Antibody-drug conjugates

Abstract

To provide an improved method for delivering a STING agonist to target cells.SOLUTION: The present disclosure provides antibody drug conjugates comprising STING modulators. Also provided are compositions comprising the antibody drug conjugates. The compounds and compositions are useful for stimulating an immune response in a subject in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] References to electronically submitted sequence listings The contents of the electronically submitted sequence listing (name: 3817_053PC03_Seqlisting_ST25; size: 7,104 bytes; creation date: April 22, 2020) are incorporated herein by reference in their entirety.

[0002] This disclosure provides an antibody-drug conjugate comprising a STING modulator. Also provided are compositions comprising the antibody-drug conjugate. The compounds and compositions are useful for stimulating an immune response in subjects requiring them. [Background technology]

[0003] Antibody-drug conjugates (ADCs), a rapidly growing class of targeted therapies, represent a promising new approach to improving drug selectivity and cytotoxic activity. These therapies consist of an antibody (or antibody fragment) that can bind to a payload drug to form an immune complex. The antibody directs the ADC to bind to target cells. The ADC then migrates internally and releases its payload, providing processing for the cells. Because the ADC is directed to its target cells, the side effects of the conjugated drug may be less severe than those encountered when the same drug is administered systemically.

[0004] The adapter protein STING (an interferon gene stimulator) has been shown to play a role in the innate immune system. Activation of the STING pathway triggers an immune response that results in the production of specific killer T cells that can shrink tumors and provide long-term immunity, thus preventing tumor recurrence. The activated STING pathway also contributes to the antiviral response by producing antiviral and pro-inflammatory cytokines that fight viruses, mobilize both the innate and adaptive immune systems, and ultimately provide long-term immunity against pathogenic viruses. The potential therapeutic benefits of enhancing both the innate and adaptive immune responses make STING an attractive target for drug discovery. Cyclic dinucleotides may also function as STING agonists and have been investigated in clinical trials. However, their anionic properties may result in low membrane permeability, which may limit their ability to engage STING within cells, often leading to undesirable distribution of these compounds in the bloodstream.

[0005] As with new STING agonists, there is still a need for improved methods to deliver them to target cells. [Overview of the project]

[0006] In the first aspect, this disclosure is formula (I):

[0007] [ka]

[0008] Provides compounds or pharmaceutically acceptable salts thereof,

[0009] During the ceremony,

[0010] a is an integer between 1 and 20;

[0011] b is an integer between 1 and 20;

[0012] m is 0, 1, 2, 3, or 4;

[0013] n is either 0 or 1;

[0014] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0015] Each R 1 These are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogen;

[0016] R 2 s is selected from C1-4C alkyl and -(CH2CH2O)3-CH3; in this case, s is an integer from 1 to 10;

[0017] R 3 and R 3’ Each is independently selected from hydrogen and C1-C3 alkyl;

[0018] L is a severable linker;

[0019] Ab stands for antibody, antibody fragment, or antigen-binding fragment.

[0020] In the first embodiment of the first aspect, a is an integer from 1 to 4, b is an integer from 1 to 10, and m is 0.

[0021] In the second embodiment of the first aspect, a is an integer from 1 to 4, m is 0, n is 0, and R 3 and R 3’ Each of these is hydrogen.

[0022] In the third embodiment of the first aspect, L is

[0023] [ka]

[0024] And,

[0025] During the ceremony:

[0026] [ka]

[0027] This is the bonding point to the nitrogen atom;

[0028] [ka]

[0029] is a connection point to Ab;

[0030] t is an integer between 1 and 10;

[0031] W does not exist, or it is a self-sacrificing element;

[0032] Z is either nonexistent or a peptide of 2-5 amino acids;

[0033] U and U' do not exist independently, or they are spacers;

[0034] Q is a heterodifunctional group;

[0035] However, this is subject to the condition that at least one of W and Z exists.

[0036] In the fourth embodiment of the first aspect, W is a self-sacrificing group selected from the following:

[0037] [ka]

[0038] During the ceremony:

[0039] [ka]

[0040] This is a bonding site to the carbonyl group;

[0041]

Chem.

[0042] is the point of attachment to Z.

[0043] In the fifth embodiment of the first aspect, W is selