobenzyl carbamate

[0599] PBS, phosphate-buffered saline

[0600] Phe, phenylalanine

[0601] bis-PNP, bis(4-nitrophenyl) carbonate

[0602] Py, pyridine

[0603] RPMI, Rosmell Park Memorial Institute medium

[0604] RT, room temperature

[0605] SEC, size exclusion chromatography

[0606] SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0607] SRB, sulforhodamine B

[0608] TCEP, tris(2-carboxyethyl)phosphine hydrochloride

[0609] TFA, trifluoroacetic acid

[0610] THF, tetrahydrofuran

[0611] Trt, triphenylmethyl

[0612] UV, ultraviolet

[0613] Val, valine

[0614] Cell lines and toxicity assays

[0615] The cell lines used herein to test the cytotoxicity of the compounds and antibody drug conjugates of the invention are referred to as:

[0616] name ATCC species organize feature OV-90 CRL-11732 Human ovaries Papillary serous adenocarcinoma NCI-N87 CRL-5822 Human Stomach Gastric cancer MDA-MB-231 HTB-26 Human breast Breast cancer

[0617] Cell line preparation

[0618] Human cell lines MDA-MB-231, NCI-N87 and OV-90 are all from American Type Culture Collection (Manassas, USA). MDA-MB-231 cells are cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100U / mL penicillin, 100pg / mL streptomycin, 2mM glutamine and 1mM sodium pyruvate. NCI-N87 cells are cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 100U / mL penicillin and 100pg / mL streptomycin. OV-90 cells are cultured in MCDB 105 culture medium / culture medium 199 (1:1) supplemented with 10% heat-inactivated fetal bovine serum. All cell lines are maintained at 37°C and in an environment with 5% CO2.

[0619] Compound preparation

[0620] Lyophilized compounds were dissolved in 100% sterile cell culture grade DMSO, aliquoted and stored at -20°C.

[0621] Cytotoxicity assay

[0622] 1 x 10 cells / well in 100 μL growth medium 3 Up to 2x10 3 Cells were seeded in 96-well white flat-bottom tissue culture plates in triplicate at a density of 10 cells and allowed to adhere and recover for 24 hours. The test compound was serially diluted in DMSO to produce a 9-point titration curve and then added to the inoculated culture with a final DMSO concentration of 0.02% per well. After incubation for another 120 hours at 37°C and 5% CO2, cell viability was determined using CellTitre-Glo 2.0 (Promega). An equal volume of CellTitre-Glo 2.0 was added to the culture medium in the wells and mixed for 10 minutes to lyse the cells. Luminescence was measured on a ThermoFisher Varioskan Lux microplate reader.

[0623] Luminescence values ​​for each test compound at different concentrations were normalized to the luminescence values ​​of cells treated with DMSO control (0.02%) and plotted against the concentration of the test compound. Sigmoidal curves and CC curves were generated in GraphPad Prism version 9.4.1 using nonlinear regression, variable slope four-parameter curve fitting of inhibitor versus response. 50 The R-squared values ​​were higher than 0.95. All the assays were performed in triplicate and were performed as two independent experiments.

[0624] Cytotoxicity of the parent compound and the synthetic compound

[0625] The following compounds were synthesized and tested for their cytotoxicity against various cell lines (++++: <1 nM; +++: <10 nM, ++: 10-200 nM; +>200 nM):

[0626]

[0627]

[0628]

[0629]

[0630] Cytotoxicity of selected antibody-drug conjugates

[0631] A range of payload compounds were used in antibody drug conjugates and tested for cytotoxicity against NCI-N87 (gastric) and MDA-MB-231 (breast) cell lines (++++: <1 nM; +++: <50 nM, ++: 50-200 nM; +>200 nM):

[0632]

[0633]

[0634]

[0635] Sac = gosartuzumab, Tra = trastuzumab, Cir = cetuximab, Pat = patrastuzumab, Cet = cetuximab, LP = linker-payload building block, LK = linker, PL = payload

[0636] Synthesis of compounds and antibody-drug conjugates

[0637] Intermediate A

[0638]

[0639] Step 1: Synthesis of benzyl (4-hydroxyphenethyl)carbamate.

[0640] To a solution of 4-(2-aminoethyl)phenol (10 g, 72.9 mmol, 1.0 eq.) in 100 mL of dichloromethane and 50 mL of saturated aqueous sodium bicarbonate was added Cbz-Cl (22.3 g, 130 mmol, 1.8 eq.) at 0 to 10 ° C. After the addition, the mixture was stirred at room temperature overnight. LCMS showed that the reaction was complete. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (100 mL×2). The combined organic layers were dried over anhydrous sodium sulfate. Then, filtered and concentrated. The crude product was recrystallized from ethyl acetate / n-heptane to give benzyl (4-hydroxyphenethyl)carbamate as a white solid (16.2 g, 81.8%). LC-MS (ESI) m / z: 272.0 [M+H] + . 1 H NMR (300MHz, CHCl3-d): δ (ppm) 7.35 (s, 5H), 7.05 (d, J = 7.8Hz, 2H), 6.77 (d, J = 7.8Hz, 2H), 5.10 (s, 2H), 4.73 (s, 1H), 3.42 (d, J = 6.7Hz, 2H), 2.74 (s, 2H).

[0641] Step 2: Synthesis of benzyl (4-hydroxy-3-nitrophenethyl)carbamate.

[0642] To a solution of benzyl (4-hydroxyphenethyl)carbamate (14 g, 51.6 mmol, 1.0 eq.) in 140 mL of acetic acid was added a mixture of 140 mL of acetic acid and 65% nitric acid (3.57 mL, 51.6 mmol, 1.0 eq.) at room temperature. After the reaction mixture was stirred for 1 hour, ethyl acetate and saturated aqueous sodium carbonate solution were added. The aqueous layer was collected and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and then concentrated. The crude product was purified by flash column chromatography using ethyl acetate: n-heptane (1:4) to give benzyl (4-hydroxy-3-nitrophenethyl)carbamate as a dark yellow solid (15.4 g, 94%). LC-MS (ESI) m / z: 317 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ (ppm) 10.49 (s, 1H), 7.93 (s, 1H), 7.48–7.31 (m, 6H), 7.10 (d, J = 8.5Hz, 1H), 5 .32(d,J=5.7Hz,1H),5.13(d,J=15.2Hz,2H),4.83(s,1H),3.45(q,J=6.8Hz,2H),2.99–2.66(m,2H).

[0643] Step 3: Synthesis of benzyl (4-methoxy-3-nitrophenethyl)carbamate.

[0644] To a solution of benzyl (4-hydroxy-3-nitrophenethyl)carbamate (12 g, 37.9 mmol, 1.0 eq.) and potassium carbonate (15.7 g, 113 mmol, 3.0 eq.) in 240 mL of acetonitrile was added iodomethane (8.08 g, 56.9 mmol, 1.5 eq.). The slurry was refluxed for 2 hours. TLC showed that the reaction was complete. The solid was filtered off and the filtrate was concentrated. 100 mL of water was added to the residue and the mixture was extracted with dichloromethane (200 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to give benzyl (4-methoxy-3-nitrophenethyl)carbamate in crude form. The crude product was used in the next step without further purification. 1 HNMR(300MHz,CHCl3-d)δ(ppm)7.80–7.61(m,1H),7.37(d,J=9.8Hz,6H),7.02(d,J=8.6Hz,1H), 5.10(s,2H),4.97–4.77(m,1H),3.95(d,J=5.5Hz,3H),3.45(q,J=6.9Hz,2H),2.97–2.68(m,2H).

[0645] Step 4: Synthesis of 5-(2-aminoethyl)-2-methoxyaniline hydrochloride (Intermediate A).

[0646] The previous crude product was diluted with 300 mL of methanol, and then 1.5 g of 5% Pd / C was added thereto under nitrogen. The suspension was vacuum degassed and purged with hydrogen three times. The mixture was stirred at room temperature overnight under hydrogen. The suspension was filtered through a celite pad, and the filter cake was washed with methanol. The filtrate was concentrated to dryness and dissolved in 10 mL of ethyl acetate. 1 eq. 2 M HCl / EtOH solution was added thereto, the precipitate was collected by filtration and vacuum dried to obtain intermediate A as a dark brown solid (7.08 g, 93%). 1 H NMR (300MHz, D2O) δ (ppm) 6.98–6.88 (m, 1H), 6.88–6.73 (m, 2H), 3.79 (d, J = 3.4Hz, 3H), 3.15 (t, J = 7.2Hz, 2H), 2.92–2.75 (m, 2H).

[0647] Intermediate B

[0648]

[0649] Step 1: Synthesis of 4-(2-(benzyloxy)ethoxy)-3-hydroxybenzaldehyde.

[0650] To a solution of 3,4-dihydroxybenzaldehyde (5.0 g, 0.036 mol, 1.0 eq.) and ((2-bromoethoxy)methyl)benzene (17.1 g, 0.08 mol, 2.2 eq.) in 50 mL of acetonitrile was added CsCO (26.0 g, 0.080 mol, 2.2 eq.). The mixture was refluxed for 1 hour and the solid was filtered off. The filtrate was concentrated, and the residue was purified by flash silica gel column chromatography (ethyl acetate:petroleum ether (1:5)) to give 4-(2-(benzyloxy)ethoxy)-3-hydroxybenzaldehyde as a yellow solid (1.30 g, 13%). 1 H NMR(400MHz,CHCl3-d)δ(ppm)9.85(d,J=2.1Hz,1H),7.45(d,J=2.0Hz,1H),7.43–7.29(m,5H),6.99(dd,J=8.2,2.0 Hz, 1H), 6.41 (s, 1H), 4.63 (d, J = 2.0Hz, 2H), 4.29 (dt, J = 6.7, 2.4Hz, 2H), 3.87 (dt, J = 6.2, 2.5Hz, 2H), 1.92 (s, 1H).

[0651] Step 2: Synthesis of 3-hydroxy-4-(2-hydroxyethoxy)benzaldehyde.

[0652] To a solution of 3,4-dihydroxybenzaldehyde (0.10 g, 0.36 mmol, 1.0 eq.) in 3 mL of dichloromethane was added dropwise BCl (1 M in dichloromethane, 3.65 mL, 3.65 mmol, 10.0 eq.) at -10 to 20°C. The solution was neutralized with saturated sodium bicarbonate, and the residue was extracted with dichloromethane. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 3-hydroxy-4-(2-hydroxyethoxy)benzaldehyde as a brown oil, which was used in the next step without further purification. 1 HNMR (300MHz, CHCl3-d) δ (ppm) 9.85 (s, 1H), 7.49–7.38 (m, 2H), 6.99 (d, J = 8.3Hz, 1H), 4.32–4.22 (m, 2H), 4.13–4.03 (m, 2H).

[0653] Step 3: Synthesis of (E)-2-(2-hydroxyethoxy)-5-(2-nitrovinyl)phenol.

[0654] A solution of 3-hydroxy-4-(2-hydroxyethoxy)benzaldehyde (45 mg, 0.25 mmol, 1.0 eq.) and ammonium acetate (9.52 mg, 0.12 mmol, 0.5 eq.) in 5 mL of nitromethane was stirred at 90°C for 4 hours. After the reaction was complete, the mixture was concentrated and the residue was dissolved in ethyl acetate and then washed with brine. The organic layer was concentrated to give (E)-2-(2-hydroxyethoxy)-5-(2-nitrovinyl)phenol as a brown oil, which was used in the next step without further purification. 1 H NMR(300MHz,DMSO-d6)δ(ppm)9.06(d,J=1.8Hz,1H),8.00(d,J=1.8Hz,2H),7.40–7.21(m,2H ),7.10–6.94(m,1H),5.01–4.74(m,1H),4.04(dd,J=6.6,3.2Hz,2H),3.74(q,J=5.3Hz,2H).

[0655] Step 4: Synthesis of 2-(2-hydroxyethoxy)-5-(2-nitroethyl)phenol.

[0656] To a solution of (E)-2-(2-hydroxyethoxy)-5-(2-nitroethinyl)phenol (50 mg, 0.22 mmol, 1.0 eq.) in 5 mL of methanol was added NaBH4 (12.48 mg, 0.33 mmol, 1.5 eq.). After completion of the reaction, the mixture was quenched with 2N HCl to a pH of 2-4. The methanol was removed under reduced pressure and the residue was dissolved in ethyl acetate and then washed with brine. The organic layer was concentrated to yield 2-(2-hydroxyethoxy)-5-(2-nitroethyl)phenol as a crude brown oil.

[0657] Step 5: Synthesis of 5-(2-aminoethyl)-2-(2-hydroxyethoxy)phenol (Intermediate B).

[0658] The crude 2-(2-hydroxyethoxy)-5-(2-nitroethyl)phenol was diluted with 10 mL of methanol, and then 10 mg of 5% Pd / C was added under N2. The mixture was stirred at room temperature overnight under H2. The suspension was filtered through a pad of celite, and the filter cake was washed with methanol. The combined filtrates were concentrated to dryness to give Intermediate B as a brown oil (26.0 mg, 59.3%). 1H NMR(300MHz,DMSO-d6)δ(ppm)6.83(dd,J=8.1,3.4Hz,1H),6.75–6.62(m,1H),6.56(d,J=8.2H z,1H),3.91(t,J=4.8Hz,2H),3.80–3.59(m,2H),2.81(t,J=7.5Hz,2H),2.60(t,J=7.7Hz,2H).

[0659] Intermediate C

[0660]

[0661] Step 1: Synthesis of 5-nitro-1H-indole-3-carbaldehyde.

[0662] To a solution of 30 mL of anhydrous DMF and 4.6 mL of POCl3 was added 5-nitro-1H-indole (5 g, 0.0308 mol, 1.0 eq.) in DMF at 0°C. The reaction mixture was warmed to 60°C and stirred for 30 minutes. The reaction was then quenched with saturated aqueous sodium carbonate. The precipitate was collected by filtration, washed with water, and dried to give 5-nitro-1H-indole-3-carbaldehyde as a gray solid (36.9 g, 68%). LC-MS (ESI) m / z: 176 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ (ppm) 12.20 (s, 1H), 10.31 (d, J = 1.7Hz, 1H), 8.08–7.97 (m,1H),7.20–7.07(m,2H),6.75(dt,J=7.2,1.5Hz,1H),3.93(d,J=1.6Hz,3H).

[0663] Step 2: Synthesis of (E)-5-nitro-3-(2-nitrovinyl)-1H-indole.

[0664] To a solution of 5-nitro-1H-indole-3-carbaldehyde (15 g, 0.079 mol, 1.0 eq.) in 75 mL of 1,4-dioxane were added ammonium acetate (6.08 g, 0.079 mol, 1.0 eq.) and nitromethane (10.1 g, 15.8 mol, 2.0 eq.). After refluxing for 4 hours, the reaction mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate and washed with water. The organic phase was dried over sodium sulfate and concentrated. The residue was purified by flash silica gel column chromatography (ethyl acetate:n-heptane (1:4)) to provide (E)-5-nitro-3-(2-nitrovinyl)-1H-indole as a yellow solid (17 g, 92%). 1H NMR (400MHz, DMSO-d6) δ (ppm) 12.71 (s, 1H), 8.91 (d, J = 2.2Hz, 1H), 8.53–8.43 (m, 2H), 8.21–8.09 (m, 2H), 7.69 (d, J = 8.9Hz, 1H).

[0665] Step 3: Synthesis of 3-(2-aminoethyl)-1H-indol-5-amine (Intermediate C).

[0666] To a solution of (E)-5-nitro-3-(2-nitrovinyl)-1H-indole (5 g, 21.25 mmol, 1.0 eq.) in 25 mL of THF was added dropwise a 2.5 M solution of LiAlH₄ in THF (1.0 eq.) at 0°C. The reaction mixture was warmed to 60°C and stirred for 2 hours. After completion of the reaction, 16 mL of water, 16 mL of a 15% aqueous NaOH solution, 48 mL of water, and a small amount of MgSO₄ were added to the mixture at 0°C. The mixture was then filtered and the filter cake was washed with THF. The filtrate was concentrated. The residue was purified by flash silica gel column chromatography (dichloromethane:methanol:NH₃·H₂O (95:5:1)) to give Intermediate C as a brown sticky solid (1.72 g, 45%). LC-MS (ESI) m / z: 176 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ (ppm) 10.28 (s, 1H), 7.01 (d, J = 8.5Hz, 1H), 6.91 (d, J = 2.5Hz, 1H), 6.6 4(d,J=2.2Hz,1H), 6.45(dd,J=8.4,2.3Hz,1H), 2.78(t,J=7.1Hz,2H), 2.64(t,J=7.1Hz,2H).

[0667] Intermediate D

[0668]

[0669] Step 1: Synthesis of benzyl (4-hydroxyphenethyl)carbamate.

[0670] To a solution of 4-(2-aminoethyl)phenol (10 g, 72.9 mmol, 1.0 eq.) in 100 mL of dichloromethane and 50 mL of saturated sodium bicarbonate was added Cbz-Cl (22.3 g, 130 mmol, 1.8 eq.) at 0-10°C. After the addition, the mixture was stirred at room temperature overnight. LCMS showed that the reaction was complete. The aqueous phase was extracted with dichloromethane (100 mL×2). The combined organic phases were dried over anhydrous sodium sulfate. Then, filtered and concentrated. The residue was dissolved in ethyl acetate and n-heptane to obtain D2 as a white crystalline solid (16.2 g, 81.8%). LC-MS (ESI) m / z: 272 [M+H] + . 1 H NMR (300MHz, CHCl3-d) δ (ppm) 7.35 (s, 5H), 7.05 (d, J = 7.8Hz, 2H), 6.77 (d, J = 7.8Hz, 2H), 5.10 (s, 2H), 4.73 (s, 1H), 3.42 (d, J = 6.7Hz, 2H), 2.74 (s, 2H).

[0671] Step 2: Synthesis of benzyl (4-hydroxy-3-nitrophenethyl)carbamate.

[0672] To a solution of benzyl (4-hydroxyphenethyl)carbamate (14 g, 51.6 mmol, 1.0 eq.) in 140 mL of acetic acid was added a mixture of 140 mL of acetic acid and 65% nitric acid (3.57 mL, 51.6 mmol, 1.0 eq.) at room temperature. The reaction mixture was stirred at room temperature for 1 hour, and then ethyl acetate and saturated sodium carbonate were added. The mixture was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate. The crude product was purified by flash silica gel column chromatography using ethyl acetate:n-heptane (1:4) to give benzyl (4-hydroxy-3-nitrophenethyl)carbamate as a dark yellow solid (15.4 g, 94%). LC-MS (ESI) m / z: 317.2 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ (ppm) 10.49 (s, 1H), 7.93 (s, 1H), 7.48–7.31 (m, 6H), 7.10 (d, J = 8.5Hz, 1H), 5 .32(d,J=5.7Hz,1H),5.13(d,J=15.2Hz,2H),4.83(s,1H),3.45(q,J=6.8Hz,2H),2.99–2.66(m,2H).

[0673] Step 3: Synthesis of benzyl (4-(2-(benzyloxy)ethoxy)-3-nitrophenethyl)carbamate.

[0674] To a solution of benzyl (4-hydroxy-3-nitrophenethyl)carbamate (1.67 g, 5.28 mmol, 1.0 eq.) in 10 mL of CHCN was added ((2-bromoethoxy)methyl)benzene (2.27 g, 10.56 mmol, 2.0 eq.) and CsCO (1.89 g, 5.8 mmol, 1.1 eq.) in a sealed tube. The slurry was refluxed for 2 hours. TLC indicated the reaction was complete. The mixture was filtered and the filtrate was concentrated. The residue was purified by flash column chromatography to give benzyl (4-(2-(benzyloxy)ethoxy)-3-nitrophenethyl)carbamate as a yellow solid (1.5 g, 63%). 1 H NMR(400MHz,CHCl3-d)δ(ppm)7.65(d,J=2.3Hz,1H),7.41–7.26(m,11H),7.01(d,J=8.6Hz,1H),5.08(s,2H),4. 86(s,1H),4.64(s,2H),4.24(t,J=4.7Hz,2H),3.91–3.82(m,2H),3.41(q,J=6.8Hz,2H),2.79(t,J=7.0Hz,2H).

[0675] Step 4: Synthesis of 2-(2-amino-4-(2-aminoethyl)phenoxy)ethan-1-ol hydrochloride (Intermediate D).

[0676] To a solution of benzyl (4-(2-(benzyloxy)ethoxy)-3-nitrophenethyl)carbamate (2.0 g, 4.4 mmol) in 40 mL of methanol was added 5% Pd / C under N2. The suspension was degassed in vacuo and purged with H2 several times. The mixture was stirred at 80°C under H2 (0.5 MPa) for 2 hours. The suspension was filtered through a pad of celite, and the filter cake was washed with methanol. The combined filtrate was concentrated to dryness and dissolved in 10 mL of ethyl acetate. 1 eq. 2M HCl / EtOH solution was added to give a salt precipitate. The precipitate was collected by filtration and dried in vacuo to give intermediate D as a gray solid (0.73 g, 70.8%). LC-MS (ESI) m / z: 197 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 6.65 (d, J = 8.0Hz, 1H), 6.45 (d, J = 2.1Hz, 1H), 6.29 (dd, J = 8.1, 2.1Hz, 1H), 4.73 ( s, 2H), 3.86 (t, J = 4.8Hz, 2H), 3.68 (dd, J = 5.3, 4.3Hz, 2H), 2.67 (dd, J = 7.9, 6.6Hz, 2H), 2.44 (t, J = 7.3Hz, 2H).

[0677] Intermediate E

[0678]

[0679] Step 1: Synthesis of benzyl 2-(4-formyl-2-hydroxyphenoxy)acetate.

[0680] To a suspension of 3,4-dihydroxybenzaldehyde (9.63 g, 42.2 mmol, 1.0 eq.) and KCO (5.94 g, 43 mmol, 1.02 eq.) in 200 mL of acetone was added benzyl 2-bromoacetate (9.86 g, 43 mmol, 1.02 eq.). After the addition, the slurry was refluxed for 2 hours and filtered. The filtrate was concentrated and dried under vacuum to afford benzyl 2-(4-formyl-2-hydroxyphenoxy)acetate as a yellow solid (15.5 g, 77.6%). 1 H NMR (400MHz, CHCl3-d) δ (ppm) 9.87 (s, 1H), 7.53 (d, J = 1.9Hz, 1H), 7.50 (d, J = 7.2Hz, 2H), 7.46–7.33 (m, 9H), 6.93 (d, J = 8.2Hz, 1H), 5.25 (d, J = 13.3Hz, 4H), 4.87 (s, 2H).

[0681] Step 2: Synthesis of (E)-benzyl 2-(2-hydroxy-4-(2-nitrovinyl)phenoxy)acetate.

[0682] To a solution of benzyl 2-(4-formyl-2-hydroxyphenoxy)acetate (0.2 g, 0.53 mmol, 1.0 eq.) in 1 mL of nitromethane was added ammonium acetate (20 mg, 0.26 mmol, 0.5 eq.) at room temperature. The reaction mixture was stirred at 90°C for 2 hours, then concentrated and purified by flash column chromatography (ethyl acetate:n-heptane (1:8)) to afford (E)-benzyl 2-(2-hydroxy-4-(2-nitrovinyl)phenoxy)acetate as a yellow solid (0.11 g, 48%). 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.26 (d, J = 13.5Hz, 1H), 8.09 (d, J = 13.5Hz, 1H), 7.71 (d, J = 2.0Hz, 1H), 7.51 (d, J =7.0Hz,3H),7.46–7.41(m,3H),7.41–7.37(m,8H),7.05(d,J=8.5Hz,1H),5.21(d,J=10.0Hz,5H),5.03(s,2H).

[0683] Step 3: Synthesis of 2-(4-(2-aminoethyl)-2-hydroxyphenoxy)acetic acid (Intermediate E).

[0684] To a solution of (E)-benzyl 2-(2-hydroxy-4-(2-nitrovinyl)phenoxy)acetate (0.5 g, 4.4 mmol) in 40 mL of methanol was added 5% Pd / C under N₂. The suspension was degassed in vacuo and purged with H₂ several times. The mixture was stirred at 80°C under H₂ (0.5 MPa) for 2 hours. The suspension was filtered through a pad of celite, and the filter cake was washed with methanol. The filtrate was concentrated and dried in vacuo to afford Intermediate E as a brown solid (0.20 g, 62%). 1 H NMR (400MHz, DMSO-d6) δ (ppm) 6.86 (dd, J=8.0, 3.2Hz, 1H), 6.66 (d, J=2.1Hz, 1H), 6.52 (dt, J=8.0,2.8Hz,1H),4.11(d,J=1.6Hz,2H),3.09–2.92(m,2H),2.74(dt,J=16.0,7.9Hz,2H).

[0685] Intermediate F

[0686]

[0687] Step 1: Synthesis of 6-nitro-1H-indole-3-carbaldehyde.

[0688] At 0°C, under argon, POCl3 (6.2 mL, 2.5 eq.) was added dropwise to 50 mL of anhydrous DMF. A solution of 5 g (0.0308 mol, 1.0 eq.) of 6-nitro-1H-indole in 5 mL of anhydrous DMF was added dropwise at room temperature, and the resulting mixture was stirred for 6 hours. The reaction mixture was poured into a mixture of ice and saturated aqueous NaHCO3 and extracted with ethyl acetate (3 x 50 mL). The combined organic solution was washed with brine, dried over anhydrous sodium sulfate, and concentrated to afford (2.4 g, 40.9% yield) 6-nitro-1H-indole-3-carbaldehyde as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ (ppm) 12.71 (s, 1H), 10.05 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 2.1Hz, 1H), 8.29 (d, J = 8.8Hz, 1H), 8.14 (dd, J = 8.8, 2.0Hz, 1H).

[0689] Step 2: Synthesis of (E)-6-nitro-3-(2-nitrovinyl)-1H-indole.

[0690] To a solution of 6-nitro-1H-indole-3-carbaldehyde (90.1 mg, 0.473 mmol, 1.0 eq.) in 20 mL of nitromethane was added ammonium acetate (80.2 mg, 1.04 mmol, 2.2 eq.). After refluxing for 2 hours, the solvent was removed under reduced pressure. The residue was washed with water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol:NH3·H2O (90:10:0.1)) to provide (E)-6-nitro-3-(2-nitrovinyl)-1H-indole (46.3 mg, 41.9% yield) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ (ppm) 12.75 (s, 1H), 8.95 (d, J = 2.2Hz, 1H), 8.52 (d, J = 13.2 Hz, 2H), 8.21 (d, J = 13.6Hz, 1H), 8.16 (dd, J = 9.0, 2.2Hz, 1H), 7.73 (d, J = 9.0Hz, 1H).

[0691] Step 3: Synthesis of 3-(2-aminoethyl)-1H-indol-6-amine (Intermediate F).

[0692] To a solution of (E)-6-nitro-3-(2-nitrovinyl)-1H-indole (750 g, 3.216 mmol, 1.0 eq.) in 20 mL of 1,4-dioxane was added 2.5 M LiAlH₄ (1.0 eq.) dropwise at 0°C. The reaction was heated to 70°C. After completion, the reaction was quenched at 0°C with 16 mL of water, 16 mL of 15% aqueous NaOH, 48 mL of water, and a small amount of MgSO₄. The solution was filtered and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol:NH₃·H₂O (95:5:1)) to give the crude product as an oil. The oil was dissolved in methanol and 6 mL of HCl / EtOH (2 M), then precipitated in ethyl acetate and filtered to give Intermediate F (125 mg, 22.0% yield) as a brown, sticky solid. 1H NMR(400MHz,D2O)δ(ppm)7.71(d,J=8.5Hz,1H),7.48(d,J=2.0Hz,1H),7.35(s, 1H), 7.07 (dd, J=8.5, 1.9Hz, 1H), 3.28 (t, J=7.1Hz, 2H), 3.12 (t, J=7.1Hz, 2H).

[0693] Intermediate G

[0694]

[0695] Step 1: Synthesis of (E)-5-nitro-3-(2-nitrovinyl)-1H-indole.

[0696] To a solution of 5-nitro-1H-indole-3-carbaldehyde (15 g, 0.079 mol, 1.0 eq.) in 75 mL of 1,4-dioxane was added ammonium acetate (6.08 g, 0.079 mol, 1.0 eq.) and nitromethane (10.1 g, 15.8 mol, 2.0 eq.) and heated to reflux. After 4 hours, the solvent was removed under reduced pressure. The residue was washed with water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:n-heptane (1:4)) to provide (E)-5-nitro-3-(2-nitrovinyl)-1H-indole (17 g, 92% yield) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ (ppm) 12.71 (s, 1H), 8.91 (d, J = 2.2Hz, 1H), 8.53–8.43 (m, 2H), 8.21–8.09 (m, 2H), 7.69 (d, J = 8.9Hz, 1H).

[0697] Step 2: Synthesis of 5-nitro-3-(1-nitropropan-2-yl)-1H-indole.

[0698] At -50 ° C, to a solution of (E) -5-nitro -3- (2-nitrovinyl) -1H- indole (260 mg, 1.11 mmol, 1.0 eq.) in 25 mL of THF was added a solution of 1M MeMgBr in THF (1.0 eq.). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled and a small amount of saturated NH4Cl solution was added to quench the reaction. The reaction mixture was extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude product. The residue was purified by silica gel column chromatography (ethyl acetate: n-heptane (1: 3)) to give 5-nitro -3- (1-nitropropan-2-yl) -1H- indole (110 mg, 36% yield) as a brown solid.1 H NMR (400MHz, CHCl3-d) δ (ppm) 8.64 (d, J = 2.2Hz, 1H), 8.18 (dd, J = 9.0, 2.2Hz, 1H), 7.46 (d, J = 9.0Hz, 1H), 7.27 (d, J = 2.3Hz, 1 H), 7.25 (d, J = 2.6Hz, 1H), 4.75 (d, J = 7.3Hz, 1H), 4.62 (dd, J = 12.0, 7.7Hz, 1H), 4.04 (h, J = 7.2Hz, 1H), 1.57 (d, J = 7.0Hz, 3H).

[0699] Step 3: Synthesis of 3-(1-aminopropan-2-yl)-1H-indol-5-amine (Intermediate G).

[0700] Under N2 atmosphere, 5% Pd / C (10 mg) was added to a solution of 5-nitro-3-(1-nitropropan-2-yl)-1H-indole (110 mg, 0.44 mmol, 1.0 eq.) in 5 mL of methanol. The suspension was degassed in vacuo and purged several times with H2. The reaction mixture was stirred at room temperature under a H2 balloon for 1.5 hours. The suspension was filtered through a celite pad, and the filter cake was washed with methanol. The filtrate was concentrated to give intermediate G as a brown oil, which was used in the next step without purification.

[0701] Intermediate H

[0702]

[0703] Step 1: Synthesis of 3-(5-methoxy-1H-indol-3-yl)propan-1-ol.

[0704] To a stirred solution of (4-methoxyphenyl)hydrazine hydrochloride (4.0 g, 22.9 mmol) in dimethylacetamide (40 mL) was added 4% H2SO4 aqueous solution (30 mL) at room temperature, and the reaction mixture was heated to 100°C. Next, 3,4-dihydropyran (2.3 mL, 25.1 mmol) was added at the same temperature. The reaction mixture was stirred at 100°C for 2 hours, and the completion of the reaction was monitored by TLC. After the reaction was complete, the solvent was removed under reduced pressure, 100 mL of water was added and extracted with ethyl acetate. The organic layer was concentrated, and the crude product was purified by column chromatography (ethyl acetate: hexane s (1: 1)) to give 3-(5-methoxy-1H-indol-3-yl)propan-1-ol as a light brown oil (2.8 g, 59% yield). LC-MS (ESI) m / z: 204.15 [MH] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.57 (s, 1H), 7.22 (d, J = 8.8Hz, 1H), 7.05 (s, 1H), 6.98 (d, J = 2.0Hz, 1H), 6.71 (dd, J = 8. 8, 2.0Hz, 1H), 4.45 (t, J = 5.2Hz, 1H), 3.71 (s, 3H), 3.48 (q, J = 6.0Hz, 2H), 2.68 (t, J = 7.2Hz, 2H), 1.80 (q, J = 7.0Hz, 1H).

[0705] Step 2: Synthesis of 3-(5-methoxy-1H-indol-3-yl)propyl methanesulfonate.

[0706] To a stirred solution of 3-(5-methoxy-1H-indol-3-yl)propan-1-ol (2.0 g, 9.75 mmol) in ethyl acetate (20 mL) was added DIPEA (3.77 mL, 29.25 mmol) at 0°C. Then, methanesulfonyl chloride (1.3 mL, 11.7 mmol) was added dropwise at the same temperature. After addition, the reaction mixture was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated, and the crude product was washed with water and extracted with ethyl acetate. The organic layer was concentrated, and the crude product was purified by column chromatography (ethyl acetate: hexane (1:1)) to give 3-(5-methoxy-1H-indol-3-yl)propyl methanesulfonate as a viscous liquid (1.5 g, 55% yield). LC-MS (ESI) m / z: 284.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 10.66 (s, 1H), 7.23 (d, J = 8.8Hz, 1H), 7.12 (d, J = 2.0Hz, 1H), 7.0 (d, J = 2.4Hz, 1H) ,6.72(dd,J=8.8,2.4Hz,1H),4.25(t,J=6.4Hz,1H),3.76(s,3H),2.76(t,J=7.6Hz,2H),2.03(t,J=7.0Hz,2H).

[0707] Step 3: Synthesis of 3-(3-azidopropyl)-5-methoxy-1H-indole.

[0708] To a stirred solution of 3-(5-methoxy-1H-indol-3-yl)propyl methanesulfonate (1.5 g, 5.3 mmol) in DMF (10.0 mL) was added NaN3 (413 mg, 6.3 mmol) at room temperature. The reaction was then heated to 80°C for 1 hour. The progress of the reaction was monitored by TLC. After completion of the reaction, H2O (30 mL) was added and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by Combi-flash column chromatography (ethyl acetate: hexane (1:1)) to give 3-(3-azidopropyl)-5-methoxy-1H-indole as a colorless liquid (700 mg, 85% yield). LC-MS (ESI) m / z: 229.1 [MH] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 10.64 (s, 1H), 7.21 (d, J = 8.8Hz, 1H), 7.09 (s, 1H), 6.98 (s, 1H), 6.71 (d, J = 8.4Hz, 1H), 3.75 (s, 3H), 3.37 (t, J = 6.6Hz, 2H), 2.71 (t, J = 7.4Hz, 2H), 1.89 (q, J = 6.8Hz, 2H).

[0709] Step 4: Synthesis of 3-(5-methoxy-1H-indol-3-yl)propan-1-amine (Intermediate I).

[0710] Under argon, 10% Pd / C (162 mg, 1.52 mmol) was added to a stirred solution of 3-(3-azidopropyl)-5-methoxy-1H-indole (700 mg, 3.04 mmol) in methanol (15.0 mL). A hydrogen balloon was then incorporated into the reaction system, and the reaction was stirred at room temperature for 2 hours. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was filtered through celite and concentrated. The crude product (760 mg) was subjected to preparative HPLC purification to give intermediate H as a colorless liquid (295 mg, 47% yield). LC-MS (ESI) m / z: 205.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.66 (s, 1H), 767 (s, 3H), 7.23 (d, J = 8.8Hz, 1H), 7.01 (d, J = 1.6Hz, 1H), 6.99 (d, J = 2.0Hz, 1H), 6.7 2(dd,J=8.8,2.4Hz,1H),4.25(t,J=6.4Hz,1H),3.76(s,3H),2.83(t,J=7.6Hz,2H),2.71(t,J=7.4Hz,2H),1.89(q,J=7.6Hz,2H).

[0711] Intermediate I

[0712]

[0713] Step 1: Synthesis of 2,2-dimethyl-5-((5-nitro-1H-indol-3-yl)methyl)-1,3-dioxane-4,6-dione.

[0714] To a stirred solution of 5-nitro-1H-indole (5 g, 30.86 mmol) and Meldrum's acid (4.8 g, 33.95 mmol) in acetonitrile (30 mL) was added 37% aqueous formaldehyde (2.7 g, 2.4 mL, 92.58 mmol) and proline (117 mg, 0.05 mmol). The reaction mixture was stirred at room temperature under argon for 18 hours. After completion of the reaction, the resulting yellow slurry was filtered and washed twice with acetonitrile and ether. The solid was then dried under vacuum to give 2,2-dimethyl-5-((5-nitro-1H-indol-3-yl)methyl)-1,3-dioxane-4,6-dione (8.1 g, 82% yield). LC-MS (ESI) m / z: 317.0 [MH] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 11.64 (s, 1H), 8.64 (d, J = 1.6Hz, 1H), 7.97 (dd, J = 9.0, 1.8Hz, 1H), 7.50 ( d,J=8.8Hz,1H),7.33(s,1H),4.85(t,J=4.6Hz,1H),3.46(d,J=4.5Hz,2H),1.79(s,3H),1.56(s,3H).

[0715] Step 2: Synthesis of ethyl 3-(5-nitro-1H-indol-3-yl)propanoate.

[0716] To a stirred solution of 2,2-dimethyl-5-((5-nitro-1H-indol-3-yl)methyl)-1,3-dioxane-4,6-dione (8.1 g, 30.86 mmol) in 50 mL of pyridine:ethanol (4:1) was added copper powder (169 mg, 2.54 mmol) at room temperature under argon, and the reaction was stirred at 120 ° C for 12 hours, stirred at room temperature for another 12 hours, and monitored by TLC for completion. After the reaction was completed, the resulting yellow slurry was filtered, washed with acetonitrile and triturated twice with ether. The solid was then dried in vacuo to give ethyl 3-(5-nitro-1H-indol-3-yl)propanoate as a yellow solid (5.3 g, 79% yield). LC-MS (ESI) m / z: 261.1 [MH] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 11.58 (s, 1H) 8.53 (d, J = 2Hz 1H),7.98(dd,J=9.0,2.2Hz,1H),7.50(d,J=8.8Hz,1H),7.40(s,1H),4.04(q,J= 7.2Hz, 2H), 3.04 (t, J = 7.4Hz, 2H), 2.68 (t, J = 7.4Hz, 2H), 1.14 (t, J = 7.0Hz, 3H).

[0717] Step 3: Synthesis of 3-(5-nitro-1H-indol-3-yl)propan-1-ol.

[0718] A stirred solution of ethyl 3-(5-nitro-1H-indol-3-yl)propanoate (5.2 g, 3.66 mmol) in dry THF (20 mL) was cooled at 0 ° C, and then a 2M LiAlH4 solution in THF (10.5 mL, 21.27 mmol) was added under argon. The reactants were stirred at the same temperature for 7 hours and monitored for completion by TLC. After completion of the reaction, the resulting reaction mixture was quenched with ethyl acetate and 1M NaOH aqueous solution, extracted with ethyl acetate, and the organic layer was concentrated under reduced pressure to give a crude product. The crude residue was triturated twice with ether. The solid was then dried in vacuo to give 3-(5-nitro-1H-indol-3-yl)propan-1-ol as a pale yellow solid (3.8 g, 88% yield). LC-MS (ESI) m / z: 219.2 [MH] + . 1H-NMR (400MHz, DMSO-d6) δ (ppm) 11.55 (s, 1H), 8.50 (d, J = 2Hz, 1H), 7.98 (dd, J = 9.0, 2.2Hz, 1H), 7.50 (d, J = 8.8 Hz,1H),7.39(bs,1H),4.48(t,J=5.2Hz,1H),3.47(q,J=6.0Hz,2H),2.79(t,J=7.6Hz,2H),1.83–1.76(m,2H).

[0719] Step 4: Synthesis of 3-(5-nitro-1H-indol-3-yl)propyl methanesulfonate.

[0720] To a stirred solution of 3-(5-nitro-1H-indol-3-yl)propan-1-ol (3.8 g, 17.27 mmol) in dichloromethane (10 mL) was added Et3N (7.16 mL, 51.81 mmol) at 0°C under argon, followed by MsCl (1.96 mL, 25.90 mmol). The reaction mixture was stirred at the same temperature under argon for 5 hours and monitored for completion by TLC. After completion of the reaction, the resulting reaction mixture was quenched with brine and extracted with ethyl acetate, and the organic layer was concentrated under reduced pressure to give a crude product. The crude product was triturated twice with pentane and ether. The solid was then dried under reduced pressure to give 3-(5-nitro-1H-indol-3-yl)propyl methanesulfonate as a yellow solid (4.1 g, 79% yield). LC-MS (ESI) m / z: 297.1 [MH] + .

[0721] Step 6: Synthesis of 3-(3-azidopropyl)-5-nitro-1H-indole.

[0722] Under argon, to a stirred solution of 3-(5-nitro-1H-indol-3-yl)propyl methanesulfonate (6, 4.1 g, 13.75 mmol) in DMF (12 mL) was added NaN3 (1.34 g, 20.63 mmol) at room temperature, the reactants were stirred at 60 ° C for 16 hours, and the completion was monitored by TLC. After the reaction was completed, the resulting reaction mixture was quenched with brine solution and extracted with ethyl acetate. The organic portion was dried over Na2SO4 and concentrated under reduced pressure to give a crude product. The crude material was purified by silica gel column to give 3-(3-azidopropyl)-5-nitro-1H-indole as a yellow solid (2.4 g, 79% yield). LC-MS (ESI) m / z: 244.12 [MH] + . 1HNMR(400MHz,DMSO-d6)δ(ppm)11.61(s,1H),8.53(d,J=1.6Hz,1H),7.98(dd,J=8.8,2.0Hz,1H),7.5 0(d,J=8.8Hz,1H),7.44(s,1H),3.42(t,J=9.6Hz,2H),2.83(q,J=7.7Hz,2H),1.91(q,J=7.2Hz,2H).

[0723] Step 7: Synthesis of 3-(3-aminopropyl)-1H-indol-5-amine (Intermediate I).

[0724] In a 100 mL autoclave, to a stirred solution of 3-(3-azidopropyl)-5-nitro-1H-indole (1.0 g, 4.08 mmol) in methanol (10 mL) was added Pd / C (432 mg, 10% w / w), followed by the introduction of H2 gas (50 psi), and the reactants were stirred at room temperature for 10 hours. The reaction was monitored for completion by TLC. After completion of the reaction, the resulting reaction mixture was filtered through celite, and the organic portion was concentrated under reduced pressure to give a crude product. The crude compound was purified by preparative HPLC to give Intermediate I as a white solid (296 mg, 38% yield). LC-MS (ESI) m / z: 190.25 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ(ppm)11.13(s,1H),9.70(brs,2H),7.75(brs,2H),7.45(s,1H),7.43-7.42(m,1H),7. 28(d,J=2.0Hz,1H),7.03(dd,J=8.4,2.0Hz,1H),2.86-2.81(m,2H),2.74(t,J=7.4Hz,2H),1.93–1.86(m,2H).

[0725] Trabectedin-related payload (PL)

[0726]

[0727] General Scheme A - Step 1: Synthesis of Tra-CN derivatives.

[0728] A mixture of M24 (e.g., disclosed in CN107739387 A) (1.0 eq.), each intermediate described above (10 eq.), and NaOAc (11 eq.) in anhydrous EtOH was stirred at 25°C overnight. After the reaction was complete, the solvent was removed under reduced pressure to obtain a solid. Ethyl acetate was added to the solid and filtered. The filtrate was purified by flash column chromatography.

[0729] General Scheme A - Step 2: Synthesis of Tra-OH derivatives.

[0730] To a solution of the Tra-CN derivative (1.0 eq.) in acetonitrile and water (3 / 2, v / v) was added silver nitrate (14 eq.). The suspension was stirred at 25°C for 24 hours. The reaction mixture was then quenched by adding a mixture of ethyl acetate / 5% ammonium hydroxide. The combined organic layers were dried over sodium sulfate and concentrated. The residue was purified by flash column chromatography.

[0731] Synthesis of PL-4 and PL-1.

[0732]

[0733] Following Step 1 of General Procedure A, a mixture of M24 (30 mg, 0.048 mmol, 1.0 eq.), Intermediate A (65.2 mg, 0.322 mmol, 10 eq.), and NaOAc (29 mg, 0.354 mmol, 11 eq.) in anhydrous EtOH was stirred at 25°C overnight. After completion of the reaction, the solvent was removed under reduced pressure to yield a solid. Ethyl acetate was added to the solid and filtered. The filtrate was purified by flash column chromatography (ethyl acetate:n-heptane (1:1)) to afford PL-4 as a light yellow solid (19 mg, 76.7%). 1 H NMR(300MHz,CHCl3-d)δ(ppm)6.60(s,1H),6.37(s,1H),6.27(s,1H),6.06(s,1H),5.99(s,1H),5.31(s, 1H),5.02(d,J=11.4Hz,1H),4.33(s,1H),4.29(d,J=4.5Hz,1H),4.19(s,1H),4.12(d,J=12.3Hz,1H),3. 82(d,J=8.3Hz,3H),3.67–3.57(m,3H),3.52(d,J=5.1Hz,1H),3.43(s,1H),3.13(s,1H),2.96(s,2H),2. 83(s,2H),2.60(s,3H),2.33(s,3H),2.32(s,1H),2.28(d,J=2.7Hz,3H),2.21(s,3H),2.11–2.02(m,4H).

[0734] According to step 2 of General Procedure A, silver nitrate (60 mg, 0.353 mmol, 14 eq.) was added to a solution of PL-4 (19 mg, 0.0246 mmol, 1.0 eq.) in acetonitrile and water (3 / 2, v / v, 1 mL). The suspension was stirred at 25°C for 24 hours. The reaction mixture was then quenched by adding a mixture of ethyl acetate and 5% ammonium hydroxide. The combined organic layers were dried over sodium sulfate and concentrated. The residue was purified by flash column chromatography (eluting with dichloromethane:methanol (20:1)) to afford PL-1 (15 mg, 80%) as a yellow solid. 1 H NMR (300MHz, CHCl3-d) δ (ppm) 6.61 (s, 1H), 6.31 (d, J = 29.4Hz, 2H), 5.99 (d, J = 22.5Hz, 2H), 5.74 (s, 1H), 5.12 (d, J = 11.4Hz, 1H), 4.82 (s, 1H), 4 .49(s,2H),4.18(s,1H),4.06(s,1H),3.80(s,4H),3.57(s,5H),3.24(s ,1H),2.89(s,2H),2.33(s,4H),2.27(s,3H),2.18(s,3H),2.03(s,3H).

[0735] Synthesis of PL-6 and PL-2.

[0736]

[0737] Following step 1 of general procedure A, a mixture of M24 (100 mg, 0.16 mmol, 1.0 eq.), 5-(2-aminoethyl)-2-(2-hydroxyethoxy)phenol (160 mg, 0.8 mmol, 5 eq.), HOAc (96.1 mg, 1.6 mmol, 10 eq.), and NaOAc (25.6 mg, 0.32 mmol, 2 eq.) in anhydrous EtOH (10 mL) was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and purified by flash column chromatography on neutral alumina (dichloromethane:methanol (50:1)) to afford PL-2 as a light yellow solid (68.0 mg, 52.8%). LC-MS (ESI) m / z: 801 [M+H] + . 1H NMR (300 MHz, deuterated chloroform) δ (ppm) 6.62 (s, 1H), 6.51 (d, J = 9.7 Hz, 2H), 6.15 (s, 1H), 6.00 (s, 1H), 5.80 (s, 1H), 5.03 (d, J = 11.6 Hz, 1H), 4.60 (s, 1H), 4.44–4.25 (m, 2H), 4.21 (s, 1H), 4.12 (d, J = 11.4 Hz ,1H),3.87(d,J=6.2Hz,4H),3.81(s,3H),3.52(s,1H),3.44(s,1H),3.13(s,1H),2.94(d,J=19 .8Hz,3H),2.86–2.38(m,3H),2.35(s,3H),2.28(s,3H),2.22(s,3H),2.16(s,1H),2.07(s,3H).

[0738] According to step 2 of general procedure A, silver nitrate (60 mg, 0.353 mmol, 14 eq.) was added to a solution of PL-2 (19 mg, 0.0246 mmol, 1.0 eq.) in acetonitrile and water (3 / 2, v / v, 1 mL). The suspension was stirred at 25 ° C for 24 hours and then treated with a mixture of 5% ammonium hydroxide in ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated. The residue was purified by preparative HPLC to give PL-6 as a yellow solid (6 mg, 31.9% yield). LC-MS (ESI) m / z: 792 [M + H] + . 1 H NMR (300MHz, CHCl3-d) δ (ppm) 6.69–6.57 (m, 1H), 6.50 (d, J = 6.5Hz, 2H), 6.12 (s, 1H), 5.96 (d,J=4.9Hz,1H),5.13(d,J=10.8Hz,1H),4.82(s,1H),4.48(d,J=3.5Hz,2H),4.18(s,2H) ,3.84(dd,J=21.5,4.6Hz,8H),3.58(s,1H),3.34(d,J=10.3Hz,1H),3.21(s,1H),3.14(s, 1H), 2.89 (s, 1H), 2.34 (d, J = 6.4Hz, 5H), 2.26 (s, 4H), 2.18 (s, 3H), 2.04 (d, J = 2.4Hz, 6H).

[0739] Synthesis of PL-7.

[0740]

[0741] According to step 1 of general procedure A, a mixture of M24 (30 mg, 0.048 mmol, 1.0 eq.), intermediate D (112.6 mg, 0.48 mmol, 10 eq.) and NaOAc (38.6 mg, 0.48 mmol, 10 eq.) in anhydrous EtOH was stirred at 25°C overnight. The solvent was removed under reduced pressure to give a solid. Ethyl acetate was added and the mixture was filtered. The filtrate was purified by flash silica gel column chromatography (dichloromethane:methanol (50:1)) to give PL-7 as a white solid (15 mg, 39%). LC-MS (ESI) m / z: 800 [M+H] + . 1 H NMR (400MHz, CHCl3-d) δ (ppm) 6.59 (s, 1H), 6.41 (s, 1H), 6.29 (s, 1H), 6.14 (d, J = 1.4Hz, 1H), 5.98 (s, 1H), 5.01 (d ,J=11.5Hz,1H),4.57(s,1H),4.32(s,1H),4.28(d,J=4.9Hz,1H),4.18(d,J=2.7Hz,1H),4.10(d,J=12.0Hz,1H),3 .91–3.80(m,4H),3.79(s,3H),3.50(d,J=4.9Hz,1H),3.41(s,1H),3.12(t,J=9.1Hz,1H),2.94(d,J=6.9Hz,2H), 2.80(d,J=12.1Hz,1H),2.42(d,J=16.1Hz,2H),2.32(s,5H),2.26(s,3H),2.19(s,3H),2.07(s,2H),2.04(s,3H).

[0742] Synthesis of PL-8.

[0743]

[0744] A mixture of M24 (20 mg, 0.032 mmol, 1.0 eq.), Intermediate E (67.96 mg, 0.32 mmol, 10 eq.), and NaOAc (5.12 mg, 0.064 mmol, 2.0 eq.) in anhydrous ethanol was stirred at 25°C overnight. The mixture was purified by preparative HPLC to give PL-8 as a white solid (9.0 mg, 34%). LC-MS (ESI) m / z: 815.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.75 (s, 1H), 6.47 (s, 1H), 6.41 (d, J = 3.9Hz, 2H), 6.22 (d, J = 11.8 Hz,2H),5.05(d,J=11.2Hz,1H),4.50(d,J=3.5Hz,2H),4.27(s,1H),4.21(d,J=4.5Hz,1H),4.15 (d,J=15.9Hz,1H),4.10–4.03(m,2H),3.65(s,3H),3.26(s,2H),3.05(s,1H),2.80(s,2H),2.70 (dd,J=3.9,2.0Hz,1H),2.37–2.35(m,1H),2.31(s,4H),2.24(s,3H),2.06(s,3H),2.01(s,3H).

[0745] Synthesis of PL-9.

[0746]

[0747] To a solution of PL-4 (30 mg, 0.04 mmol, 1.0 eq.) and glycolic acid (12.16 mg, 0.16 mmol, 4.0 eq.) in 2 mL of DMF was added HATU (45.6 mg, 0.12 mmol, 3.0 eq.). After stirring for 5-10 minutes, DIPEA (25.8 mg, 0.2 mmol, 5.0 eq.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was purified by preparative HPLC to give PL-9 (20 mg, 61.9% yield) as a white solid. LC-MS (ESI) m / z: 828.3 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ(ppm)9.03(s,1H),8.75(s,1H),7.90(s,1H),6.47(s,2H),6.18(s,2H),6.13(t ,J=5.5Hz,1H),5.07(d,J=11.6Hz,1H),4.52(d,J=2.8Hz,2H),4.22(d,J=4.8Hz,1H),4.18–4.05(m,2H), 3.96(d,J=5.5Hz,2H),3.66(s,3H),3.61(s,3H),3.30(s,1H),3.01(s,1H),2.78(s,2H),2.70(t,J=1.9 Hz,1H),2.39–2.34(m,1H),2.32(s,3H),2.24(s,3H),2.18(d,J=14.6Hz,1H),2.07(s,4H),2.00(s,3H).

[0748] Synthesis of PL-11.

[0749]

[0750] A mixture of M24 (5 mg, 0.08 mmol, 1.0 eq.), 3-(2-aminoethyl)phenol hydrochloride (13.84 mg, 0.08 mmol, 5.0 eq.), and NaOAc (7.2 mg, 0.088 mmol, 10.0 eq.) in anhydrous EtOH (1 mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure and the product was purified by neutral alumina column chromatography (dichloromethane:methanol (50:1)) to yield 5 mg (83.9%) of PL-11 as a brown solid. LC-MS (ESI) m / z: 741 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.32 (s, 1H), 8.79 (s, 1H), 6.74 (d, J = 8.7Hz, 1H), 6.46 (d ,J=10.7Hz,2H),6.38(s,1H),6.29(s,1H),6.18(s,1H),5.08(d,J=11.9Hz,1H),4.48(s ,1H),4.22(s,1H),4.08(s,1H),3.99(d,J=11.4Hz,1H),3.65(s,3H),3.24(s,1H),2.81 (s,2H),2.31(s,3H),2.25(s,3H),2.06(s,3H),2.03(s,1H),2.02(s,1H),2.01(s,3H).

[0751] Synthesis of PL-12.

[0752]

[0753] Following step 1 of General Procedure A, a mixture of M24 (10 mg, 0.016 mmol, 1.0 eq), 3-(2-aminoethyl)aniline (138 mg, 0.08 mmol, 5.0 eq), and NaOAc (12.8 mg, 0.016 mmol, 10.0 eq) in anhydrous EtOH (10 mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was purified by preparative TLC (dichloromethane:methanol (10:1)) to afford PL-12 (6.1 mg, 51.2%) as a light yellow solid. LC-MS (ESI) m / z: 740.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.76 (s, 1H), 7.68–7.55 (m, 2H), 6.58 (d, J = 8.5Hz, 1H), 6.47 (s, 1H), 6.28 ( d,J=1.0Hz,1H),6.23(d,J=9.2Hz,1H),6.17(d,J=1.3Hz,2H),5.06(d,J=11.6Hz,1H),4.94(s,1H),4.47 (s,2H),4.22(s,1H),4.08(s,1H),3.97(d,J=11.5Hz,1H),3.65(s,3H),2.81(d,J=5.3Hz,2H),2.70(s,1 H), 2.36 (s, 1H), 2.31 (s, 3H), 2.24 (s, 3H), 2.06 (s, 3H), 2.02 (d, J = 6.6Hz, 2H), 2.01 (s, 3H), 1.94 (s, 1H).

[0754] Synthesis of PL-13.

[0755]

[0756] A solution of PL-4 (10 mg, 0.013 mmol, 1.0 eq.) and (((9H-fluoren-9-yl)methoxy)carbonyl)glycine (38.6 mg, 0.13 mmol, 10.0 eq.), HOBt (35 mg, 0.26 mmol, 20.0 eq.), EDCI (49 mg, 0.25 mmol, 20 eq.), and HATU (98.8 mg, 0.26 mmol, 20.0 eq.) in 3 mL of dichloromethane was stirred at room temperature for 2 hours. LC-MS confirmed complete conversion of the reaction. The reaction mixture was washed with water, and the organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was used in the next step without purification.

[0757] A solution of crude N-Fmoc-PL-4 in 4 mL of acetonitrile and 1.6 mL of Et2N was stirred at room temperature for 1 hour. Complete conversion of the reaction was confirmed by LC-MS. The reaction mixture was concentrated and dissolved in dichloromethane, and the solution was washed with water and dried over anhydrous sodium sulfate. The organic layer was concentrated and the residue was purified by preparative HPLC to give PL-12 (4.6 mg, 43% yield) as a white solid. LC-MS (ESI) m / z: 827 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.41 (s, 2H), 7.93 (s, 1H), 6.46 (d, J = 8.3Hz, 2H), 6.18 (s, 2H), 5.3 5(t,J=4.8Hz,1H),5.07(d,J=11.5Hz,1H),4.52(s,1H),4.23(s,1H),4.14(s,1H),4.08(d,J=11. 7Hz,1H),3.66(s,3H),3.60(s,3H),3.23(s,2H),3.02(s,2H),2.85–2.72(m,2H),2.32(s,3H),2. 24(s,3H),2.18(d,J=14.5Hz,1H),2.07(s,3H),2.05(d,J=7.4Hz,2H),2.02(s,1H),2.00(s,3H).

[0758] Synthesis of PL-14.

[0759]

[0760] To a solution of Ecteinascidin 743 (CAS: 114899-77-3) (20 mg, 0.026 mmol, 1.0 eq.) and (9H-fluoren-9-yl)methyl (2-hydroxyethyl)carbamate (73.42 mg, 0.26 mmol, 10.0 eq.) in 2 mL of THF was added PPh3 (204 mg, 0.78 mmol, 30.0 eq.) and DIAD (157 mg, 0.78 mmol, 30.0 eq.). After addition, the mixture was stirred at room temperature overnight. LC-MS showed that the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give N-Fmoc-PL-14 (2 mg, 7.4% yield) as a white solid. LC-MS (ESI) m / z: 1036 [M+H] + .

[0761] A solution of N-Fmoc-PL-14 (2 mg, 0.00193 mmol, 1.0 eq.) in 1 mL of DMF and 0.2 mL of Et3N was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by preparative HPLC to afford PL-14 (0.77 mg, 49% yield) as a white solid. LC-MS (ESI) m / z: 814 [M+H] + .

[0762] Synthesis of PL-16.

[0763]

[0764] To a solution of PL-12 (20 mg, 0.027 mmol, 1.0 eq.) and (9H-fluoren-9-yl)methyl (2-chloro-2-oxoethyl)carbamate (59.7 mg, 0.189 mmol, 7.0 eq.) in 2 mL of DMF was added pyridine (15 mg, 0.189 mmol, 7.0 eq.) and the reaction mixture was stirred at room temperature overnight. After complete consumption of PL-12, triethylamine (0.1 mL) was added and the reaction mixture was stirred at room temperature for another 4 hours. The reaction mixture was purified by preparative HPLC to give PL-16 (11 mg, 55% yield) as a white solid. LC-MS (ESI) m / z: 797.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.78–8.70 (m, 1H), 8.30 (s, 1H), 7.30 (s, 1H), 7.25 (d, J = 8.2Hz, 1H),6.85(d,J=8.6Hz,1H),6.47(s,1H),6.27(s,1H),6.17(s,1H),5.07(d,J=11.3Hz,1H),4.4 6(s,1H),4.20(d,J=4.4Hz,1H),4.08(s,1H),4.01(d,J=11.2Hz,1H),3.64(s,3H),3.26(s,5H ),3.23(s,2H),2.80(s,2H),2.68(s,1H),2.29(s,3H),2.23(s,3H),2.05(s,4H),1.99(s,4H).

[0765] Synthesis of PL-18.

[0766]

[0767] A solution of M24 (20 mg, 0.032 mmol, 1.0 eq.), 4-(2-aminoethyl)aniline (21.76 mg, 0.16 mmol, 5.0 eq.), and p-TsOH (5.5 mg, 0.032 mmol, 1.0 eq.) in anhydrous n-BuOH (1 mL) was stirred at 110° C. for 1.5 hours. The reaction mixture was concentrated and purified by preparative HPLC to give compound PL-18 (1.3 mg, 5.4% yield) as a brown solid. LC-MS (ESI) m / z: 740 [M+H] + .

[0768] Synthesis of PL-19.

[0769]

[0770] To a solution of PL-12 (20 mg, 0.026 mmol, 1.0 eq.) and 2-((tert-butyldimethylsilyl)oxy)acetic acid (8 mg, 0.105 mmol, 4.0 eq.) in 2 mL of DMF were added HATU (29.64 mg, 0.078 mmol, 3.0 eq.) and DIPEA (16.77 mg, 0.13 mmol, 5.0 eq.). After the addition, the mixture was stirred at room temperature overnight. After the reaction was complete, 70% HF / pyridine solution (12.87 mg, 0.13 mmol, 5.0 eq.) was added. Stirring was continued at room temperature for another hour. The reaction mixture was concentrated and purified by preparative HPLC to give PL-19 (3.4 mg, 16% yield) as a white solid. LC-MS (ESI) m / z: 798 [M+H] + . 1 HNMR (400MHz, DMSO-d6) δ (ppm) 9.52 (s, 1H), 8.76 (s, 1H), 7.39 (d, J = 2.3Hz, 1H), 7.31 (dd, J = 8.7, 2.3Hz, 1H), 6.85 (d, J=8.7Hz,1H),6.48(s,1H),6.32–6.25(m,1H),6.18(s,1H),5.65(s,1H),5.08(d,J=11.5Hz,1H),4.47(d,J=2.9Hz,2H ),4.25–4.20(m,1H),4.09(s,1H),4.06–4.00(m,1H),3.94(d,J=4.5Hz,2H),3.65(s,3H),3.27–3.22(m,1H),3.22–3. 13(m,1H),2.81(d,J=5.2Hz,2H),2.69(s,1H),2.51–2.41(m,2H),2.30(s,3H),2.25(s,3H),2.06(s,4H),2.00(s,3H).

[0771] Synthesis of PL-22.

[0772]

[0773] PL-9 (6 mg, 0.0073 mmol, 1.0 eq.) and AgNO₃ (12.4 mg, 0.0073 mmol, 10.0 eq.) were suspended in acetonitrile:H₂O (3:2, v / v). The solution was stirred at room temperature in the dark for 24 hours. The suspension was quenched with a mixture of saturated brine and saturated sodium bicarbonate and filtered. The resulting solution was purified by preparative HPLC to afford PL-22 (0.97 mg, 16.3% yield) as a white solid. LC-MS (ESI) m / z: 819 [M+H] + .

[0774] Lubitel-related payload (PL)

[0775]

[0776] General Scheme B - Step 1: Synthesis of Lur-CN derivatives.

[0777] A mixture of M24 (1.0 eq.), each intermediate described above (10.0 eq.), AcOH (10 eq.) and NaOAc (2 eq.) in anhydrous EtOH was stirred for 2 hours at 60° C. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography on neutral alumina.

[0778] General Scheme B - Step 2: Synthesis of Lur-OH derivatives.

[0779] To a solution of Lur-CN derivative (1.0 eq.) in acetonitrile and water (3 / 2, v / v) was added silver nitrate (15 eq.). The suspension was stirred at 25 ° C for 24 hours and then treated with a mixture of saturated sodium bicarbonate and brine. The precipitated solid was filtered off and the filtrate was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated. The residue was purified by preparative HPLC.

[0780] Synthesis of PL-5 and PL-3.

[0781]

[0782] According to step 1 of general procedure B, a mixture of M24 (100 mg, 0.16 mmol, 1.0 eq.), intermediate C (282.2 mg, 1.6 mmol, 10.0 eq.), AcOH (96.1 mg, 1.6 mmol, 10 eq.), and NaOAc (25.6 mg, 0.32 mmol, 2 eq.) in anhydrous EtOH (10 mL) was stirred at 60°C for 2 hours. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography on neutral alumina (dichloromethane:methanol = 50:1) to give PL-5 as a light yellow solid (80 g, 63.8%). LC-MS (ESI) m / z: 779 [M+H] + . 1 H NMR(300MHz,CHCl3-d)δ(ppm)7.54(s,1H),7.04(d,J=8.5Hz,1H),6.76–6.64(m,2H),6.56(dd,J=8.5,2.3Hz,1H),6.2 8–6.18(m,1H),6.09–6.01(m,1H),5.08(d,J=11.5Hz,1H),4.56(s,1H),4.30(d,J=11.0Hz,2H),4.25–4.15(m,2H),3. 82(s,3H),3.49–3.38(m,2H),3.12(d,J=13.2Hz,1H),3.04(s,1H),2.96(d,J=9.0Hz,1H),2.86–2.77(m,1H),2.55(d, J=5.7Hz, 2H), 2.49 (s, 1H), 2.38 (d, J= 4.7Hz, 4H), 2.28 (d, J= 6.1Hz, 4H), 2.23 (d, J= 3.7Hz, 3H), 2.07 (d, J= 4.1Hz, 3H).

[0783] According to step 2 of general procedure B, silver nitrate (212 mg, 1.248 mmol, 15 eq.) was added to a solution of PL-5 (65 mg, 0.0833 mmol, 1.0 eq.) in acetonitrile and water (3 / 2, v / v, 1 mL). The suspension was stirred at 25°C for 24 hours and then treated with a mixture of saturated sodium bicarbonate and brine. The precipitated solid was filtered off and the filtrate was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated. The residue was purified by preparative HPLC to give PL-3 as a yellow solid (2.6 mg, 4%). LC-MS (ESI) m / z: 376.8 [(M-H2O) / 2+H] + . 1H NMR(300MHz,CD3OD-d4)δ(ppm)8.10(s,1H),7.15(d,J=8.6Hz,1H),6.94(s,2H),6.74(q,J=14.0Hz,3H),6.28(s,1H),6.09(d,J=6.7H z,1H),5.35(t,J=4.8Hz,3H),3.80(d,J=7.7Hz,5H),2.60(s,2H),2.52(s,2H),2.40(s,3H),2.32(d,J=6.6Hz,9H),2.25–2.17(m,6H).

[0784] Synthesis of PL-10.

[0785]

[0786] To a solution of PL-5 (20 mg, 0.026 mmol, 1.0 eq.) and glycolic acid (7.81 mg, 0.103 mmol, 4.0 eq.) in 2 mL of dichloromethane were added HATU (29.6 mg, 0.078 mmol, 3.0 eq.) and DIPEA (16.8 mg, 0.13 mmol, 5.0 eq.). After the addition, the mixture was stirred at room temperature overnight. The reaction was purified by preparative HPLC to give compound PL-10 as a white solid (1.53 mg, 7.1%). LC-MS (ESI) m / z: 837.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 10.08 (s, 1H), 9.34 (s, 1H), 8.76 (s, 1H), 7.69 (s, 1H), 7.23 (d, J = 1.3Hz, 2H), 6. 47(s,1H),6.22(d,J=14.4Hz,2H),5.57(t,J=6.0Hz,1H),5.07(d,J=11.1Hz,1H),4.46(s,2H),4.19(s,1H),4 .06(d,J=10.8Hz,2H),3.93(d,J=5.7Hz,2H),3.64(s,3H),3.19(s,1H),2.85–2.74(m,1H),2.68–2.65(m,1H) ,2.61(d,J=15.4Hz,1H),2.41(s,2H),2.34–2.30(m,2H),2.28(s,3H),2.25(s,3H),2.04(s,4H),1.97(s,3H).

[0787] Synthesis of PL-15.

[0788]

[0789] A mixture of M24 (10 mg, 0.016 mmol, 1.0 eq.), intermediate F (24 mg, 0.16 mmol, 10.0 eq.), AcOH (96.1 mg, 1.6 mmol, 10.0 eq.), and NaOAc (145 mg, 0.176 mmol, 11.0 eq.) in anhydrous EtOH (1 mL) was stirred at 40°C for RT. The solvent was removed under reduced pressure and purified by neutral alumina column chromatography (dichloromethane:methanol (30:1)) to give PL-15 (2.5 mg, 20% yield) as a light yellow solid. LC-MS (ESI) m / z: 779.2 [M+H] + . 1 H NMR(400MHz,CHCl3-d)δ(ppm)7.46(s,1H),7.19(d,J=8.5Hz,1H),6.69(s,1H),6.60(s,1H),6.50(d,J=8.4Hz,1H),6.26(s,1H), 6.07(s,1H),5.83(s,1H),5.12(d,J=11.7Hz,1H),4.59(s,1H),4.36(s,1H),4.31(d,J=4.7Hz,1H),4.26(s,1H),4.21(d,J=11.7 Hz,1H),3.85(s,3H),3.46(d,J=11.3Hz,2H),3.18(s,1H),3.10(d,J=18.0Hz,1H),2.97(dd,J=18.1,9.0Hz,1H),2.84(d,J=12.4 Hz,1H),2.68–2.59(m,1H),2.54(d,J=15.5Hz,2H),2.41(s,4H),2.35(d,J=14.4Hz,1H),2.31(s,3H),2.26(s,4H),2.11(s,3H).

[0790] Synthesis of PL-17.

[0791]

[0792] A mixture of M24 (10 mg, 0.016 mmol, 1.0 eq.), Intermediate G (28 mg, 0.16 mmol, 10.0 eq.), and AcOH (9.6 mg, 0.16 mmol, 10.0 eq.) in anhydrous EtOH (10 mL) was stirred at 60°C for 2 hours. The solvent was removed under reduced pressure and purified by neutral alumina column chromatography (dichloromethane:methanol (50:1)) to afford PL-17 (1 mg, 7.8% yield) as a light yellow solid. LC-MS (ESI) m / z: 793 [M+H] + .

[0793] Synthesis of PL-20.

[0794]

[0795] A mixture of M24 (20 mg, 0.032 mmol, 1.0 eq.), intermediate H (50.88 mg, 0.16 mmol, 5.0 eq.), and p-TsOH (5.5 mg, 0.032 mmol, 1.0 eq.) in anhydrous n-BuOH (1 mL) was stirred at 110° C. for 1.5 hours. The solution was concentrated and purified by preparative HPLC to afford PL-20 (1.6 mg, 6.1% yield) as a brown solid. LC-MS (ESI) m / z: 808 [M+H] + .

[0796] Synthesis of PL-21

[0797]

[0798] A mixture of M24 (20 mg, 0.032 mmol, 1.0 eq.), Intermediate I (50.88 mg, 0.16 mmol, 5.0 eq.), and p-TsOH (5.5 mg, 0.032 mmol, 1.0 eq.) in anhydrous n-BuOH (1 mL) was stirred at 110° C. for 1.5 hours. The solution was concentrated and purified by preparative HPLC to afford PL-21 (0.8 mg, 3% yield) as a brown solid. LC-MS (ESI) m / z: 793 [M+H] + .

[0799] Linker (LK)

[0800] Synthesis of LK-1.

[0801]

[0802] To a solution of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid (572 g, 1.32 mmol) and HATU (652 mg, 1.72 mmol) in DMF (4 mL) was added (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (500 mg, 1.32 mmol) and N,N-diisopropylethylamine (503 mg, 3.96 mmol) at 0°C. After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by reverse phase flash column chromatography (0-60% acetonitrile in H2O) to afford 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-carboxamide as a yellow solid (400 mg, 38% yield). LC-MS: (ESI) m / z = 795.8 [M+H] + .

[0803] To a solution of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-amide (400 mg, 0.504 mmol) in DMF (2 mL) at 0° C. was added bis(4-nitrophenyl)carbonate (766 mg, 2.52 mmol) and N,N-diisopropylethylamine (320 mg, 2.52 mmol). After the addition, the mixture was stirred at room temperature for 3 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated to give the crude product, which was purified by preparative HPLC to give 4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosamido)benzyl carbonate (4-nitrophenyl) as a white solid (220 mg, 45% yield). LC-MS: (ESI) m / z: 960.4 [M+H] + . 1HNMR (400MHz, DMSO-d6): δ (ppm) 9.94 (s, 1H), 8.31-8.34 (m, 2H), 7.98 (d, J = 8.0Hz, 1H), 7.76 (d, J = 8.0 Hz,1H),7.67(d,J=8.0Hz,2H),7.57-7.59(m,2H),7.43(d,J=8.0Hz,2H),7.01(s,2H),5.91-5.94(m,1 H),5.32(s,2H),5.28(s,2H),4.43-4.45(m,1H),4.23-4.27(m,1H),3.48-3.67(m,26H),2.98-3.07(m ,2H),2.40-2.51(m,2H),2.01-2.05(m,1H),1.76-1.78(m,2H),1.44-1.66(m,2H),0.90-0.85(m,6H).

[0804] Synthesis of LK-2.

[0805]

[0806] To a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (1.00 g, 2.35 mmol) and HATU (1.34 g, 3.53 mmol) in DMF (10 mL) at 0° C. was added 2,5,8,11,14,17,20,23-octaoxapentacosan-25-amine (901 mg, 2.35 mmol) and N,N-diisopropylethylamine (910 mg, 7.05 mmol). After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the crude product, which was purified by flash silica gel column chromatography (0-10% methanol in dichloromethane) to afford (S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontane-31-oic acid tert-butyl ester as a yellow solid (1.50 g, 81% yield). LC-MS: (ESI) m / z: 791.4 [M+H] + .

[0807] To a solution of (S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontane-31-oic acid tert-butyl ester (1.50 g, 1.89 mmol) in dichloromethane (6 mL) was added trifluoroacetic acid (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated in vacuo to give (S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontane-31-oic acid as a crude yellow oil (1.50 g, crude), which was used in the next reaction without further purification. LC-MS: (ESI) m / z: 735.4 [M+H] + .

[0808] To a solution of (S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontane-31-oic acid (1.50 g, crude) and HATU (1.55 g, 4.08 mmol) in DMF (10 mL) at 0° C. were added (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (774 mg, 2.04 mmol) and N,N-diisopropylethylamine (791 mg, 6.13 mmol). After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by reverse phase flash column chromatography (0-50% acetonitrile in H2O) to afford (28S,33S,36S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-33-isopropyl-27,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptatriacontane-37-oic acid 4-(hydroxymethyl)phenyl ester (1.10 g, crude) as a white solid. LC-MS: (ESI) m / z: 1097.6 [M+H] + .

[0809] To a solution of (28S,33S,36S)-28-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-33-isopropyl-27,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptatriacontane-37-oic acid 4-(hydroxymethyl)phenyl ester (900 mg, 0.82 mmol) in DMF (10 mL) was added triethylamine (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuo to afford crude (28S,33S,36S)-28-amino-33-isopropyl-27,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptatriacontane-37-oic acid 4-(hydroxymethyl)phenyl ester as a white solid (1.30 g, crude), which was used in the next reaction without further purification. LC-MS: (ESI) m / z: 874.4 [M+H] + .

[0810] To a solution of (28S,33S,36S)-28-amino-33-isopropyl-27,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptatriacontane-37-oic acid 4-(hydroxymethyl)phenyl ester (1.30 g, crude) and 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid 2,5-dioxopyrrolidin-1-yl ester (396 mg, 1.48 mmol) in DMF (6 mL) was added N,N-diisopropylethylamine (576 mg, 4.46 mmol) at 0°C. After addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by reverse phase flash column chromatography (0-70% acetonitrile in H2O) to afford (S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N5-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-N1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)pentanediamide as a white solid (400 mg, crude). LC-MS: (ESI) m / z: 1025.4 [M+H] + .

[0811] To a solution of (S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-N5-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-N1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)pentanediamide (400 mg, 0.390 mmol) and bis(4-nitrophenyl)carbonate (593 mg, 1.95 mmol) in DMF (6 mL) was added N,N-diisopropylethylamine (201 mg, 1.56 mmol) at 0°C. After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the crude product, which was purified by preparative HPLC to give 4-((28S,33S,36S)-28-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-33-isopropyl-27,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptatriacontane-37-amido)benzyl carbonate (4-nitrophenyl) as a white solid (118 mg, 25% yield). LC-MS: (ESI) m / z: 1191.6 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 10.04 (s, 1H), 8.33-8.29 (m, 2H), 8.14-8.10 (m, 2H), 7.91-7.88 (m, 1H), 7.79 (d, J = 8.8Hz, 1H), 7.65 (d,J=8.8Hz,2H),7.58-7.54(m,2H),7.40(d,J=8.4Hz,2H),6.99(s,2H),5.99-5.96(m,1H),5.41(s,2H),5.24(s,2H),4.41-4.35( m,1H),4.21-4.12(m,2H),3.61(t,J=7.2Hz,2H),3.50-3.49(m,26H),3.43-3.38(m,4H),3.23-3.18(m,5H),3.02-2.95(m,2H),2.4 3-2.38(m,2H),2.18-2.14(m,2H),2.01-1.94(m,1H),1.84-1.81(m,1H),1.71-1.58(m,3H),1.46-1.36(m,2H),0.87-0.82(m,6H).

[0812] Synthesis of LK-3.

[0813]

[0814] 4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propionylamino)-3-methylbutyrylamino)-5-ureidopentanylamino)benzyl (4-nitrophenyl) carbonate. LC-MS: (ESI) m / z: 740.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 10.07 (s, 1H), 8.34 (d, J = 9.2Hz, 2H), 8.14 (d, J = 7.2Hz, 1H), 7.87 (d, J = 8.8Hz,1H),7.67(d,J=8.4Hz,2H),7.62(d,J=8.4Hz,2H),7.43(d,J=8.6Hz,2H),7.03(s,2H),6.00(s, 1H),5.43(s,2H),5.27(s,2H),4.44-4.45(m,1H),4.28-4.18(m,1H),3.65-3.47(m,6H),3.11-2.83(m ,2H),2.49-2.30(m,2H),2.02-1.93(m,1H),1.75-1.56(m,2H),1.50-1.31(m,2H),0.90-0.81(m,6H).

[0815] Synthesis of LK-4.

[0816]

[0817] 4-((2S,5S)-14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12-dioxa-3,6-diazatetradecanoylamino)benzyl (4-nitrophenyl) carbonate. LC-MS: (ESI) m / z: 770.2 [M+H] + . 1H NMR (400MHz, DMSO-d6): δ (ppm) 10.13 (s, 1H), 8.32-8.36 (m, 3H), 7.67 (d, J = 8.0Hz, 2H), 7.57-7.60(m,2H),7.42-7.44(m,3H),7.01(s,2H),6.03-5.97(m,1H),5.44(s,2H),5.2 7(s,2H),4.34-4.42(m,1H),4.30-4.32(m,1H),3.93(s,2H),3.56-3.58(m,8H),2.98-3 .04(m,2H),2.00-2.03(m,1H),1.71-1.98(m,2H),1.40-1.63(m,2H),0.82-0.90(m,6H).

[0818] Synthesis of LK-5.

[0819]

[0820] 4-((14S,17S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16-diazaoctadecanoylamino)benzyl (4-nitrophenyl) carbonate. LC-MS: (ESI) m / z: 850.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 10.08 (s, 1H), 8.33 (d, J = 9.0Hz, 2H), 8.18-8.12 (m, 1H), 7.88 (d, J = 8. 6Hz,1H),7.68(d,J=8.0Hz,2H),7.59(d,J=9.0Hz,2H),7.43(d,J=8.4Hz,2H),7.04(s,2H),5.99(t,1H ),5.43(s,2H),5.26(s,2H),4.45-4.35(m,1H),4.29–4.15(m,1H),3.68–3.44(m,14H),3.10–2.85(m ,2H),2.50–2.29(m,2H),2.05-1.95(m,1H),1.80-1.60(m,2H),1.41-1.35(m,2H),0.91-0.84(m,6H).

[0821] Synthesis of LK-6.

[0822]

[0823] 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazaheneicosanoylamino)benzyl (4-nitrophenyl) carbonate. LC-MS: (ESI) m / z: 872.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 10.06 (s, 1H), 8.31 (d, J = 9.2Hz, 2H), 8.13 (d, J = 7.2Hz, 1H), 7.87 (d, J = 8. 4Hz, 1H), 7.65 (d, J = 8.4Hz, 2H), 7.57 (d, J = 9.2Hz, 2H), 7.41 (d, J = 8.4Hz, 2H), 7.02 (s, 2H), 5.98 (t, J = 4. 8Hz,1H),5.41(S,2H),5.24(s,2H),4.53-4.30(m,1H),4.30-4.15(m,1H),3.56-3.45(m,18H),3.03-2.9 4(m,2H),2.48-2.32(m,2H),2.0-1.95(m,1H),1.75-1.55(m,2H),1.50-1.30(m,2H),0.89-0.81(m,6H).

[0824] Synthesis of LK-7.

[0825]

[0826] 4-((20S,23S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-20-isopropyl-18,21-dioxo-23-(3-ureidopropyl)-3,6,9,12,15-pentaoxa-19,22-diazatetracosanoylamino)benzyl (4-nitrophenyl) carbonate. LC-MS: (ESI) m / z: 916.4 [M+H] + . 1H NMR (400MHz, DMSO-d6): δ (ppm) 10.08 (s, 1H), 8.34 (d, J = 12.0Hz, 2H), 8.15 (d, J = 8.0Hz, 1H), 7.89 (d, J = 8.0H) z,1H),7.67(d,J=8.0Hz,2H),7.58-6.60(m,2H),7.43(d,J=8.0Hz,2H),7.04(s,2H),6.01-5.90(m,1H),5.43 (s,2H),5.26(s,2H),4.40-4.41(m,1H),4.23-4.25(m,1H),3.48-3.50(m,4H),3.46-3.47(m,18H),2.97-3.0 4(m,2H),2.38-2.49(m,2H),1.98-2.00(m,1H),1.61-1.72(m,2H),1.26-1.47(m,2H),0.88(q,J=6.8Hz,6H).

[0827] Synthesis of LK-8.

[0828]

[0829] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-oic acid (11.5 g, 23.0 mmol) and DIEA (11.9 g, 92.0 mmol) in DMF (100 mL) was added EDCI (11.9 g, 92.0 mmol) and HOBt (6.2 g, 46.0 mmol) at 0°C, followed by the addition of benzyl 2-aminoacetate (9.60 g, 46.0 mmol). After addition, the mixture was stirred at room temperature for 12 hours. LCMS showed that the reaction was complete. The mixture was partitioned between H2O (100 mL) and ethyl acetate (200 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, and concentrated to afford the crude product, which was purified by flash silica gel chromatography (0-15% methanol in dichloromethane) to afford (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid benzyl ester (2.6 g, 17% yield) as a yellow oil. LC-MS: (ESI) m / z: 649.2 [M+H] + .

[0830] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid benzyl ester (4.50 g, 6.94 mmol) in MeOH (50 mL) and DMF (50 mL) was added Pd / C (500 mg, 10% wt.) under H₂. The mixture was stirred at room temperature under H₂ for 12 hours. LCMS indicated the reaction was complete. The mixture was filtered, and the filtrate was concentrated to afford (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid (4.00 g, quantitative) as a yellow solid. LC-MS: (ESI) m / z: 559.2 [M+H] + .

[0831] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid (3.00 g, 5.38 mmol) and DIEA (1.73 g, 13.4 mmol) in DMF (20 mL) was added HATU (3,88 g, 10.2 mmol) and (4-aminophenyl)methanol (790 mg, 6.45 mmol) at 0°C. After addition, the mixture was stirred at room temperature for 12 hours. LCMS showed that the reaction was complete. The mixture was partitioned between H2O (20 ml) and ethyl acetate (40 ml). The organic phase was separated, dried over anhydrous NaSO, filtered, and concentrated to give the crude product, which was purified by flash silica gel chromatography (0-10% methanol in dichloromethane) to afford (S)-(9H-fluoren-9-yl)methyl(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (2.4 g, 69% yield) as a white solid. LC-MS: (ESI) m / z: 646.2 [M+H] + .

[0832] To a solution of (S)-(9H-fluoren-9-yl)methyl (2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (2.4 g, 3.62 mmol) in DMF (20 mL) was added TEA (4 mL) at room temperature. After the addition, the mixture was stirred at room temperature for 12 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (1.97 g, quantitative) as a white solid. LC-MS: (ESI) m / z: 442.0 [M+H] + .

[0833] To a solution of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)-3-phenylpropanamide (1.97 g, 4.47 mmol) in DMF (20 mL) at 0° C. were added DIEA (1.15 g, 8.94 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.07 g, 6.71 mmol). After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the crude product, which was purified by flash silica gel chromatography (0-17% methanol in dichloromethane) to afford (S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (820 mg, 34.3% yield) as a white solid. LC-MS: (ESI) m / z: 635.3 [M+H] + .

[0834] To a solution of (S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (800 mg, 1.26 mmol) in DMF (4 mL) was added DIEA (650 mg, 5.04 mmol) and bis(4-nitrophenyl)carbonate (1.92 g, 6.30 mmol) at 0°C. After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by C18 (0-55% acetonitrile in H2O) to give LK-8 (250 mg, 34% yield) as a white solid. LC-MS: (ESI) m / z: 617.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 9.92 (s, 1H), 8.40 (t, 1H), 8.30 (d, J = 8.4Hz, 2H), 8.18 (d, J = 7.2Hz, 1H), 8.15–8.1 0(m,1H),8.08–8.02(m,2H),7.67(d,J=8.4Hz,2H),7.55(d,J=8.4Hz,2H),7.43(d,J=8.4Hz,2H),7.26(d,J=4.4H z,4H),7.20-7.19(m,1H),6.99(s,2H),6.95–6.89(m,1H),5.25(s,2H),4.56-4.46(m,1H),4.00–3.52(m,4H),3. 38-3.32(m,2H),3.16–3.01(m,1H),2.90–2.78(m,1H),2.15-2.05(m,2H),1.54-1.40(m,4H),1.20-1.15(m,2H).

[0835] Synthesis of LK-9.

[0836]

[0837] To a solution of 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (20.0 g, 56.5 mmol) in THF (600 mL) and toluene (200 mL) was added Pb(OAc)4 (34.7 g, 78.3 mmol) and pyridine (5.4 mL) at room temperature. After the addition, the mixture was stirred at 75 ° C for 12 hours. LCMS showed that the reaction was complete. The mixture was filtered and concentrated to give the crude product, which was purified by flash silica gel chromatography (0-100% ethyl acetate in petroleum ether) to give (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (13.6 g, 66% yield) as a white solid. LC-MS: (ESI) m / z: 390.9 [M+Na] + .

[0838] To a solution of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (6.60 g, 17.9 mmol) in THF (150 mL) at 0°C was added benzyl 2-hydroxyacetate (5.95 g, 35.8 mmol) and PTSA (1.04 g, 6.05 mmol). After addition, the mixture was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The mixture was partitioned between H2O (150 mL) and ethyl acetate (150 mL x 3). The organic phase was separated, washed with brine (80 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product, which was purified by silica gel chromatography (0-80% ethyl acetate in petroleum ether) to give 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (4.9 g, 58% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.79-8.73 (m, 1H), 7.89 (d, J = 8.0Hz, 2H), 7.71 (d, J = 8.0Hz, 2H), 7.60-7.50 (m, 1H), 7. 33-7.42(m,9H),5.75(s,1H),4.63(d,J=8.0Hz,2H),4.15-4.30(m,5H),3.63(d,J=8.0Hz,2H),3.18(d,J=4.0Hz,1H).

[0839] To a solution of benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (4.00 g, 8.40 mmol) in DMF (20 mL) was added piperidine (4 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to give benzyl 2-((2-aminoacetamido)methoxy)acetate (5.0 g, crude) as a white solid, which was used in the next step without further purification.

[0840] To a solution of 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (50.0 g, 141 mmol) in THF (800 mL) was added 4-nitrophenol (49.1 g, 353 mmol) and DCC (72.7 g, 353 mmol) at room temperature. After the addition, the mixture was stirred at room temperature for 12 hours. LCMS showed that the reaction was complete. The mixture was filtered to give 4-nitrophenyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (42.0 g, 63% yield) as a white solid, which was used in the next step without further purification. LC-MS: (ESI) m / z: 476.2 [M+H] + .

[0841] To a solution of 4-nitrophenyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (22.0 g, 46.3 mmol) in THF (300 mL) was added a solution of NaCO (7.94 g, 92.6 mmol) in HO (99 ml) at room temperature, followed by the addition of (S)-2-amino-3-phenylpropionic acid (6.11 g, 37.1 mmol). The resulting mixture was stirred at room temperature for 12 hours. LCMS showed the reaction was complete. The mixture was partitioned between HO (100 mL) and ethyl acetate (200 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, and concentrated to afford the crude product, which was purified by flash silica gel chromatography (0-15% methanol in dichloromethane) to afford (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-oic acid (11 g, 59% yield) as a white solid. LC-MS: (ESI) m / z: 501.9 [M+H] + .

[0842] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecane-12-oic acid (5.00 g, 9.98 mmol) and HATU (7.58 g, 19.9 mmol) in DMF (20 mL) at 0°C were added benzyl 2-((2-aminoacetamido)methoxy)acetate (5.01 g, 19.9 mmol) and N,N-diisopropylethylamine (5.06 g, 39.9 mmol). After the addition, the mixture was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by flash silica gel chromatography (0-10% MeOH in DCM) to give (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosano-20-oic acid benzyl ester (1.00 g, 14% yield) as a yellow solid. LC-MS: (ESI) m / z: 569.9 [M+H] + .

[0843] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid benzyl ester (1.00 g, 1.36 mmol) in MeOH (20 mL) was added Pd / C (10%, aqueous, 100 mg) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and concentrated in vacuo to afford (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid (900 mg, crude) as a yellow solid, which was used in the next step without further purification. LC-MS: (ESI) m / z: 569.8 [M+H] + .

[0844] To a solution of (S)-benzyl (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid (900 mg, 1.39 mmol) in DMF (10 mL) was added TEA (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and concentrated in vacuo to give (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-1-oic acid (1.50 g, crude) as a yellow solid, which was used in the next step without further purification. LC-MS: (ESI) m / z: 424.2 [M+H] + .

[0845] To a solution of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-1-oic acid (1.0 g, 2.36 mmol) in DMF (5 mL) was added 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (1.08 g, 3.54 mmol) and N,N-diisopropylethylamine (1.20 g, 9.45 mmol) at 0 ° C. After addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by C18 (0-70% acetonitrile in H2O) to give LK-9 (201 mg, 14% yield) as a white solid. LC-MS: (ESI) m / z: 617.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.60-8.50 (m, 1H), 8.32-8.27 (m, 1H), 8.15-7.97 (m, 3H), 7.31-7.11 (m, 5H), 7.00 (s, 2H), 4.61 (d, J = 6.4Hz, 1H), 4.5 4-4.45(m,1H),3.98(s,2H),3.81-3.53(m,6H),3.10-3.00(m,1H),2.84- 2.74(m,2H),2.11(t,J=7.4Hz,2H),1.55-1.40(m,5H),1.25-1.00(m,3H).

[0846] Synthesis of LK-10.

[0847] Mc-Gly-Gly-Phe-Gly (CAS# 2413428-36-9, also known as Mc-GGFG) was purchased from a commercial supplier and used without further purification.

[0848] Synthesis of LK-11.

[0849]

[0850] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-oic acid benzyl ester (3.00 g, 4.63 mmol) in DMF (20.0 mL) was added diethylamine (508 mg, 6.95 mmol), and the reaction mixture was stirred at room temperature for 2 hours. Then, the reaction mixture was filtered, and the filtrate was concentrated to give crude (S)-(2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate as a yellow oil (2.50 g, crude), which was used in the next step without further purification. LC-MS: (ESI) m / z: 426.9 [M+H] + .

[0851] To a solution of benzyl (S)-2-(2-(2-(2-aminoacetamido)acetamido)-3-phenylpropionamido)acetate (2.50 g, crude) and HATU (4.46 g, 11.7 mmol) in DMF (30.0 mL) were added (S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (2.96 g, 8.78 mmol) and N,N-diisopropylethylamine (3.03 g, 23.5 mmol) at 0° C. After the addition, the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by flash silica gel chromatography (0-10% methanol in dichloromethane) to give (5S,14S)-5-benzyl-14-(((benzyloxy)carbonyl)amino)-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptadecan-1,17-dioic acid 1-benzyl ester 17-tert-butyl ester as a yellow solid (1.20 g, 27%). LC-MS: (ESI) m / z: 745.7 [M+H] + .

[0852] To a solution of (7S,16S)-7-benzyl-16-(((benzyloxy)carbonyl)amino)-3,6,9,12,15-pentaoxo-1-phenyl-2-oxa-5,8,11,14-tetraazanonadecan-19-oic acid (1.20 g, crude) and HATU (1.32 g, 3.47 mmol) in DMF (20 mL) was added MPEG12 -NH2 (778 mg, 1.39 mmol) and N,N-diisopropylethylamine (898 mg, 6.96 mmol) were added. After addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by reverse phase flash column chromatography (0-65% acetonitrile in H2O) to give (42S,51S)-51-benzyl-42-(((benzyloxy)carbonyl)amino)-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentadecanoic-55-oic acid benzyl ester as a white solid (645 mg, 30%). LC-MS: (ESI) m / z: 616.3 [M+H] + .

[0853] To a solution of ((S)-42-(((benzyloxy)carbonyl)amino)-39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-azatricarboxan-43-yl)glycylglycyl-L-phenylalanylglycine benzyl ester (645 mg, 0.52 mmol) in methanol (10.0 mL) was added Pd / C (500 mg, 10% wt.) under N2. After replacing N2 with H2, the reaction mixture was stirred at room temperature for 12 hours. The reaction was filtered and the filtrate was concentrated to give (42S,51S)-42-amino-51-benzyl-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentapentadecan-55-oic acid as a crude yellow oil (320 mg, crude), which was used in the next step without further purification. LC-MS: (ESI) m / z: 1006.9 [M+H] + .

[0854] To a solution of (42S,51S)-42-amino-51-benzyl-39,43,46,49,52-pentaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38,44,47,50,53-pentaazapentadecane-55-oic acid (320 mg, 0.31 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (147 mg, 0.48 mmol) in DMF (4.00 mL) was added N,N-diisopropylethylamine (164 mg, 1.27 mmol) at 0°C. After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by preparative HPLC (0-70% acetonitrile in H2O) to give LK-11 as a yellow oil (100 mg, 26%). LC-MS: (ESI) m / z: 1201.6 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ8.36-8.38(m,1H),8.10-8.13(m,2H),8.01-8.03(m,2H),7.85-7 .87(m,1H),7.19-7.33(m,5H),7.01(s,2H),4.52-4.58(m,1H),4.19-4.23(m,1H),3.59-3 .79(m,44H),3.36-3.46(m,9H),3.25(s,3H),3.01-3.08(m,1H),2.75-2.81(m,1H),2.10- 2.12(m,5H),1.86-1.89(m,2H),1.70-1.75(m,1H),1.47-1.51(m,5H),1.19-1.22(m,2H).

[0855] Synthesis of LK-12.

[0856]

[0857] To a solution of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanic acid (2.10 g, 4.23 mmol) in DMF (20.00 mL) was added diethylamine (6.12 g, 8.46 mmol) at 0°C. After addition, the mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to give (S)-2-((S)-2-amino-3-methylbutyrylamino)-5-ureidopentanic acid (2.00 g, quantitative) as a white solid. LC-MS (ESI) m / z: 274.9 [M+H] + .

[0858] To a solution of (S)-2-((S)-2-amino-3-methylbutyrylamino)-5-ureidopentanic acid (2.00 g, 4.23 mmol, crude) in DMF (20 mL) was added 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid 2,5-dioxopyrrolidin-1-yl ester (1.12 g, 2.12 mmol) and DIPEA (657 mg, 5.09 mmol) at 0 ° C. After addition, the mixture was stirred at 25 ° C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to give the product, which was purified by C 18 Purification with 0-35% ACN in H2O, 0.1% TFA afforded LK-12 (242 mg, 35% yield) as a white solid. LC-MS (ESI) m / z: 689.7 [M+H] + . 1 H NMR(400MHz,CH3OD)δ(ppm)6.86(s,2H),4.37–4.06(m,3H),3.79–3.70(m,2H),3.75–3.58(m,20H),3.50–3.40(m,1H),3.23–3 .04(m,3H),2.66–2.53(m,2H),2.23–2.08(m,1H),1.95–1.81(m,1H),1.72–1.65(m,1H),1.62–1.46(m,2H),0.99–0.90(m,6H).

[0859] Synthesis of LK-13.

[0860]

[0861] To a solution of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid benzyl ester (10.0 g, 13.6 mmol) in DMF (20.0 mL) was added diethylamine (1.98 g, 27.2 mmol) at 0°C. After addition, the mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to give (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid benzyl ester (8.00 g, crude) as a yellow oil, which was used in the next step without further purification. LC-MS (ESI) m / z: 515.3 [M+H] + .

[0862] To a solution of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid benzyl ester (8.00 g, crude) in DMF (10 mL) was added (S)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (7.70 g, 22.8 mmol) at 0°C, followed by HATU (21.0 g, 57.4 mmol) and DIPEA (9.80 g, 75.9 mmol). After the addition, the mixture was stirred at 25°C under N2 for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated, and the residue was purified by C 18 Purification with 0-45% ACN in H2O, 0.1% TFA afforded 1-benzyl 22-(tert-butyl)-10-benzyl-19-(((benzyloxy)carbonyl)amino)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazadocosanedioate (4.0 g, 35% yield over two steps) as a white solid. LC-MS (ESI) m / z: 832.6 [M+H] + .

[0863] To a solution of 22-(tert-butyl) (10S,19S)-10-benzyl-19-(((benzyloxy)carbonyl)amino)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazadocosanedioate (1.0 g, 1.20 mmol) in dichloromethane (10 mL) was added TFA (2 mL) at 0°C. After the addition, the mixture was stirred at 25°C for 2 hours. LC-MS showed the reaction was complete. The mixture was concentrated to give (12S,21S)-12-benzyl-21-(((benzyloxy)carbonyl)amino)-3,8,11,14,17,20-hexaoxo-1-phenyl-2,5-dioxa-7,10,13,16,19-pentaazatetracosano-24-oic acid (800 mg, crude) as a yellow oil. LC-MS (ESI) m / z: 798.5 [M+Na] + .

[0864] To a solution of (12S,21S)-12-benzyl-21-(((benzyloxy)carbonyl)amino)-3,8,11,14,17,20-hexaoxo-1-phenyl-2,5-dioxa-7,10,13,16,19-pentaazatetracosano-24-oic acid (800 mg, crude) in DMF (2 mL) was added MPEG 12 -NH2 (672 mg, 1.20 mmol), HATU (912 mg, 2.40 mmol), HOBt (162 mg, 1.20 mmol) and DIPEA (610 mg, 4.80 mmol). The mixture was stirred at 25 ° C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated and the residue was purified by C 18 Purification with 0-35% ACN in H2O, 0.1% TFA afforded (42S,51S)-51-benzyl-42-(((benzyloxy)carbonyl)amino)-39,43,46,49,52,55-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,58-trideca-38,44,47,50,53,56-hexaazahexacontan-60-oic acid benzyl ester (220 mg, 14% yield over two steps) as a white solid. LC-MS (ESI) m / z: 1339.4 [M+Na] + .

[0865] To a solution of (42S,51S)-51-benzyl-42-(((benzyloxy)carbonyl)amino)-39,43,46,49,52,55-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,58-trideca-38,44,47,50,53,56-hexaazahexacontan-60-oic acid benzyl ester (220 mg, 0.17 mmol) in DMF (2 mL) was added Pd / C (20 mg, 10% wt.%, 55% HO) at 25° C. The mixture was stirred at 25° C. under a nitrogen atmosphere for 12 hours. LC-MS showed the reaction was complete. The mixture was filtered and concentrated to give (42S,51S)-42-amino-51-benzyl-39,43,46,49,52,55-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,58-trideca-38,44,47,50,53,56-hexaazahexadecanoic-60-oic acid (200 mg, crude) as a yellow oil. LC-MS (ESI) m / z: 1094.0 [M+Na] + .

[0866] To a solution of 1-benzyl (42S, 51S) -42-amino-51-benzyl -39,43,46,49,52,55- hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,58- trideca-38,44,47,50,53,56- hexaazahexacontan-60-oic acid (200 mg, crude) in DMF (2 mL) was added 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (102 mg, 0.330 mmol) followed by DIPEA (42.4 mg, 0.330 mmol) at 0 ° C. The mixture was stirred at 25 ° C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated and the residue was purified by C 18 Purification (0-40% ACN in H2O, 0.1% TFA) afforded LK-13 (55 mg, 26% yield, two steps) as a white solid. LC-MS (ESI) m / z: 1287.3 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ(ppm)8.81–8.17(m,5H),7.89–7.86(m,1H),7.24–7.15(m,5H),7.00(s,2H),4.71–4.6 1(m,2H),4.41–4.35(m,1H),4.21–4.15(m,1H),3.90–3.60(m,7H),3.50–3.45(m,37H),3.42–3.37(m,5H),3.2 4(s,3H),3.17–3.15(m,2H),3.07-3.00(m,1H),2.90–2.80(m,2H),2.65-2.60(m,1H),2.15–2.09(m,4H),2.00 –1.90(m,1H),1.93–1.83(m,1H),1.80–1.69(m,1H),1.65–1.56(m,1H),1.49–1.40(m,5H),1.30–1.12(m,3H).

[0867] Synthesis of LK-14.

[0868]

[0869] To a solution of 1-benzyl (42S,51S)-42-amino-51-benzyl-39,43,46,49,52,55-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35,58-trideca-38,44,47,50,53,56-hexaazahexacontan-60-oic acid (180 mg, 0.160 mmol) in DMF (2 mL) at 0°C was added 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oic acid 2,5-dioxopyrrolidin-1-yl ester (87 mg, 0.160 mmol) followed by DIPEA (41.0 mg, 0.32 mmol). The mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated to give a crude product, which was purified by preparative HPLC (C 18 ; 20 to 70% ACN in H2O, 0.1% TFA) to give LK-14 (50 mg, 20% yield) as a yellow solid. LC-MS (ESI) m / z: 1531.3 [M+Na] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)8.55–8.52(m,1H),8.36–8.29(m,1H),8.17–8.07(m,3H),8.02–7.99(m,1H),7. 84–7.81(m,1H),7.22–7.15(m,5H),7.02(s,2H),4.61(d,J=6.5Hz,2H),4.51–4.45(m,1H),4.22–4.12(m,1H), 3.98(s,2H),3.80–3.64(m,7H),3.63–3.42(m,69H),3.24(s,3H),3.18–3.10(m,2H),3.05–2.90(m,1H),2.80 –2.70(m,2H),2.43–2.30(m,3H),2.10–2.08(m,2H),2.03–1.94(m,1H),1.87–1.80(m,1H),1.73–1.65(m,1H).

[0870] Synthesis of LK-15.

[0871]

[0872] To a solution of Fmoc-Gly-Gly-Gly-Gly (1.0 g, 2.13 mmol, 1.0 eq.), HOBt (862 mg, 6.39 mmol, 3.0 eq.) and EDCI (1.22 g, 6.39 mmol, 3.0 eq.) in 10 mL of anhydrous DMF was added Val-Cit-PAB-OH (887 mg, 2.34 mmol, 1.1 eq.). After addition, the solution was stirred for 1 hour until the reaction was confirmed to be complete by LC-MS. The reaction mixture was separated by C 18 Purification by chromatography (ACN / H2O, from 10% to 100%) and lyophilization afforded Fmoc-Gly-Gly-Gly-Gly-Val-Cit-PAB-OH (1.4 g, 80% yield) as a white powder. LC-MS (ESI) m / z: 830 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.91 (s, 1H), 8.20 (d, J = 8.9Hz, 2H), 8.15 (s, 2H), 7.92 (d, J = 7.5Hz, 2H), 7.86 (d, J = 8.7Hz, 1H), 7.75 (d ,J=7.5Hz,2H),7.60(s,1H),7.57(d,J=8.4Hz,2H),7.45(t,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.26(d,J=8.3Hz,2H),6.01(s,1H) ,5.45(s,2H),5.13(s,1H),4.45(d,J=5.6Hz,2H),4.40(d,J=6.4Hz,1H),4.32(d,J=6.9Hz,2H),4.26(d,J=6.5Hz,2H),3.79(dt,J=1 3.1, 6.6Hz, 7H), 3.70 (d, J = 6.0Hz, 2H), 2.01 (d, J = 6.4Hz, 1H), 1.68 (d, J = 35.7Hz, 2H), 0.89 (d, J = 6.7Hz, 3H), 0.85 (d, J = 6.8Hz, 3H).

[0873] Fmoc-Gly-Gly-Gly-Gly-Val-Cit-PAB-OH (200 mg, 0.24 mmol, 1.0 eq.) was dissolved in 5 mL of Et2N / DMSO (1:1) and stirred for 1 hour. After the reaction was complete, the solution was concentrated and purified by C 18 Purification by column chromatography (ACN / H2O, from 10% to 100%) and lyophilization gave Gly-Gly-Gly-Gly-Val-Cit-PAB-OH (139 mg, 95% yield) as a white powder. LC-MS (ESI) m / z: 608 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.06 (s, 1H), 8.61 (s, 1H), 8.41 (t, J = 6.0Hz, 1H), 8.30–8.18 (m, 2H), 7.88 (d, J = 8.5Hz, 1 H),7.60(d,J=8.2Hz,2H),7.25(d,J=8.2Hz,2H),6.23(s,1H),5.52(s,2H),4.45(s,2H),4.40(d,J=6.8Hz,1H),4.24(t ,J=7.7Hz,1H),3.86–3.72(m,6H),3.19(s,1H),2.99(d,J=7.0Hz,3H),2.57(s,10H),2.04(dd,J=13.3,6.4Hz,1H),1.7 4(s,1H),1.64(d,J=10.2Hz,1H),1.48(s,1H),1.40(s,1H),1.22(dd,J=16.9,9.7Hz,2H),0.87(dd,J=14.7,6.7Hz,6H).

[0874] To a solution of Gly-Gly-Gly-Gly-Val-Cit-PAB-OH (180 mg, 0.296 mmol, 1.0 eq.) in 15 ml of DMSO was added N-succinimidyl maleimidoacetate (224 mg, 0.889 mmol, 3.0 eq.), and the solution was stirred at room temperature for 1 hour. After the reaction was complete, the solution was concentrated and filtered through C 18 Purification by column chromatography (ACN / 0.1% TFA aqueous solution, from 10% to 100%) and lyophilization gave LK-15 (160 mg, 73% yield) as a white powder. LC-MS (ESI) m / z: 745 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.92 (s, 1H), 8.48 (s, 1H), 8.22 (s, 3H), 7.84 (d, J = 8 .7Hz,1H),7.55(d,J=7.9Hz,2H),7.23(d,J=8.2Hz,2H),7.09(s,1H),7.02(s,2H), 5.44(s,1H),5.11(t,J=5.7Hz,1H),4.42(d,J=5.5Hz,2H),4.15–4.10(m,3H),3.82 –3.69(m,9H),3.16(d,J=5.1Hz,5H),0.86(d,J=6.7Hz,3H),0.82(d,J=6.9Hz,3H).

[0875] Synthesis of LK-16.

[0876]

[0877] To a solution of 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosane-21-oate (626 mg, 1.18 mmol) in DMF (4.00 mL) at 0° C. was added (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (500 mg, crude) and N,N-diisopropylethylamine (609 mg, 4.72 mmol). After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated and the residue was purified by preparative HPLC (0-60% acetonitrile in water) to give LK-16 (90 mg, 9% yield) as a yellow oil. LC-MS (ESI) m / z: 861.4 [M+Na] + . 1 H NMR (400MHz, DMSO-d6): δ (ppm) 8.51-8.57 (m, 1H), 8.26-8.29 (m, 1H), 8.12-8.15 (m, 1H), 8.08-8.10 (m, 1H), 7.96-7.99 (m, 1H), 7.15-7.27 (m, 6H), 7.01(s,2H),4.61(d,J=6.8Hz,2H),4.47-4.52(m,1H),3.98(s,2H),3.49 -3.73(m,32H),3.03-3.08(m,1H),2.77-2.83(m,1H),2.37-2.41(m,2H).

[0878] Linker-Payload Building Block (LP)

[0879] Synthesis of LP-1.

[0880]

[0881] To a solution of PL-1 (16 mg, 0.0210 mmol, 1.0 eq.) and Mc-Val-Cit-PABC-PNP (46.5 mg, 0.0631 mmol, 3.0 eq.) in 0.25 mL of pyridine and 1 mL of anhydrous DMF was added HOAt (2.86 mg, 0.0210 mmol, 1.0 eq.) and DIPEA (43.4 mg, 0.3364 mmol, 16.0 eq.) under argon. The mixture was stirred at 40 ° C overnight. After completion of the reaction, the mixture was purified by preparative HPLC to give LP-1 as a white solid (1.5 mg, 5.2% yield). LC-MS (ESI) m / z: 1340 [M-OH] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.99 (s, 1H), 8.55 (s, 1H), 8.07 (d, J = 7.5Hz, 1H), 7 .80(d,J=8.4Hz,1H),7.57(d,J=8.2Hz,2H),7.44(d,J=8.3Hz,1H),7.29(d,J=8. 5Hz,2H),6.98(d,J=5.6Hz,2H),6.63(s,1H),6.31(s,1H),6.13(d,J=10.7Hz,1H ),6.02(d,J=29.5Hz,1H),5.41(s,1H),5.06(s,1H),5.00(s,1H),4.90(s,1H),4. 65(s,1H),4.50(d,J=13.6Hz,1H),4.35(s,1H),4.23–4.04(m,2H),3.84(d,J=46 .7Hz,2H),3.64(d,J=4.3Hz,4H),3.48(s,6H),2.92(s,9H),2.30–2.23(m,5H),2 .22–2.05(m,4H),1.95(d,J=5.5Hz,5H),1.66(s,1H),1.51–1.39(m,6H),1.22(s ,3H),1.15(q,J=7.1Hz,3H),1.04(t,J=7.0Hz,2H),0.82(dt,J=12.4,6.0Hz,8H).

[0882] Synthesis of LP-2.

[0883]

[0884] To a solution of PL-5 (80 mg, 0.10 mmol, 1.0 eq.) and Mc-Val-Cit-PABC-PNP (227 mg, 0.308 mmol, 3.0 eq.) in 0.25 mL of pyridine and 1 mL of anhydrous DMF were added HOAt (14 mg, 0.103 mmol, 1.0 eq.) and DIPEA (212 mg, 1.64 mmol, 16.0 eq.). After addition, the mixture was stirred at 40 ° C overnight. The reaction mixture was purified by preparative HPLC to give LP-2 (55 mg, 38.8% yield) as a white solid. LC-MS (ESI) m / z: 689 [M / 2 + H] + . 1 H NMR(400MHz,DMSO-d6)δ(ppm)10.76(s,1H),10.00(s,1H),9.54(s,1H),9.19(s,1H) ,8.08(d,J=7.4Hz,1H),7.81(d,J=8.6Hz,1H),7.61(d,J=8.7Hz,3H),7.34(d,J=8.2 Hz,2H),7.27(d,J=9.0Hz,1H),7.12(d,J=8.5Hz,1H),6.99(s,2H),6.59(s,1H),6.2 6(d,J=25.7Hz,2H),6.03(s,1H),5.28(d,J=11.4Hz,1H),5.05(s,2H),4.58(s,2H), 4.33(dd,J=26.5,6.7Hz,4H),4.18(d,J=8.9Hz,3H),3.69(s,7H),3.36(t,J=7.1Hz, 5H),3.32(d,J=4.3Hz,2H),3.17(s,4H),2.88(tdd,J=40.9,23.0,9.1Hz,12H),2.32 (d,J=15.2Hz,7H),2.22–2.09(m,7H),2.07(s,8H),1.99(s,5H),1.75–1.55(m,4H), 1.47(p,J=7.4Hz,6H), 1.19(dd,J=19.5,11.6Hz,5H), 0.83(dd,J=12.4,6.7Hz,7H).

[0885] Synthesis of LP-3.

[0886]

[0887] To a solution of PL-1 (20 mg, 0.064 mmol, 1.0 eq.) and LK-1 (76 mg, 0.0792 mmol, 3.0 eq.) in 1 mL of anhydrous DMF and 1 mL of pyridine were added HOAt (3.57 mg, 0.52 mmol, 1.0 eq.) and DIPEA (13.57 mg, 0.42 mmol, 16 eq.). After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-3 as a white solid (7.2 mg, 17.3% yield). LC-MS (ESI) m / z: 782 [(M-H2O) / 2+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 10.00 (s, 1H), 9.54 (s, 1H), 8.55 (s, 1H), 8.12 (s, 1H), 7.88 (d, J=8.4Hz, 1H), 7.59 (d, J= 8.1Hz,2H),7.31(d,J=9.0Hz,3H),7.01(s,2H),6.64(s,1H),6.34(s,1H),6.14(d,J=11.2Hz,2H),6.03(s,1H),5.05( d,J=17.7Hz,3H),4.91(s,1H),4.67(s,1H),4.21(s,2H),4.08(s,1H),3.80(s,2H),3.66(s,7H),3.55(s,8H),3.48(s ,20H),3.14–2.92(m,7H),2.28(d,J=9.6Hz,6H),2.21(s,1H),1.97(s,4H),1.23(s,2H),0.84(dd,J=12.5,6.7Hz,8H).

[0888] Synthesis of LP-4.

[0889]

[0890] To a solution of PL-1 (20 mg, 0.026 mmol, 1.0 eq.) and LK-7 (18 mg, 0.0197 mmol, 0.75 eq.) in 1 mL of pyridine and 4 mL of anhydrous DMF were added HOAt (3.5 mg, 0.026 mmol, 1.0 eq.) and DIPEA (13.57 mg, 0.42 mmol, 16 eq.). After the addition, the mixture was stirred at 40 °C overnight. The reaction mixture was purified by preparative HPLC to give LP-4 as a white solid (2.2 mg, 7.2% yield). LC-MS (ESI) m / z: 760 [(M-H2O) / 2+H] + .1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.99 (s, 1H), 9.60 (s, 1H), 8.63 (s, 1H), 8.11 (d, J = 7.3Hz, 1H), 7.88 (d, J = 8.6Hz, 1H), 7.59 (d, J = 8.2Hz, 2H), 7. 38(s,1H),7.31(d,J=8.2Hz,2H),7.00(s,2H),6.66(s,1H),6.29(s,1H),6.16(d,J=15.5Hz,2H),6.01(s,1H),5.08(d,J=11.8Hz,1H),5.03( s,2H),4.93(s,1H),4.68(s,2H),4.36(s,1H),4.24–4.11(m,2H),3.9 2(s,1H),3.80(s,1H),3.66(s,5H),3.17(s,2H),3.12(s,3H),2.97(d, J=22.7Hz,2H),2.67(s,1H),2.47(s,3H),2.28(d,J=8.6Hz,6H),1.97(s,5H),1.72–1.39(m,1H),1.23(s,2H),0.84(dd,J=12.6,6.7Hz,8H).

[0891] Synthesis of LP-5.

[0892]

[0893] To a solution of PL-5 (20 mg, 0.026 mmol, 1.0 eq.) and LK-1 (74.88 mg, 0.078 mmol, 3.0 eq.) in 1 mL of pyridine and 3 mL of anhydrous DMF was added HOAt (3.54 mg, 0.026 mmol, 1.0 eq.) and DIPEA (53.66 mg, 0.416 mmol, 16.0 eq.) under argon. The mixture was stirred at 40 ° C overnight. After completion of the reaction, the mixture was purified by preparative HPLC to give LP-5 as a white solid (16 mg, 38.9% yield). LC-MS (ESI) m / z: 800 [M / 2 + H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.02 (d, J = 14.3Hz, 2H), 9.39 (s, 1H), 8.77 (s, 1H), 8.12 (d, J = 7.2Hz, 1H), 7.88 (d, J = 8.3Hz, 1H), 7.6 1(d,J=8.1Hz,2H),7.49(s,1H),7.34(d,J=8.1Hz,2H),7.21(d,J=8.8Hz,1H),7.02(s,2H),6.48(s,1H),6.22(d,J=13.0Hz,2H),5.9 8(s,1H),5.41(s,2H),5.06(d,J=17.0Hz,3H),4.46(s,1H),4.21(d,J=8.8Hz,2H),4.07(d,J=10.8Hz,2H),3.65(s,3H),3.55(d,J= 5.2Hz,2H),3.50(s,3H),3.47(s,4H),3.46(s,5H),2.27(d,J=12.7Hz,6H),2.05(s,3H),1.98(s,3H),0.84(dd,J=13.2,6.7Hz,8H).

[0894] Synthesis of LP-6.

[0895]

[0896] Under argon, PL-5 (20 mg, 0.0263 mmol, 1.0 eq.) was added to a solution of LK-10 (13.6 mg, 0.0263 mmol, 1.0 eq.), EDCI (99.68 mg, 0.528 mmol, 20.0 eq.), and HOBt (70.26 mg, 0.528 mmol, 20.0 eq.) in 3 mL of anhydrous DMF. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was purified by preparative HPLC to give LP-6 as a white solid (14 mg, 42.2% yield). LC-MS (ESI) m / z: 645 [M / 2+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.11 (s, 1H), 9.59 (s, 1H), 8.38 (s, 1H), 8.19 (d, J = 7.9Hz, 1H), 8.08 (d, J = 6.1Hz, 2H), 7.68(s,1H),7.25(d,J=4.7Hz,6H),7.20–7.11(m,2H),6.98(s,2H),6.48(s,1H),6.22(d,J=14.0Hz,2H),5.07(d,J= 11.1Hz,1H),4.46(s,2H),4.20(s,1H),4.08(d,J=12.5Hz,2H),3.84(s,2H),3.65(s,6H),3.19(s,2H),5.48–1.57(m ,462H),2.27(d,J=13.5Hz,7H),2.09(t,J=7.5Hz,3H),2.05(s,5H),1.98(s,4H),1.46(q,J=7.6Hz,5H),1.23(s,8H).

[0897] Synthesis of LP-7.

[0898]

[0899] To a solution of PL-5 (20 mg, 0.026 mmol, 1.0 eq.) and LK-9 (16.2 mg, 0.026 mmol, 1.0 eq.) in 5 mL of anhydrous DMF were added HOBt (71.36 mg, 0.52 mmol, 20 eq.) and EDCI (101.2 mg, 0.52 mmol, 20 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-7 as a white solid (8.8 mg, 24.7% yield). LC-MS (ESI) m / z: 1377.8 [M+H] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)9.57(s,1H),8.70(s,1H),8.33(s,1H),8.13(d,J=8.2Hz,1H),8.07(s,1H),8.01(s,1H),7.83(s,1H),7.28(dd,J =17.7,8.3Hz,3H),7.21(d,J=13.8Hz,6H),6.98(s,2H),6.57(s,1H),6 .29(s,1H),6.22(s,1H),4.66(d,J=6.3Hz,2H),4.56(s,2H),4.48(s,1H ),4.29(s,2H),4.18(s,1H),4.02(s,2H),3.74(d,J=8.3Hz,4H),3.67(d,J=11.2Hz,6H),3.34(d,J=7.3Hz,7H),3.31(s,3H),2.83(d,J=12.3Hz ,4H),2.67(s,1H),2.33(s,3H),2.30(s,3H),2.09(d,J=5.1Hz,5H),1.9 9(s,4H),1.46(d,J=8.2Hz,5H),1.24(s,6H),1.18(s,3H),0.86(s,1H).

[0900] Synthesis of LP-8.

[0901]

[0902] To a solution of PL-5 (20 mg, 0.026 mmol, 1.0 eq.) and LK-2 (91.67 mg, 0.077 mmol, 3.0 eq.) in 0.4 mL of pyridine and 1.6 mL of anhydrous DMF were added HOAt (3.63 mg, 0.0267 mmol, 1.0 eq.) and N,N-diisopropylethylamine (55.1 mg, 0.427 mmol, 16.0 eq.). After the addition, the mixture was stirred at room temperature overnight. LC-MS showed that the reaction was complete. The reaction mixture was purified by preparative HPLC to give LP-8 as a white solid (7.9 mg, 16.8% yield). LC-MS (ESI) m / z: 915 [M / 2+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.04 (d, J = 8.1Hz, 2H), 9.40 (s, 1H), 8.80 (s, 1H), 8.39 ( s,1H),8.14(d,J=8.1Hz,2H),7.93(d,J=5.7Hz,1H),7.81(d,J=8.5Hz,1H),7.60(d,J= 8.3Hz,2H),7.48(s,1H),7.33(d,J=8.3Hz,2H),7.21(d,J=8.7Hz,1H),7.02(s,1H),6. 98(s,2H),6.48(s,1H),6.22(d,J=12.0Hz,2H),6.02(s,1H),5.43(s,2H),5.06(d,J=16 .8Hz,3H),4.42(d,J=33.4Hz,3H),4.22–4.03(m,5H),3.65(s,3H),3.60(t,J=7.4Hz,3 H),3.49(d,J=1.5Hz,32H),3.21(d,J=15.0Hz,6H),3.05–2.73(m,1H),2.70–2.56(m,1H ),2.40(t,J=7.2Hz,2H),2.27(d,J=12.2Hz,5H),2.15(t,J=7.9Hz,1H),2.05(s,3H),1 .97(s,3H),1.83(d,J=8.7Hz,1H),1.69(d,J=9.0Hz,2H),0.83(dd,J=13.9,6.7Hz,8H).

[0903] Synthesis of LP-9.

[0904]

[0905] To a solution of PL-6 (20 mg, 0.026 mmol, 1.0 eq.) and Mc-Val-Cit-PAB-PNP (38.3 mg, 0.052 mmol, 2.0 eq.) in 1 mL of DMSO was added DIPEA (13.4 g, 0.104 mmol, 4.0 eq.). After addition, the mixture was stirred at 40°C overnight. LC-MS showed the reaction was complete. The reaction was purified by preparative HPLC to give LP-9 (4.5 mg, 12.8% yield) as a white solid. LC-MS (ESI) m / z: 700 [M / 2+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.10 (d, J = 6.3Hz, 1H), 8.41 (s, 1H), 8.14 (d, J = 7.1Hz ,1H),7.85(dd,J=8.6,4.3Hz,1H),7.63(d,J=8.1Hz,2H),7.34(d,J=8.2Hz,2H),7.0 0(d,J=1.8Hz,2H),6.85(s,1H),6.48(d,J=23.6Hz,2H),6.19(d,J=14.5Hz,2H),6.0 7(s,1H),5.45(s,2H),5.15(s,2H),5.06(d,J=12.2Hz,1H),4.51(s,2H),4.38(s,1H) ,4.24–4.16(m,2H),4.09(d,J=24.5Hz,2H),3.70(d,J=6.4Hz,1H),3.64(s,3H),3.6 1(d,J=6.1Hz,4H),3.28(d,J=5.0Hz,1H),3.00(q,J=9.2,6.4Hz,2H),2.79(d,J=18. 1Hz,1H),2.30(s,2H),2.22(s,2H),2.15(td,J=14.6,7.1Hz,2H),2.06(s,2H),1.99 (s,3H),1.49(q,J=8.0,7.6Hz,4H),1.29–1.14(m,4H),0.85(dd,J=12.8,6.7Hz,7H).

[0906] Synthesis of LP-10.

[0907]

[0908] To a solution of PL-4 (20 mg, 0.026 mmol, 1.0 eq.) and LK-9 (16.2 mg, 0.026 mmol, 1.0 eq.) in 5 mL of anhydrous DMF were added HOBt (71.36 mg, 0.52 mmol, 20.0 eq.) and EDCI (101.2 mg, 0.52 mmol, 20.0 eq.). After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-10 as a white solid (4.6 mg, 12.9% yield). LC-MS (ESI) m / z: 1368.8 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.86 (s, 1H), 8.64 (s, 1H), 8.30 (s, 1H), 8.12 (d, J = 8.4Hz, 1H), 8.06 (s, 1H), 8.00 (s, 1H), 7 .88(s,1H),7.23(d,J=8.5Hz,4H),6.98(s,2H),6.51(s,1H),6.32(s,1H),6.19(d,J=7.9Hz,2H),4.67(s,2H),4.56(s,1 H),4.24(s,2H),4.04(s,2H),3.71(s,2H),3.66(d,J=6.2Hz,6H),3.59(s,4H),3.35(s,6H),2.31(s,3H),2.24(s,3H),2 .10(t,J=7.5Hz,2H),2.02(s,3H),1.97(s,3H),1.46(d,J=7.7Hz,5H),1.24(s,6H),1.19(d,J=8.4Hz,3H),0.85(s,1H).

[0909] Synthesis of LP-11.

[0910]

[0911] To a solution of PL-5 (10 mg, 0.013 mmol, 1.0 eq.) and LK-10 (32.6 mg, 0.013 mmol, 1.0 eq.) in 5 mL of anhydrous DMF were added HOBt (35.7 mg, 0.26 mmol, 20.0 eq.) and EDCI (101.2 mg, 0.52 mmol, 20.0 eq.). After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-11 as a white solid (2.5 mg, 12.9% yield). LC-MS (ESI) m / z: 981.3 [M / 2+H] + . 1H NMR(400MHz,DMSO-d6)δ(ppm)10.09(s,1H),9.55(s,1H),8.34(s,4H),8.13(s,2H),7.84(s,1H),7.67(s, 1H),7.24(d,J=4.6Hz,6H),7.16(d,J=14.0Hz,4H),6.98(s,2H),6.48(s,1H),6.22(d,J=14.2Hz,3H),4.4 5(s,2H),4.19(s,3H),4.06(s,2H),3.69(s,2H),3.65(s,3H),3.49(d,J=3.0Hz,74H),2.67(d,J=2.8Hz,4 H), 2.33 (d, J = 2.7Hz, 3H), 2.28 (s, 2H), 2.25 (s, 3H), 2.05 (s, 4H), 1.98 (s, 4H), 1.45 (s, 7H), 0.86 (s, 2H).

[0912] Synthesis of LP-12.

[0913]

[0914] To a solution of PL-6 (25 mg, 0.031 mmol, 1.0 eq.) and LK-8 (50 mg, 0.062 mmol, 2.0 eq.) in 1 mL of DMSO was added DIPEA (16.14 g, 0.125 mmol, 4.0 eq.). After addition, the mixture was stirred at 40°C overnight. LC-MS showed the reaction was complete. The reaction was purified by preparative HPLC to give LP-12 (10 mg, 21.9% yield) as a white solid. LC-MS (ESI) m / z: 731 [M+H] + / 2. 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.95 (s, 1H), 8.38 (d, J = 21.4Hz, 2H), 8.19 (d, J = 7.9 Hz,1H),8.13–8.06(m,2H),7.64(d,J=8.4Hz,2H),7.37(d,J=8.5Hz,2H),7.28(d,J =4.4Hz,5H),7.23–7.18(m,1H),6.97(s,2H),6.85(s,1H),6.48(d,J=21.3Hz,2H) ,6.18(d,J=16.5Hz,2H),5.16(s,2H),5.06(d,J=11.1Hz,1H),4.93(s,1H),4.50(s ,3H),4.21(s,1H),4.10(d,J=23.1Hz,2H),3.90(dd,J=9.4,5.9Hz,2H),3.80(d,J =5.8Hz,3H),3.76(d,J=6.2Hz,3H),3.37(t,J=7.0Hz,4H),3.28(s,1H),3.14–3.06 (m,2H),2.89–2.72(m,3H),2.71–2.67(m,1H),2.37–2.34(m,1H),2.30(s,3H),2. 23(s,3H),2.12(t,J=7.5Hz,3H),2.06(s,3H),1.99(s,4H),1.48(q,J=8.5Hz,6H).

[0915] Synthesis of LP-13.

[0916]

[0917] To a solution of PL-12 (20 mg, 0.027 mmol, 1.0 eq.) and LK-16 (22.8 mg, 0.027 mmol, 1.0 eq.) in 1 mL of anhydrous DMF were added HOBt (73 mg, 0.54 mmol, 20.0 eq.) and EDCI (103.6 mg, 0.54 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-13 (11.5 mg, 27.3% yield) as a white solid. LC-MS (ESI) m / z: 780.9 [1 / 2 M+H] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)9.61(s,1H),8.75(s,1H),8.69(t,J=6.5Hz,1H),8.32(dd,J=11.5,5.5Hz,1H),8.19–8.09(m,1H),8.00(t,J=5.8Hz,1H), 7.34–7.14(m,7H),7.01(s,2H),6.84(d,J=8.7Hz,1H),6.45(s,1H),6.24(s ,1H),6.14(s,1H),5.05(d,J=11.7Hz,1H),4.61(d,J=6.7Hz,2H),4.45(s,3H ),4.19(d,J=4.5Hz,1H),4.06(s,1H),4.00(d,J=13.5Hz,3H),3.76–3.66(m ,5H),3.63(s,3H),3.62–3.50(m,10H),3.48(d,J=2.2Hz,10H),3.46(s,15H) ,3.24–3.00(m,2H),2.78(d,J=8.6Hz,2H),2.68(d,J=35.2Hz,1H),2.46–2. 34(m,3H),2.27(s,3H),2.22(s,3H),2.04(s,3H),2.01(s,2H),1.98(s,3H).

[0918] Synthesis of LP-14.

[0919]

[0920] To a solution of PL-16 (5 mg, 0.0063 mmol, 1.0 eq) and LK-8 (10 mg, 0.0126 mmol, 1.0 eq.) in 1 mL of anhydrous pyridine:DMF (1:4) were added HOAt (0.85 mg, 0.252 mmol, 1.0 eq.) and DIEA (13.15 mg, 0.1 mmol, 16.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-14 (3.8 mg, 41.0% yield) as a white solid. LC-MS (ESI) m / z: 729.0 [1 / 2 M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.85 (d, J = 7.6Hz, 2H), 8.76 (s, 1H), 8.38 (s, 1H), 8.17(t,J=8.8Hz,1H),8.06(dd,J=12.6,6.2Hz,2H),7.59(d,J=8.1Hz,2H),7.45 (t,J=6.1Hz,1H),7.33–7.14(m,10H),6.97(s,2H),6.84(d,J=8.7Hz,1H),6.45( s,1H),6.25(s,1H),6.16(s,1H),5.05(d,J=11.4Hz,1H),4.95(s,2H),4.47(d,J =21.7Hz,3H),4.19(d,J=4.6Hz,1H),4.09–3.97(m,2H),3.88(dd,J=10.9,5.5H z,2H),3.80–3.65(m,6H),3.63(s,4H),3.22–3.15(m,2H),3.15–3.04(m,2H),2. 89–2.81(m,1H),2.79(s,2H),2.67(s,1H),2.27(s,3H),2.22(s,3H),2.10(t,J= 7.4Hz, 2H), 2.03 (s, 3H), 1.98 (s, 3H), 1.45 (p, J = 7.7Hz, 5H), 1.22–1.13 (m, 2H).

[0921] Synthesis of LP-15.

[0922]

[0923] To a solution of PL-16 (10 mg, 0.0126 mmol, 1.0 eq.) and LK-10 (6.7 mg, 0.0126 mmol, 1.0 eq.) in 1 mL of anhydrous DMF were added HOBt (34 mg, 0.252 mmol, 20.0 eq.) and EDCI (48 mg, 0.252 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-15 (4.96 mg, 30.0% yield) as a white solid. LC-MS (ESI) m / z: 654.5 [1 / 2 M+H] + . 1H NMR (400MHz, DMSO-d6) δ9.73(s,1H),8.76(s,1H),8.35(d,J=14.4Hz,1H),8.17–7.95(m,4H),7.29–7.12(m,8H),6.97(s,2H),6.84(d,J=8.7 Hz,1H),6.45(s,1H),6.24(s,1H),6.15(s,1H),5.05(d,J=11.1Hz,1H),4.45(s,3H),4.19(s,1H),4.10–3.95(m,2H),3.83(d,J=5.7Hz,2H),3 .73(s,2H),3.69(s,1H),3.65(d,J=5.8Hz,2H),3.63(s,3H),3.60(d,J=5.6Hz,1H),3.55(d,J=4.9Hz,1H),3.21(d,J=4.3Hz,1H),3.16(s,1H) ,3.08–3.01(m,1H),2.83–2.75(m,3H),2.66(s,1H),2.42(s,2H),2.27 (s,3H),2.22(s,3H),2.09(t,J=7.5Hz,2H),2.03(s,4H),1.98(s,3H).

[0924] Synthesis of LP-16.

[0925]

[0926] To a solution of PL-4 (20 mg, 0.026 mmol, 1.0 eq.), LK-16 (43.62 mg, 0.052 mmol, 2.0 eq.), DMAP (1.6 mg, 0.013 mmol, 0.5 eq.), and HOAt (70.78 mg, 0.52 mmol, 20.0 eq.) in 1 mL of anhydrous DMF was added EDCI (53.9 mg, 0.52 mmol, 20.0 eq.) in portions. After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-16 (15 mg, 34.5% yield) as a white solid. LC-MS (ESI) m / z: 795.6 [1 / 2 M+H] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)8.73(d,J=5.5Hz,2H),8.65(t,J=6.7Hz,1H),8.32(t,J=5.9Hz,1H),8.20–8.09(m,2H),8.00(t,J=5.8Hz,1H),7.74(s, 1H),7.29–7.14(m,6H),7.01(s,2H),6.43(d,J=7.0Hz,2H),6.13(s,2H),5 .04(d,J=11.5Hz,1H),4.66(d,J=7.0Hz,2H),4.48(d,J=3.1Hz,3H),4.19(d ,J=4.6Hz,1H),4.11(s,1H),4.09–3.99(m,3H),3.79–3.66(m,5H),3.63(s ,3H),3.62–3.53(m,9H),3.52–3.49(m,2H),3.49–3.43(m,23H),3.27(d,J= 5.6Hz,2H),3.07–2.92(m,3H),2.85–2.65(m,4H),2.38(t,J=6.5Hz,3H),2 .29(s,3H),2.21(s,3H),2.15(d,J=14.4Hz,1H),2.04(s,4H),1.97(s,3H).

[0927] Synthesis of LP-17.

[0928]

[0929] A mixture of PL-12 (18 mg, 0.024 mmol, 1.0 eq.), Fmoc-Val-Cit-COOH (15 mg, 0.036 mmol, 1.5 eq.), and HATU (16 mg, 0.04 mmol, 1.7 eq.) in DMF was stirred at room temperature for 10 minutes, and then DIPEA (13 μL, 0.072 mmol, 3.0 eq.) was added to the mixture and stirred for 4 hours. LC-MS showed the desired product, and the mixture was immediately used in the next step. LC-MS (ESI) m / z: 1132.4 [M+1] + .

[0930] Fmoc removal was promoted by adding TEA (150 μL), and the mixture was stirred at room temperature for 3 hours. LC-MS showed the desired product, which was purified by preparative HPLC to obtain the desired product (4.3 mg, 24.6% yield) as a white solid. LC-MS (ESI) m / z: 910.3 [M+1] + . 1H NMR(400MHz, DMSO-d6)d(ppm)10.33(s,1H),9.13(s,1H),8.85-8.73(m,1H),8.12(s,3H),7.48(d,J=16Hz,1H),7.34-7.32(d,J=8Hz 1H),6.90(s,1H),6.56(s,1H),6.27-6.20(m,2H),5.23(d,J=8Hz,1H),4.58(s,2H),4.56-4.43(m,1H),4.28(s,2H),4.21( s,1H),3.69(s,3H),2.72-2.68(m,2H),2.30(s,6H),2.07-1.99(m,7H),1.32-1.24(m,3H),0.94-0.93(m,6H),0.83(s,2H).

[0931] A solution of the product from the previous step (4.3 mg, 0.0047 mmol, 1.0 eq.), Mal-PEG6-NHS (6 mg, 0.0095 mmol, 2.0 eq.), and DIPEA (2.3 μL, 0.0141 mmol, 6.0 eq.) in DMF (400 μL) was stirred at room temperature overnight. LC-MS indicated the desired product. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to afford LP-17 (1.2 mg, 18.2% yield) as a white solid. LC-MS (ESI) m / z: 1419.6 [M+23] + . 1 H NMR(400MHz,DMSO-d6)δ(ppm)9.88-9.75(m,1H),8.39-8.16(m,1H),8.04-7.87(m,2H),7.30(s, 1H),7.26-7.23(m,1H),7.00(s,1H),6.87(s,1H),6.49(s,1H),6.26(s,1H),6.19(s,1H),5.12(s ,1H),4.74-4.72(m,1H),4.49(s,4H),4.45-4.05(m,10H),3.65-3.3(m,22H),3.17-3.14(m,3H) ,2.83-2.60(m,8H),2.39-2.25(m,9H),2.04-1.99(m,6H),1.26-1.24(m,3H),0.85-0.80(m,6H).

[0932] Synthesis of LP-18.

[0933]

[0934] To a solution of PL-4 (10 mg, 0.013 mmol, 1.0 eq.), LK-13 (33.4 mg, 0.026 mmol, 2.0 eq.), DMAP (7 mg, 0.006 mmol, 0.5 eq.), and HOAt (35.4 mg, 0.26 mmol, 20.0 eq.) in 1 mL of anhydrous DMF was added EDCI (27 mg, 0.26 mmol, 20.0 eq.) in portions. After the addition, the mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to give LP-18 (7.7 mg, 29.0% yield) as a white solid. LC-MS (ESI) m / z: 828.1 [1 / 2 M+H] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 8.74 (s, 2H), 8.65 (s, 1H), 8.33 (s, 1H), 8.12 (d, J= 7.2Hz,2H),8.08–7.98(m,2H),7.86(d,J=5.6Hz,1H),7.73(s,1H),7.40(d,J=8. 3Hz,1H),7.27–7.16(m,5H),6.96(d,J=6.2Hz,2H),6.43(d,J=7.2Hz,2H),6.13( s,2H),5.33(s,1H),5.03(d,J=11.7Hz,1H),4.66(d,J=6.8Hz,2H),4.48(s,3H),4 .18(t,J=7.0Hz,2H),4.11(s,1H),4.05(d,J=10.9Hz,1H),4.01(s,2H),3.75(s, 1H),3.70(d,J=5.9Hz,5H),3.63(s,4H),3.55(s,6H),3.50(s,52H),3.23(s,3H), 3.20–3.14(m,2H),3.08–2.94(m,2H),2.83–2.69(m,3H),2.29(s,3H),2.21(s,3 H),2.17–2.06(m,5H),2.04(s,3H),1.96(s,3H),1.46(s,5H),1.21–1.12(m,2H).

[0935] Synthesis of LP-19.

[0936]

[0937] To a solution of PL-4 (20 mg, 0.026 mmol, 1.0 eq.), LK-14 (78.46 mg, 0.052 mmol, 2.0 eq.), DMAP (1.6 mg, 0.013 mmol, 0.5 eq.), and HOAt (70.78 mg, 0.52 mmol, 20.0 eq.) in 1 mL of anhydrous DMF was added EDCI (53.9 mg, 0.52 mmol, 20.0 eq.) in portions. After addition, the mixture was stirred at room temperature overnight. The reaction mixture was concentrated, and the residue was purified by preparative HPLC to give LP-19 (13.84 mg, 24% yield) as a brown oil. LC-MS (ESI) m / z: 1131 [1 / 2 M+2H] + .

[0938] Synthesis of LP-20.

[0939]

[0940] To a solution of PL-5 (5 mg, 0.0064 mmol, 1.0 eq.) and LK-7 (17.64 mg, 0.0197 mmol, 3.0 eq.) in 0.1 mL of pyridine and 0.4 mL of anhydrous DMF were added HOAt (0.87 mg, 0.00642 mmol, 1.0 eq.) and DIPEA (13.25 mg, 0.102 mmol, 16 eq.). After addition, the mixture was stirred at 40 ° C overnight. The reaction mixture was purified by preparative HPLC to give LP-20 (1.28 mg, 12.8% yield) as a white solid. LC-MS (ESI) m / z: 778.5 [1 / 2 M + H] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)10.05(d,J=18.8Hz,2H),8.43(s,3H),7.92(d,J=8.2Hz,1H),7.62(d,J=8.4Hz,2H),7 .36(d,J=8.3Hz,2H),7.23(d,J=8.5Hz,1H),7.02(s,2H),6.50(s,1H),6.24(d,J=10.5Hz,2H),6.05(s,1H),5.45(s ,2H),5.06(s,3H),4.48(s,1H),4.21(s,2H),4.10(d,J=18.4Hz,2H),3.68(s,5H),3.62(s,10H),3.20(s,1H),2.31 (s,2H),2.28(s,3H),2.09(s,7H),2.08(s,3H),2.00(s,4H),1.08(t,J=7.0Hz,2H),0.86(dd,J=12.9,6.5Hz,10H).

[0941] Synthesis of LP-21.

[0942]

[0943] To a solution of PL-5 (5 mg, 0.0064 mmol, 1.0 eq.) and Mal-di-PEG-Val-Cit-PNP (16.46 mg, 0.0197 mmol, 3.0 eq.) in 0.1 mL of pyridine and 0.4 mL of anhydrous DMF were added HOAt (0.87 mg, 0.00642 mmol, 1.0 eq.) and DIPEA (13.25 mg, 0.102 mmol, 16.0 eq.). After addition, the mixture was stirred at 40 ° C overnight. The reaction mixture was purified by preparative HPLC to give LP-21 (2.26 mg, 23.5% yield) as a white solid. LC-MS (ESI) m / z: 747.9 [1 / 2 M + H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.05 (d, J = 14.8Hz, 2H), 8.81 (s, 1H), 8.38 (s, 2H), 7.92 (s, 1H), 7.63 (d, J = 8.3Hz, 2H) ,7.36(d,J=8.5Hz,2H),7.24(d,J=9.0Hz,2H),7.02(s,2H),6.51(s,1H),6.26(s,1H),6.23(s,1H),5.78(s,1H),5.4 4(s,2H),5.07(s,2H),4.49(s,1H),4.23(s,2H),4.09(s,2H),3.68(s,3H),3.61(t,J=7.2Hz,5H),3.16(d,J=5.6Hz, 1H),2.92(s,1H),2.76(s,1H),2.31(s,3H),2.28(s,3H),2.08(s,4H),2.04(s,1H),2.00(s,4H),0.91–0.83(m,13H).

[0944] Synthesis of LP-22.

[0945]

[0946] To a solution of PL-16 (5 mg, 0.0063 mmol, 1.0 eq.) and LK-1 (18.15 mg, 0.0189 mmol, 3.0 eq.) in 1 mL of anhydrous DMF:pyridine (4:1) was added HOAt (0.9 mg, 0.252 mmol, 1.0 eq.) and DIPEA (13.15 mg, 0.1 mmol, 16.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-22 (5 mg, 49.1% yield) as a white solid. LC-MS (ESI) m / z: 809.8 [1 / 2 M+1] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.84 (s, 1H), 8.76–8.74 (m, 1H), 7.58 (d, J = 8.6Hz, 1H), 7.26 (s, 2H), 7.01–6 .99(m,2H),6.45(d,J=1.8Hz,1H),6.26–6.24(m,1H),6.17–6.14(m,1H),5.45–5.37(m,2H),5.33–5.27(m, 1H),4.94(s,2H),4.44(d,J=1.3Hz,1H),4.06(d,J=2.2Hz,1H),3.62(s,2H),3.51(s,2H),3.47(d,J=9.7H z,30H),3.17(s,1H),2.79(s,1H),2.27(s,2H),2.22(s,3H),2.03(s,4H),1.98(s,4H),0.87–0.82(m,7H).

[0947] Synthesis of LP-23.

[0948]

[0949] To a solution of PL-4 (5 mg, 0.0065 mmol, 1.0 eq) and LK-10 (5.2 mg, 0.0095 mmol, 1.5 eq.) in 2 mL of anhydrous DMF were added HOBt (9.5 mg, 0.13 mmol, 20.0 eq.) and EDCI (13.5 mg, 0.22 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to afford LP-23 (0.8 mg, 6.8% yield) as a white solid. LC-MS (ESI) m / z: 641.4 [1 / 2 M+H] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 8.98 (s, 1H), 8.42 (s, 3H), 8.15 (s, 1H), 7.68 (s, 1H), 7.26 (d, J = 4. 4Hz,4H),6.99(s,2H),6.46(s,1H),6.43(s,1H),6.16(d,J=6.2Hz,2H),5.34(s,1H),4.51(s,2H ),4.13(s,1H),3.88(s,1H),3.69–3.64(m,5H),3.56(s,3H),2.56(s,2H),2.31(s,3H),2.23(s, 3H), 2.14–2.08 (m, 2H), 2.07 (s, 3H), 2.02 (d, J = 8.7Hz, 3H), 1.99 (s, 4H), 1.48 (d, J = 7.6Hz, 5H).

[0950] Synthesis of LP-24.

[0951]

[0952] To a solution of PL-16 (5.0 mg, 0.0063 mmol, 1.0 eq.) and LK-8 (10 mg, 0.0126 mmol, 1.0 eq.) in 1 mL of anhydrous DMF:pyridine (4:1) was added HOAt (0.85 mg, 0.252 mmol, 1.0 eq.) and DIPEA (13.15 mg, 0.1 mmol, 16.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to afford LP-24 (3.8 mg, 41% yield) as a white solid. LC-MS (ESI) m / z: 729 [M+2] / 2. 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.85 (d, J = 7.6Hz, 2H), 8.76 (s, 1H), 8.38 (s, 1H), 8.17(t,J=8.8Hz,1H),8.06(dd,J=12.6,6.2Hz,2H),7.59(d,J=8.1Hz,2H),7.45 (t,J=6.1Hz,1H),7.33–7.14(m,10H),6.97(s,2H),6.84(d,J=8.7Hz,1H),6.45( s,1H),6.25(s,1H),6.16(s,1H),5.05(d,J=11.4Hz,1H),4.95(s,2H),4.47(d,J =21.7Hz,3H),4.19(d,J=4.6Hz,1H),4.09–3.97(m,2H),3.88(dd,J=10.9,5.5H z,2H),3.80–3.65(m,6H),3.63(s,4H),3.22–3.15(m,2H),3.15–3.04(m,2H),2. 89–2.81(m,1H),2.79(s,2H),2.67(s,1H),2.27(s,3H),2.22(s,3H),2.10(t,J= 7.4Hz, 2H), 2.03 (s, 3H), 1.98 (s, 3H), 1.45 (p, J = 7.7Hz, 5H), 1.22–1.13 (m, 2H).

[0953] Synthesis of LP-25.

[0954]

[0955] To a solution of PL-16 (10 mg, 0.0126 mmol, 1.0 eq.) and LK-9 (7.8 mg, 0.0126 mmol, 1.0 eq.) in 1 mL of anhydrous DMF were added HOBt (34 mg, 0.252 mmol, 20.0 eq.) and EDCI (34 mg, 0.252 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was purified by preparative HPLC to give LP-25 (4.3 mg, 24.5% yield) as a white solid. LC-MS (ESI) m / z: 855.0 [1 / 2 M+H] + . 11H NMR (400 MHz, DMSO-d6) δ (ppm) 9.88 (s, 1H), 8.75 (s, 1H), 8.62 (t, J = 6.6 Hz, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 8.06 (t, J = 5.8 Hz, 1H), 8.00 (t, J = 5.7 Hz, 1H), 7.94 (t, J = 5.9 Hz, 1H), 7.28–7.13 (m, 7H), 6.98 (s, 2H), 6.84 (d, J = 8.6 Hz, 1H), 6.46 (s, 1H), 6.25 (s, 1H), 6.16 (s, 1H), 5.06 (d, J = 11.6 Hz, 1H), 4.63 (d, J = 6.7 Hz, 2H), 4.51–4.42 (m, 3H), 4.20 (d, J = 4.6 Hz, 1H), 4.07 (s, 1H), 4.04–3.97 (m, 1H), 3.91 (s, 2H), 3.87 (d, J = 5.7 Hz, 2H), 3.75 (ddd, J = 19.1, 9.4, 5.0 Hz, 3H), 3.67 (d, J = 5.7 Hz, 2H), 3.63 (s, 4H), 3.58 (d, J = 5.4 Hz, 1H), 3.22 (s, 2H), 3.14 (s, 2H), 3.05 (dd, J = 13.8, 4.5 Hz, 2H), 2.91–2.82 (m, 1H), 2.81–2.77 (m, 2H), 2.44 (d, J = 15.2 Hz, 1H), 2.28 (s, 3H), 2.23 (s, 3H), 2.10 (t, J = 7.4 Hz, 2H), 2.04 (s, 4H), 1.98 (s, 3H), 1.47 (h, J = 7.1 Hz, 4H), 1.23–1.15 (m, 2H).

[0956] Synthesis of LP-26.

[0957]

[0958] To a solution of PL-13 (4 mg, 0.00484 mmol, 1.0 eq.) and Mc-Val-Cit-PAB-PNP (38.28 mg, 0.0145 mmol, 3.0 eq.) in 1 mL of pyridine:DMF (1:4) was added DIPEA (10 mg, 0.0774 mmol, 16.0 eq.) and HOAt (0.65 mg, 0.00484 mmol, 1.0 eq.) and the reaction mixture was stirred at 40 ° C overnight. LC-MS showed that the reaction was complete. The reaction mixture was concentrated and purified by preparative HPLC to give LP-26 (0.9 mg, 13% yield) as a white solid. LC-MS (ESI) m / z: 713 [1 / 2 M + 2] + . 1 H NMR (400MHz, DMSO-d6) δ (ppm) 10.03 (s, 1H), 9.08 (s, 1H), 8.12 (d, J = 6.4Hz, 1H), 7.86 (d, J = 8.2Hz, 1H), 7.65 (s, 1H), 7 .60(d,J=8.6Hz,2H),7.30(d,J=8.3Hz,2H),6.99(s,2H),6.44(d,J=12.3Hz,2H),6.16(d,J=6.9Hz,2H),6.05(s,1H),5 .46(s,2H),5.34(s,1H),5.04(s,1H),4.98(s,2H),4.50(s,1H),4.20(s,2H),4.13(s,1H),4.07(s,1H),3.29(s,1H), 2.31(d,J=4.1Hz,3H),2.23(s,2H),2.14(s,1H),2.05(d,J=11.4Hz,3H),1.99(s,6H),1.49(s,7H),0.86–0.81(m,6H).

[0959] Synthesis of LP-27.

[0960]

[0961] To a solution of PL-5 (5 mg, 0.0064 mmol, 1.0 eq.) and LK-3 (15 mg, 0.019 mmol, 3.0 eq.) in 1 mL of anhydrous pyridine:DMF (1:4) was added HOAt (0.87 mg, 0.0064 mmol, 1.0 eq.) and DIPEA (13.2 mg, 0.10 mmol, 16.0 eq.) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and purified by preparative HPLC to give LP-27 (2.03 mg, 23% yield) as a white solid. LC-MS (ESI) m / z: 690 [1 / 2 M+2] + . 1 HNMR(400MHz,DMSO-d6)δ(ppm)10.04(d,J=18.5Hz,2H),8.80(s,1H),7.89(s,1H),7.64(s,1H),7.62(s,1H),7.3 6(d,J=8.3Hz,2H),7.23(d,J=8.4Hz,2H),7.03(s,2H),6.50(s,1H),6.26(s,1H),6.23(s,1H),5.43(s,2H),5.35 (s,1H),5.06(s,2H),4.49(s,1H),4.22(s,2H),4.08(s,2H),3.68(s,3H),3.58(s,2H),3.56(s,1H),3.54(s,1H) ,2.31(s,4H),2.28(s,3H),2.08(s,4H),2.03(s,1H),2.00(s,4H),0.87(d,J=7.0Hz,6H),0.83(d,J=6.7Hz,4H).

[0962] Synthesis of LP-28.

[0963]

[0964] To a solution of PL-5 (5 mg, 0.0064 mmol, 1.0 eq.) and LK-5 (15 mg, 0.019 mmol, 3.0 eq.) in 1 mL of anhydrous pyridine:DMF (1:4) was added HOAt (0.87 mg, 0.0064 mmol, 1.0 eq.) and DIPEA (13.2 mg, 0.10 mmol, 16.0 eq.) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and purified by preparative HPLC to give LP-28 (3.3 mg, 35% yield) as a white solid. LC-MS (ESI) m / z: 734 [1 / 2 M+2] + . 1HNMR(400MHz,DMSO-d6)δ(ppm)10.04(d,J=18.5Hz,2H),8.80(s,1H),7.89(s,1H),7.64(s,1H),7.62(s,1H),7.3 6(d,J=8.3Hz,2H),7.23(d,J=8.4Hz,2H),7.03(s,2H),6.50(s,1H),6.26(s,1H),6.23(s,1H),5.43(s,2H),5.35 (s,1H),5.06(s,2H),4.49(s,1H),4.22(s,2H),4.08(s,2H),3.68(s,3H),3.58(s,2H),3.56(s,1H),3.54(s,1H) ,2.31(s,4H),2.28(s,3H),2.08(s,4H),2.03(s,1H),2.00(s,4H),0.87(d,J=7.0Hz,6H),0.83(d,J=6.7Hz,4H).

[0965] Synthesis of LP-29.

[0966]

[0967] Under argon, PL-12 (5 mg, 0.00676 mmol, 1.0 eq.) was added to a solution of LK-10 (3.58 mg, 0.00676 mmol, 1.0 eq.), EDCI (25.92 mg, 0.135 mmol, 20.0 eq.), and HOBt (18.27 mg, 0.135 mmol, 20.0 eq.) in 1 mL of anhydrous DMF and stirred overnight at room temperature. After completion, the reaction was concentrated and purified by preparative HPLC to afford LP-29 (0.6 mg, 5% yield) as a white solid. LC-MS (ESI) m / z: 626 [1 / 2 M+2] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 9.74 (s, 1H), 8.83 (s, 1H), 8.36 (d, J = 5.2Hz, 2H), 8.11–8.06 (m, 1H), 7.31 (s ,1H),7.25(s,5H),7.20(s,1H),6.98(s,2H),6.47(s,1H),6.26(s,1H),6.17(s,1H),5.34(s,1H),4.46(s ,2H),4.08(s,1H),3.24–3.14(m,1H),2.81(s,2H),2.29(s,3H),2.24(s,3H),2.11(s,3H),2.06(s,3H),2 .03(s,2H),2.00(d,J=3.0Hz,6H),1.48(d,J=7.8Hz,2H),1.19(s,2H),1.07(d,J=7.0Hz,1H),0.87(s,2H).

[0968] Synthesis of LP-30.

[0969]

[0970] To a solution of PL-5 (10 mg, 0.013 mmol, 1.0 eq.) and LK-4 (30 mg, 0.039 mmol, 3.0 eq.) in 2 mL of anhydrous pyridine:DMF (1:4) was added HOAt (1.8 mg, 0.013 mmol, 1.0 eq.) and DIPEA (26.8 mg, 0.21 mmol, 16.0 eq.) and stirred at room temperature overnight. The reaction mixture was concentrated and purified by preparative HPLC to give compound LP-30 (6.5 mg, 36% yield) as a white solid. LC-MS (ESI) m / z: 705 [1 / 2 M+2] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.08 (d, J = 5.2Hz, 2H), 8.83 (s, 1H), 8.35 (s, 2 H),8.03(d,J=9.1Hz,1H),7.62(d,J=8.4Hz,2H),7.56(d,J=8.5Hz,1H),7.46( d,J=9.2Hz,2H),7.36(d,J=8.2Hz,2H),7.25(t,J=8.9Hz,2H),7.02(s,1H),7 .00(s,2H),6.50(s,1H),6.25(d,J=11.4Hz,2H),6.03(s,2H),5.45(s,3H),5. 07(s,2H),4.49(s,1H),4.45(s,1H),4.31(t,J=7.7Hz,1H),4.22(s,1H),4.0 8(s,1H),3.93(s,3H),3.68(s,3H),3.58(s,4H),3.56(s,3H),3.53(s,3H),3. 19(s,2H),2.92(s,2H),2.57(s,5H),2.31(s,3H),2.28(s,3H),2.08(s,4H), 2.02(d,J=8.0Hz,2H),2.00(s,3H),0.88(d,J=6.5Hz,6H),0.85–0.80(m,5H).

[0971] Synthesis of LP-31.

[0972]

[0973] To a solution of PL-5 (10 mg, 0.013 mmol, 1.0 eq.) and LK-6 (34 mg, 0.039 mmol, 3.0 eq.) in 2 mL of anhydrous pyridine:DMF (1:4) was added HOAt (1.8 mg, 0.013 mmol, 1.0 eq.) and DIPEA (26.8 mg, 0.21 mmol, 16.0 eq.) and stirred at room temperature overnight. The reaction mixture was concentrated and purified by preparative HPLC to afford compound LP-31 (3.3 mg, 17% yield) as a white solid. LC-MS (ESI) m / z: 756 [1 / 2 M+2] + . 1H NMR (400MHz, DMSO-d6) δ (ppm) 10.03 (d, J = 13.7Hz, 2H), 8.79 (s, 1H), 8.41 (s, 1H), 8.13 (d, J = 7.0Hz, 1H), 7.93 (d, J = 9.1Hz, 1H), 7.88 (d, J =8.8Hz,1H),7.60(d,J=8.4Hz,2H),7.34(d,J=8.0Hz,2H),7.20(d,J=8.9Hz,1H),7.02(s,1H),7.01(s,2H),6.48(s,2H),6.22(d,J=12.3 Hz,2H),5.99(s,1H),5.75(s,1H),5.42(s,2H),5.04(s,2H),4.46(s,1H),4.21(d,J=8.5Hz,2H),4.05(s,2H),3.65(s,3H),3.54(d,J=5. 2Hz, 3H), 3.50 (d, J = 5.1Hz, 5H), 3.47 (d, J = 2.7Hz, 12H), 2.27 (d, J = 12.7Hz, 6H), 2.05 (s, 3H), 1.98 (d, J = 4.1Hz, 5H), 0.88–0.79 (m, 11H).

[0974] Synthesis of LP-32.

[0975]

[0976] To a solution of Mal-Gly-Gly-Gly-Gly-Gly-Val-Cit-PAB-OH (10 mg, 0.0133 mmol, 1.0 eq.) and bis(4-nitrophenyl) carbonate (6.12 mg, 0.2 mmol, 1.5 eq.) in 0.5 mL of DMSO was added DIPEA (8.39 mg, 0.065 mmol, 5.0 eq.) and the reaction was stirred at 40°C overnight. PL-5 (5 mg, 0.00642 mmol) was added and maintained at 40°C for another 18 hours. The reaction was concentrated and purified by preparative HPLC to give LP-32 (1.03 mg, 10% yield) as a white solid. LC-MS (ESI) m / z: 775 [1 / 2 M+2] + . 1H NMR(400MHz,DMSO-d6)δ9.96(s,1H),9.92(s,1H),8.31(s,4H),7.50(s,3H),7.26(s,1H),7. 24(s,1H),7.11(s,1H),7.00(s,2H),6.40(s,1H),6.15(s,1H),6.12(s,1H),5.35(s,2H),5.2 4(s,2H),4.95(s,2H),4.02(s,2H),3.97(s,2H),3.69(s,2H),3.68(s,2H),3.57(s,3H),2.20 (s,3H),2.17(s,4H),1.99(s,1H),1.97(s,4H),1.93(d,J=7.6Hz,4H),0.74(d,J=9.1Hz,6H).

[0977] Synthesis of LP-33.

[0978]

[0979] To a solution of PL-5 (10 mg, 0.0129 mmol, 1.0 eq.) and LK-12 (8.9 mg, 0.0129 mmol, 1.0 eq.) in 2 mL of anhydrous DMF was added HOBt (50 mg, 0.258 mmol, 20.0 eq.) and EDCI (35 mg, 0.258 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction was concentrated and purified by preparative HPLC to afford LP-33 (0.82 mg, 4.3% yield) as a white solid. LC-MS (ESI) m / z: 726.1 [1 / 2 M+H] + .

[0980] Synthesis of LP-34.

[0981]

[0982] To a solution of PL-12 (20 mg, 0.027 mmol, 1.0 eq.) and LK-9 (16.64 mg, 0.027 mmol, 3.0 eq.) in 5 mL of DMF were added HOBt (72.96 mg, 0.54 mmol, 20.0 eq.) and EDCI (103.5 mg, 0.54 mmol, 20.0 eq.). After addition, the mixture was stirred at room temperature overnight. The reaction mixture was concentrated and purified by preparative HPLC to give compound LP-34 (8 mg, 22.1% yield) as a white solid. LC-MS (ESI) m / z: 670.0 [1 / 2 M+2] + .1 H NMR (400MHz, DMSO-d6) δ (ppm) 9.66 (s, 1H), 8.40 (d, J = 19.0Hz, 4H), 8.18 (d, J = 8.0Hz, 1H), 8.11 (s, 1H), 8.05 (s, 1H), 7.38–7.29 (m, 3H), 7.26 (d ,J=7.0Hz,6H),7.22(s,2H),7.01(s,2H),6.87(d,J=8.5Hz,1H),6.48(s ,1H),6.27(s,1H),6.18(s,1H),5.35(s,1H),5.08(d,J=12.0Hz,1H),4. 64(d,J=6.5Hz,2H),4.48(s,4H),4.22(s,1H),4.10(s,1H),4.05(s,1H ),4.01(s,3H),3.78(s,1H),3.70(s,1H),3.68(s,1H),3.66(s,4H),2.8 2(s,3H),2.31(s,3H),2.25(s,3H),2.11(d,J=9.2Hz,3H),2.07(s,4H), 2.04 (s, 1H), 2.01 (s, 4H), 1.49 (q, J = 7.7Hz, 6H), 0.87 (d, J = 6.7Hz, 4H).

[0983] Synthesis of LP-35.

[0984]

[0985] LP-35 was prepared according to the process of LP-34. LC-MS (ESI) m / z: 684.6 [1 / 2M+H] + . 1H NMR(400MHz, DMSO-d6)δ(ppm)8.74(s,2H),8.65(t,J=6.6Hz,1H),8.31(t,J=5.9Hz,1H),8.13(d,J=7.9Hz,1H),8.07(t,J=5.8Hz,1H),8.01( t,J=5.7Hz,1H),7.73(s,1H),7.26–7.16(m,6H),6.96(s,2H),6.43(s,2H),6.41(s,2H),6.12(s,1H),5.03(d,J=8.0Hz,1H),4.66(d,J=6.7Hz ,2H),4.48–4.43(m,3H),4.19(d,J=4.6Hz,1H),4.10(s,1H),4.06(s,1 H),4.04(m,1H),4.01(s,3H),3.76–3.65(m,5H),3.62(s,4H),3.05–2. 98(m,2H),2.82–2.74(m,2H),2.44(m,3H),2.20(s,3H),2.16(s,3H),2 .09(m,3H),2.04(s,3H),1.96(s,3H),1.45(m,5H),1.18–1.16(m,2H).

[0986] Antibody Drug Conjugates

[0987] General Process C

[0988] The monoclonal antibody (1 eq.) was placed in a 0.5 mL centrifuge tube and diluted to 5 mg / mL with 30 mM His-HAc pH 5.5 buffer. A 100 mM EDTA aqueous solution was then added to the mixture, representing 5% of the total reaction volume. After vortexing, a 2 mg / mL TCEP aqueous solution (1.5 eq. to 9 eq.) was added to reduce the antibody. After vortexing, the reaction was placed on a thermomixer at 20°C for 1.5 hours. A 10 mg / mL DMSO solution of the linker payload building block (LP) (4.0 eq. to 10 eq.) was added, followed by DMSO added to 20% of the final reaction volume. After vortexing, the solution was placed on a thermomixer at 20°C for 0.5 hours for coupling.

[0989] The unbound residual linker payload in the reaction solution was removed by treatment with 300 mg / mL dextran-coated activated carbon (Sigma). The dextran-coated activated carbon suspension was added to the reaction solution at 10% volume. After vortexing, the mixture was shaken at 4°C for 2 hours, and then the supernatant was taken to detect the residual linker payload content. If the free linker payload content was higher than 1%, the above process was repeated several times until the free linker payload content reached the standard (3 treatments were sufficient). After treatment, the mixture was centrifuged, the supernatant was collected using a syringe, and the activated carbon was filtered out using a hydrophilic membrane filter. The buffer of the desired ADC was replaced with a suitable storage buffer by 4 ultrafiltrations and stored at -80°C.

[0990] General Characterization Protocol for Antibody Drug Conjugates

[0991] (a) Measurement of ADC DAR values ​​by RP-HPLC analysis

[0992] HPLC instrument: Waters e2695 high performance liquid chromatography system. Column: RP mAb polyphenylene ion (4.6 × 100 mm, 2.7 μm) (Waters). Mobile phases: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN. The following elution schedule (35%-45%) was used: from 0 to 3 minutes, the volume of mobile phase A was 90%-90%, and the volume of mobile phase B was 10%-10%. From 3 to 5 minutes, the volume of mobile phase A was 90%-65%, and the volume of mobile phase B was 10%-35%. From 5 to 20 minutes, the volume of mobile phase A was 5%-0%, and the volume of mobile phase B was 95%-100%. From 24 to 26 minutes, the volume of mobile phase A was 0-100%, and the volume of mobile phase B was 100%-0%. At 26-30 minutes, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.

[0993] Detection conditions: mobile phase flow rate was set at 1 mL / min, detection wavelength was 280 nm, and column temperature was 65 °C.

[0994] Experimental Procedure: Take 50 μg of ADC sample (converted to volume based on concentration), add 5 μL of 1 M DTT, and add storage buffer to a final volume of 50 μL. After vortexing, incubate at 37°C for 30 minutes. After warming to room temperature, centrifuge the sample at 12,000 rpm for 5 minutes. Load 20 μL of the supernatant onto the HPLC, perform elution according to the above elution procedure, and record the chromatogram.

[0995] DAR value calculation formula: DAR = (L0 peak area ratio x 0 + L1 peak area ratio x 1 + H0 peak area ratio x 0 + H1 peak area ratio x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100x 2.

[0996] (b) Measurement of ADC DAR values ​​by HIC-HPLC analysis

[0997] HPLC instrument: Waters e2695 high performance liquid chromatography system. Column: MabPac TM HIC-Butyl 5μm 4.6×100mm (Thermo). Mobile phase: Mobile phase A (MPA): 1.5M (NH₄)₂SO₄ + 50mM potassium phosphate (pH 7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0) / isopropanol (75:25 v / v). The elution profile was 5%–95%, with the following elution schedule: from 0 to 2 minutes, the volume of mobile phase A was 100%–95%, and the volume of mobile phase B was 0%–5%. From 2 to 22 minutes, the volumes of mobile phases A and B were 95%–5% and 5%–95%, respectively. From 22 to 24 minutes, the volume of mobile phase A was 5%–0%, and the volume of mobile phase B was 95%–100%. From 24 to 26 minutes, the volume of mobile phase A was 0–100%, and the volume of mobile phase B was 100%–0%. At 26-30 minutes, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.

[0998] Detection conditions: mobile phase flow rate was set at 1 mL / min, detection wavelength was 280 nm, and column temperature was 30 °C.

[0999] Experimental procedure: 50 μg of ADC sample (converted to volume based on concentration) was loaded onto HPLC, eluted according to the above elution procedure, and the chromatogram was recorded.

[1000] The DAR value calculation formula is: DAR = Σ (correlation peak area × amount of loaded drug) / 100.

[1001] (c) Measurement of ADC DAR values ​​by SEC-HPLC analysis

[1002] HPLC instrument: 1260Agilent high performance liquid chromatography system. Column: Waters BEH200SEC (7.8×300 mm, 3.5 μm). Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, elution according to the following schedule, 0-30 minutes, the volume of mobile phase A is 100%-100%.

[1003] Detection conditions: The flow rate of the mobile phase was set at 0.5 mL / min, the detection wavelengths were 280 nm and 254 nm, and the column temperature was 26 °C.

[1004] Experimental procedure: 20 μg ADC sample (converted to volume based on sample concentration) was loaded onto HPLC, eluted according to the above elution procedure, and the chromatogram was recorded.

[1005] Calculation formula: SEC-DAR=C 药物 / C mAb Among them, the sum of the absorbance of small molecule drugs and antibodies at 280nm is Total absorbance of small molecule drugs at the maximum absorption point λ (254nm) in and L is the path length.

[1006] (d) Through C 18 -HPLC analysis to measure free linker payload (mol / mol%)

[1007] HPLC instrument: Waters e2695 high performance liquid chromatography system. Column: C18 3.5μm 4.6×150mm (Waters). Mobile phase: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN. The following elution schedule (5%-95%) was used: from 0 to 30 minutes, the volume of mobile phase A was 90%-20%, and the volume of mobile phase B was 10%-80%. From 30 to 31 minutes, the volume of mobile phase A was 20%-90%, and the volume of mobile phase B was 80%-10%. From 31 to 35 minutes, the volume of mobile phase A was 90%-90%, and the volume of mobile phase B was 10%-10%.

[1008] Detection conditions: the flow rate of the mobile phase was set to 0.5 mL / min, the detection wavelength was set to 254 nm, and the column temperature was set to 30 °C.

[1009] Preparation of Reagent I: A solution of 10 g of sodium chloride in 30 mL of anhydrous methanol and 50 mL of acetonitrile was stirred at room temperature for more than one hour and then allowed to stand for one hour. The supernatant was filtered through a 0.22 μm organic membrane and stored at room temperature for three months.

[1010] Preparation of Reagent II: A mixture of 100 mL of Reagent I, 15 mL of DMSO, and 85 mL of ADC storage buffer was thoroughly mixed and stored at room temperature (expiry date is 2 months).

[1011] (e) Sample solution preparation

[1012] Free linker payload reference: Dissolve the ADC-related linker payload in the above reagent II to obtain a concentration of 1.0 mg / mL.

[1013] Prepare a 100 μg / mL solution by adding 10 μL of the above free linker payload reference at a concentration of 1.0 mg / mL to 90 μL of Reagent II. Then, dilute the sample with Reagent II according to the table below to prepare the desired sample concentration.

[1014] Sample number Sample concentration Sample volume Reagent II volume Required concentration for sample preparation 1 100 μg / mL 40 μL 360μL 10 μg / mL 2 10 μg / mL 180 μL 180 μL 5.0 μg / mL 3 5.0 μg / mL 120 μL 180 μL 2.0 μg / mL 4 2.0 μg / mL 180 μL 180 μL 1.0 μg / mL 5 1.0 μg / mL 180 μL 180 μL 0.5 μg / mL 6 0.5 μg / mL 120 μL 180 μL 0.2 μg / mL 7 0.2 μg / mL 180 μL 180 μL 0.1 μg / mL

[1015] Note: The samples of desired concentration prepared above were loaded onto HPLC in order from low to high concentration.

[1016] Test sample: Mix 85 μg of ADC with 3 μL of DMSO for 5 minutes, then add 60 μL of Reagent I. Mix thoroughly for 5-10 minutes and centrifuge at 2000 rpm for 2 minutes. Finally, load 20 μL of the supernatant onto the HPLC and elute according to the above elution procedure. Record the chromatogram.

[1017] The amount of free linker payload in the ADC was calculated according to the standard bivariate linear regression curve equation: Free linker payload (mol / mol%) = molar concentration of residual linker payload / molar concentration of antibody × 100.

[1018] (f) Measurement of ADC aggregation by SEC-HPLC analysis

[1019] HPLC instrument: 1260Agilent high performance liquid chromatography system. Column: Waters BEH200 SEC (7.8 × 300 mm, 3.5 μm). Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, according to the following elution schedule, 0–30 min, mobile phase A volume 100%–100%;

[1020] Detection conditions: the flow rate of the mobile phase was set to 0.5 mL / min, the detection wavelength was set to 280 nm, and the column temperature was set to 26 °C.

[1021] Experimental procedure: 20 μg ADC sample (converted to volume based on sample concentration) was loaded onto HPLC, eluted according to the above elution procedure, and the chromatogram was recorded.

[1022] Calculation formula: monomer purity (%) = A monomer / total amount of A × 100%; aggregate purity (%) = A aggregate / total amount of A × 100%.

[1023] Synthesis of ADC-1.

[1024] According to the general coupling protocol for preparing ADCs, gosartuzumab (3.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-1 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-1 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.46, SEC purity: 82.69%, and free linker payload (mol / mol%): 0.42%.

[1025] Synthesis of ADC-2.

[1026] According to the general coupling protocol for preparing ADCs, gosartuzumab (3.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-2 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-2 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.40, SEC purity: 89.68%, and free linker payload (mol / mol%): 1.25%.

[1027] Synthesis of ADC-3.

[1028] According to the general coupling protocol for preparing ADCs, gosartuzumab (3.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-3 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times and filtering through a hydrophilic membrane filter, followed by ultrafiltration four times to obtain ADC-3 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.68, SEC purity: 74.94%, and free linker payload (mol / mol%): -0.07%.

[1029] Synthesis of ADC-4.

[1030] According to the general coupling protocol for preparing ADCs, gosartuzumab (3.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-4 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times and filtering through a hydrophilic membrane filter, followed by ultrafiltration four times, to obtain ADC-4 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.69, SEC purity: 80.76%, and free linker payload (mol / mol%): -0.95%.

[1031] Synthesis of ADC-5.

[1032] According to the general coupling protocol for preparing ADCs, trastuzumab (3.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-1 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-5 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.58, SEC purity: 83.30%, and free linker payload (mol / mol%): 0.37%.

[1033] Synthesis of ADC-6.

[1034] According to the general coupling protocol for preparing ADCs, trastuzumab (3.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-2 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-6 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 2.38, SEC purity: 92.51%, and free linker payload (mol / mol%): 0.91%.

[1035] Synthesis of ADC-7.

[1036] According to the general coupling protocol for preparing ADCs, trastuzumab (3.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-3 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times and filtering through a hydrophilic membrane filter, followed by ultrafiltration four times to obtain ADC-7 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.76, SEC purity: 83.43%, and free linker payload (mol / mol%): -0.04%.

[1037] Synthesis of ADC-8.

[1038] According to the general coupling protocol for preparing ADCs, trastuzumab (5 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 2.6 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-3 (10 mg / mL, 7.5 eq.) in DMSO at 20°C for 0.5 hours. The mixture was treated three times with dextran-coated activated carbon, filtered through a hydrophilic membrane filter, and further purified by SEC chromatography with a fraction collector to obtain ADC-8 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: RP-DAR: 3.82, SEC purity: 84.9%, and free linker payload (mol / mol%): 0.52%.

[1039] Synthesis of ADC-9.

[1040] According to the general coupling protocol for preparing ADCs, trastuzumab (3.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-4 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-9 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.51, SEC purity: 81.55%, and free linker payload (mol / mol%): -1.11%.

[1041] Synthesis of ADC-10.

[1042] According to the general coupling protocol for preparing ADCs, ciclosporin (3.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-2 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating once with dextran-coated activated carbon, filtering through a hydrophilic membrane filter, and then ultrafiltration five times to obtain ADC-10 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 2.50, RP-DAR: 1.77, SEC purity: 95.73%, and free linker payload (mol / mol%): 0.68%.

[1043] Synthesis of ADC-11.

[1044] According to the general coupling procedure for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 3.2 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-5 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-11 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.78, SEC purity: 85.39%, and free linker payload (mol / mol%): 0.28%.

[1045] Synthesis of ADC-12.

[1046] According to the general coupling protocol for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 2.4 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-6 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain AT01-1A12D2.1-b1 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 2.14, SEC purity: 94.35%, free linker payload (mol / mol%): -0.49%.

[1047] Synthesis of ADC-13.

[1048] According to the general coupling protocol for preparing ADCs, ciclosporin (3.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 3.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-7 (10 mg / mL, 12 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-13 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 3.48, SEC purity: 95.99%, free linker payload (mol / mol%): -0.31%.

[1049] Synthesis of ADC-14.

[1050] According to the general coupling protocol for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-8 (10 mg / mL, 4 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-14 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.47, SEC purity: 96.33%, and free linker payload (mol / mol%): 0.95%.

[1051] Synthesis of ADC-15.

[1052] According to the general coupling protocol for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating twice with dextran-coated activated carbon, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-15 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.53, SEC purity: 81.16%, and free linker payload (mol / mol%): 0.63%.

[1053] Synthesis of ADC-16.

[1054] According to the general coupling protocol for preparing ADCs, pertuzumab (1.0 mg, 3.77 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-5 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-16 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.32, SEC purity: 91.74%, and free linker payload (mol / mol%): 0.37%.

[1055] Synthesis of ADC-17.

[1056] According to the general coupling protocol for preparing ADCs, pertuzumab (1.0 mg, 3.77 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treatment with dextran-coated activated carbon, filtration through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-17 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.72, SEC-DAR: 1.39, SEC purity: 96.22%, and free linker payload (mol / mol%): 0.14%.

[1057] Synthesis of ADC-18.

[1058] According to the general coupling protocol for preparing ADCs, patrastuzumab (1.0 mg, 3.77 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours and coupled with LP-14 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-18 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.67, SEC-DAR: 2.09, SEC purity: 98.61%, free linker payload (mol / mol%): not detected.

[1059] Synthesis of ADC-19.

[1060] According to the general coupling protocol for preparing ADCs, gosartuzumab (1.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treatment with dextran-coated activated carbon, filtration through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-19 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.79, SEC-DAR: 1.48, SEC purity: 95.8%, free linker payload (mol / mol%): not detected.

[1061] Synthesis of ADC-20.

[1062] According to the general coupling protocol for preparing ADCs, gosartuzumab (1.0 mg, 11.26 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-14 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-20 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.45, SEC-DAR: 1.90, SEC purity: 97.61%, free linker payload (mol / mol%): not detected.

[1063] Synthesis of ADC-21.

[1064] According to the general coupling procedure for preparing ADCs, gosartuzumab (0.7 mg, 3.09 mg / mL, 1.0 eq.) in PBS pH 7.4 was partially reduced by adding TCEP (1 mg / mL, 2.0 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-5 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by ultrafiltration three times to obtain ADC-21 (C ADC (mg / mL): 3.07, V (mL): 0.14, yield: 61.4%). The following characterization values ​​were obtained according to the commonly used ADC characterization protocol: HIC-DAR: 1.58, SEC purity: 94.6%.

[1065] Synthesis of ADC-22.

[1066] According to the general coupling procedure for preparing ADCs, gosartuzumab (0.45 mg, 3.09 mg / mL, 1.0 eq.) in PBS pH 7.4 was partially reduced by adding TCEP (1 mg / mL, 2.0 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-6 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by ultrafiltration three times to obtain ADC-22 (C ADC (mg / mL): 2.80, V (mL): 0.11, yield: 68.4%). The following characterization values ​​were obtained according to the common ADC characterization protocol: HIC-DAR: 1.11, SEC purity: 100%.

[1067] Synthesis of ADC-23.

[1068] According to the general coupling procedure for preparing ADCs, gosartuzumab (0.45 mg, 3.09 mg / mL, 1.0 eq.) in PBS pH 7.4 was partially reduced by adding TCEP (1 mg / mL, 2.0 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-8 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by ultrafiltration three times to obtain ADC-23 (C ADC(mg / mL): 2.07, V (mL): 0.14, yield: 64.4%). The following characterization values ​​were obtained according to the commonly used ADC characterization protocol: HIC-DAR: 0.78, SEC purity: 93.2%.

[1069] Synthesis of ADC-24.

[1070] According to the general coupling protocol for preparing ADCs, metuximab (1.0 mg, 6.70 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treatment with dextran-coated activated carbon, filtration through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-24 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.89, SEC-DAR: 1.92, SEC purity: 95.38%, and free linker payload (mol / mol%): 0.3%.

[1071] Synthesis of ADC-25.

[1072] According to the general coupling protocol for preparing ADCs, metuximab (1.0 mg, 6.70 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-14 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating twice with dextran-coated activated carbon, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-25 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.55, SEC-DAR: 2.37, SEC purity: 98.41%, free linker payload (mol / mol%): not detected.

[1073] Synthesis of ADC-26.

[1074] According to the general coupling protocol for preparing ADCs, cetuximab (1.0 mg, 6.47 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-5 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-25 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.18, SEC purity: 97.37%, and free linker payload (mol / mol%): 0.91%.

[1075] Synthesis of ADC-27.

[1076] According to the general coupling protocol for preparing ADCs, cetuximab (1.0 mg, 6.47 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-27 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.23, SEC-DAR: 1.14, SEC purity: 98.44%, free linker payload (mol / mol%): not detected.

[1077] Synthesis of ADC-28.

[1078] According to the general coupling protocol for preparing ADCs, cetuximab (1.0 mg, 6.47 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-28 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.32, SEC-DAR: 2.35, SEC purity: 98.92%, free linker payload (mol / mol%): not detected.

[1079] Synthesis of ADC-29.

[1080] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-5 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-29 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 1.04, SEC purity: 85.13%, and free linker payload (mol / mol%): <1%.

[1081] Synthesis of ADC-30.

[1082] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-6 (10 mg / mL, 4 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-30 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 1.21, SEC purity: 95.11%, and free linker payload (mol / mol%): <1%.

[1083] Synthesis of ADC-31.

[1084] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-34 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-31 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 0.71, SEC purity: 98.99%, free linker payload (mol / mol%): <1%.

[1085] Synthesis of ADC-32.

[1086] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-7 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-32 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 2.0, SEC purity: 99.95%, free linker payload (mol / mol%): not detected.

[1087] Synthesis of ADC-33.

[1088] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-8 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-33 (C ADCThe following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 1.01, SEC purity: 84.42%, and free linker payload (mol / mol%): <1%.

[1089] Synthesis of ADC-34.

[1090] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-34 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.52, SEC-DAR: 1.69, SEC purity: 97.17%, free linker payload (mol / mol%): not detected.

[1091] Synthesis of ADC-35.

[1092] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. Reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-12 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times and filtering through a hydrophilic membrane filter, followed by ultrafiltration twelve times, to obtain ADC-35 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 2.4, SEC purity: 94.72%, and free linker payload (mol / mol%): 0.71%.

[1093] Synthesis of ADC-36.

[1094] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-11 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-36 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.9, SEC purity: 99.92%, free linker payload (mol / mol%): not detected.

[1095] Synthesis of ADC-37.

[1096] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-30 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-37 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 0.5, SEC purity: 99.95%, and free linker payload (mol / mol%): 0.23%.

[1097] Synthesis of ADC-38.

[1098] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.7 mg / mL, 1.0 eq.) in 50 mM PBS pH 6.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-14 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-38 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.56, SEC-DAR: 3.04, SEC purity: 99.35%, free linker payload (mol / mol%): not detected.

[1099] Synthesis of ADC-39.

[1100] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-29 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-39 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.9, SEC purity: 96.8%, free linker payload (mol / mol%): not detected.

[1101] Synthesis of ADC-40.

[1102] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-33 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-40 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 0.5, SEC purity: 95.0%, free linker payload (mol / mol%): not detected.

[1103] Synthesis of ADC-41.

[1104] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-13 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-41 (C ADC The following characterization values ​​were obtained using the commonly used ADC characterization protocol: HIC-DAR: 1.73, SEC purity: 99.92%, and free linker payload (mol / mol%): 0.83%.

[1105] Synthesis of ADC-42.

[1106] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-23 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-42 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.3, SEC purity: 96.50%, free linker payload (mol / mol%): not detected.

[1107] Synthesis of ADC-43.

[1108] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. Reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-22 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-43 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 2.2, SEC purity: 88.7%, free linker payload (mol / mol%): not detected.

[1109] Synthesis of ADC-44.

[1110] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-15 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-44 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 2.8, SEC purity: 99.4%, free linker payload (mol / mol%): not detected.

[1111] Synthesis of ADC-45.

[1112] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-24 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-45 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.2, SEC purity: 78.8%, free linker payload (mol / mol%): not detected.

[1113] Synthesis of ADC-46.

[1114] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-25 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-46 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.0, SEC purity: 98.7%, free linker payload (mol / mol%): not detected.

[1115] Synthesis of ADC-47.

[1116] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-16 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-47 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 2.2, SEC purity: 97.9%, free linker payload (mol / mol%): not detected.

[1117] Synthesis of ADC-48.

[1118] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (2 mg / mL, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-18 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-48 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 2.1, SEC purity: 99.3%, free linker payload (mol / mol%): not detected.

[1119] Synthesis of ADC-49.

[1120] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 1.8 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-19 (10 mg / mL, 4.0 eq.) in DMSO at 22°C for 1 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration twelve times to obtain ADC-49 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 0.9, SEC purity: 99.39%, free linker payload (mol / mol%): not detected.

[1121] Synthesis of ADC-50.

[1122] According to the general coupling protocol for preparing ADCs, ciclosporin (3.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-1 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-50 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.60, SEC purity: 88.63%, and free linker payload (mol / mol%): -0.17.

[1123] Synthesis of ADC-51.

[1124] According to the general coupling protocol for preparing ADCs, ciclosporin (3.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-3 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-51 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 0.69, RP-DAR: 1.32, SEC purity: 89.75%, and free linker payload (mol / mol%): -2.09.

[1125] Synthesis of ADC-52.

[1126] According to the general coupling protocol for preparing ADCs, ciclosporin (3.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-4 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-52 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: SEC-DAR: 1.40, SEC purity: 87.71%, and free linker payload (mol / mol%): -0.62.

[1127] Synthesis of ADC-53.

[1128] According to the general coupling protocol for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 1.0 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-9 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon four times, filtering through a hydrophilic membrane filter, and then ultrafiltration four times to obtain ADC-53 (C ADCThe following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 0.47, SEC-DAR: 0.86, SEC purity: 95.31%, and free linker payload (mol / mol%): 1.07.

[1129] Synthesis of ADC-54.

[1130] According to the general coupling protocol for preparing ADCs, ciclosporin (1.0 mg, 4.21 mg / mL, 1.0 eq.) in 50 mM PBS pH 8.0 was partially reduced by adding TCEP (2 mg / mL, 2.5 eq.) in water. Reduction was carried out at 20°C for 1.5 hours, followed by coupling with LP-14 (10 mg / mL, 4.0 eq.) in DMSO at 20°C for 0.5 hours. The mixture was purified by treating with dextran-coated activated carbon twice, filtering through a hydrophilic membrane filter, and then ultrafiltration three times to obtain ADC-54 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 1.78, SEC purity: 91.99%, free linker payload (mol / mol%): not detected.

[1131] Synthesis of ADC-55.

[1132] According to the general coupling protocol for preparing ADCs, trastuzumab (1.0 mg, 38.9 mg / mL, 1.0 eq.) in 50 mM PBS pH 7.4 was partially reduced by adding TCEP (10 mM, 15.0 eq.) in water. The reduction was carried out at 22°C for 2.0 hours, followed by coupling with LP-18 (10 mg / mL, 15.0 eq.) in DMSO at 22°C for 1.0 hour. The mixture was purified by treating with dextran-coated activated carbon three times, filtering through a hydrophilic membrane filter, and then ultrafiltration eight times to obtain ADC-55 (C ADC The following characterization values ​​were obtained using a common ADC characterization protocol: HIC-DAR: 8.0, SEC purity: 100%, free linker payload (mol / mol%): not detected.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: in -R1 is a substituent having a negative inductive effect; -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl; -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; -R4 is selected from -H, -CH2OR R4 、-CH2NHR R4 and -CH2C(=O)OR R4 , where R R4 Is -H or -C 1-4 alkyl; and -R5 is -H or -C 1-4 alkyl; Provided that compounds of formula (I) having the following combinations of R2, R3, R4 and R5 are not included: -R2 is -OCH3, R3 is -OH, R4 is -H and R5 is -H.

2. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 1, wherein R1 is -OH or -C≡N.

3. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 or 2, wherein -R2 is -H, -OR R2 、-OCH2CH2OR R2 、-OCH2C(=O)OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(=O)OH or -NH2; and / or -R3 is -H, -OR R3 、-OCH2CH2NHR R3 、-NHR R3 、-NHC(=O)CH2OR R3 or -NHC(=O)CH2NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H, -OH, OCH2CH2NH2, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2.

4. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 3, wherein -R4 is -H, -CH2OR R4 or -CH2NHR R4 , where R R4 Is -H or -C 1-4 Alkyl, preferably R4 is -H; and / or -R5 is -H or -C 1-4 Alkyl, preferably R5 is -H.

5. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 4, wherein -R1 is -OH or -C≡N; -R2 is -H, -OCH3, -OCH2CH2OH, -OCH2C(=O)OH or -NH2; -R3 is -H, -OH, -OCH2CH2NH2, -NH2, -NHC(=O)CH2OH or -NHC(=O)CH2NH2; -R4 is -H; and -R5 is -H.

6. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 1, wherein formula (I) is:

7. A compound of formula (VII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: in -R1 is a substituent having a negative inductive effect; -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl; -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and -R4 is -H or -C 1-4 alkyl; Provided that compounds of formula (VII) having the following combinations of R2, R3 and R4 are not included: -R2 is -H, R3 is -H and R4 is -H; and -R2 is -OCH3, R3 is -H and R4 is -H.

8. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 7, wherein R1 is -OH or -C≡N.

9. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 7 or 8, wherein -R2 is -H, -NHR R2 or -NHC(=O)CH2OR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -H, -NH2 or -NHC(=O)CH2OH; and / or -R3 is -H or -NHR R3 , where R R3 Is -H or -C 1-4 Alkyl, preferably R3 is -H or -NH2.

10. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 7 to 9, wherein -R4 is -H or -CH3.

11. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 7 to 10, wherein -R1 is -OH or -C≡N; -R2 is -H, -NH2 or -NHC(=O)CH2OH; -R3 is -H or -NH2; and -R4 is -H or -CH3.

12. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 7, wherein formula (VII) is:

13. A compound of formula (XIII) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: in -R1 is a substituent having a negative inductive effect; -R2 is -H or -X(CH2) p C(=O) 0-1 (CH2) q (C(R R2 )2) 0-1 (X) 0-1 R R2 , wherein q and p are independently integers from 0 to 10, wherein X is independently O or NH, and R R2 are independently -H or -C 1-4 alkyl; -R3 is -H or -X(CH2) m C(=O) 0-1 (CH2) n (C(R R3 )2) 0-1 (X) 0-1 R R3 , wherein m and n are independently integers from 0 to 10, wherein X is independently O or NH, and R R3 are independently -H or -C 1-4 alkyl; and -R4 is -H or -CH2NHR R4 , where R R4 Is -H or -C 1-4 alkyl.

14. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 13, wherein R1 is -OH or -C≡N.

15. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 13 or 14, wherein -R2 is -OR R2 or -NHR R2 , where R R2 Is -H or -C 1-4 Alkyl, preferably R2 is -OCH3 or -NH2; and / or -R3 is -H.

16. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 13 to 15, wherein -R4 is -H.

17. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 13 to 16, wherein -R1 is -OH or -C≡N; -R2 is -OCH3 or -NH2; -R3 is -H; and -R4 is -H.

18. The compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 13, wherein formula (XIII) is:

19. Antibody drug conjugate of formula (XIX): in -Ab represents an antibody, an antigen-binding fragment, or an immunologically active portion thereof; -L represents a linker; and -D represents a compound selected from trabectedin, rubectedin and a compound according to any one of claims 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer, preferably a compound according to any one of claims 1 to 18, a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer; in - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and -z is an integer from 1 to 20. 20 . The antibody drug conjugate according to claim 19 , wherein z is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, and still more preferably 2 or 4.

21. The antibody drug conjugate according to any one of claims 19 or 20, wherein D is covalently linked to L through any one of R2, R3 or R4, preferably a nitrogen or oxygen present in any one of R2 or R3.

22. The antibody drug conjugate according to any one of claims 19 to 21, wherein the antibody is: -Anti-ROR1 antibody, preferably cimetuzumab; -Anti-TROP2 antibodies, preferably gosartuzumab; -Anti-HER2 antibody, preferably trastuzumab; -Anti-HER3 antibodies, preferably patrastuzumab; - an anti-FRα antibody, preferably metuximab; or -Anti-EGFR antibody, preferably cetuximab.

23. The antibody drug conjugate according to any one of claims 19 to 22, wherein L has the following structure: in - T and U are independently spacer units, each of which has 1 to 24 chain carbon atoms, wherein t and u are independently 0 or 1, preferably t+u≥1; - each A is independently an amino acid unit, wherein a is an integer from 0 to 12; -H is a hydrophilic unit, wherein h is 0 or 1; and -Y is a coupling group covalently linked to Ab; in, Preferably, the coupling group Y is covalently linked to the sulfur atom present in Ab.

24. The antibody drug conjugate according to claim 23, wherein Y is such that L is one of the following formulae: in, If present, R6 is -H or -C 1-4 Alkyl, preferably -H or -CH3.

25. The antibody drug conjugate according to any one of claims 23 or 24, wherein -U is one of the following: in, Preferably n is an integer from 5 to 10, more preferably an integer from 6 to 8, and most preferably 7; and / or -T is one of the following: wherein n is preferably an integer of 1 to 8, more preferably an integer of 1 to 6, and most preferably 2 or 5; or wherein preferably m is 1, n is an integer from 2 to 8, more preferably an integer from 4 to 7, and most preferably 5 or 6; and / or -H is derived from a PEGylated amino acid, preferably one of the following formulas: Among them, n is more preferably an integer of 5 to 10, still more preferably an integer of 6 to 8, and most preferably 7; or Among them, n is more preferably an integer of 5 to 10, still more preferably an integer of 6 to 8, and most preferably 7; or H has the following formula: Among them, n is preferably an integer of 2 to 8, more preferably an integer of 4 to 7, and most preferably 5 or 6.

26. The antibody drug conjugate according to any one of claims 23 to 25, wherein each A has the following structure: in, R7 is independently selected from -H, -CH3, -C(H)(CH3)2, benzyl, p-hydroxybenzyl and -(CH2)3NHC(C=O)NH2; Preferably, R7 is independently selected from -H, -C(H)(CH3)2, benzyl and -(CH2)3NHC(C=O)NH2; and / or a is an integer of 1 to 10, more preferably an integer of 1 to 5, still more preferably an integer of 1 to 4, and most preferably 2 or 4.

27. The antibody drug conjugate according to claim 23, wherein H and h, A and a, and U and u are such that L is one of the following formulae:

28. The antibody drug conjugate according to any one of claims 19 to 22, wherein L is such that formula (XIX) is one of the following:

29. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 18 for use as a medicament.

30. A compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to claim 31 for use in the treatment of cancer, preferably ovarian cancer, gastric cancer or breast cancer.

31. The antibody drug conjugate according to any one of claims 19 to 28 for use as a medicament.

32. The antibody drug conjugate according to claim 31, for use in treating cancer, preferably ovarian cancer, gastric cancer or breast cancer.

33. Method for preparing an antibody drug conjugate of formula (XIX): The method comprises coupling an antibody, an antigen-binding fragment or an immunologically active portion thereof (Ab) to a compound (D) via a linker (L), wherein -D is a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 18; -L is a linker according to any one of claims 19 or 23 to 27; - D is covalently linked to L through the nitrogen or oxygen present in D, and L is covalently linked to Ab; and -z is an integer from 1 to 20.

34. The method according to claim 35, wherein z is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, and still more preferably 2 or 4.

35. The method according to any one of claims 34 or 35, wherein the antibody is: -Anti-ROR1 antibody, preferably cimetuzumab; -Anti-TROP2 antibodies, preferably gosartuzumab; -Anti-HER2 antibody, preferably trastuzumab; -Anti-HER3 antibodies, preferably patrastuzumab; - an anti-FRα antibody, preferably metuximab; or -Anti-EGFR antibody, preferably cetuximab.

36. A pharmaceutical dosage form comprising a therapeutically effective amount of a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 18, or a therapeutically effective amount of an antibody drug conjugate according to any one of claims 19 to 28.

37. A method of treating cancer, wherein the method comprises administering to a subject a therapeutically effective amount of a compound, pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer according to any one of claims 1 to 18, a therapeutically effective amount of an antibody drug conjugate according to any one of claims 19 to 28, or a pharmaceutical dosage form according to claim 36.

38. The method of claim 37, wherein the cancer is ovarian cancer, gastric cancer, or breast cancer.

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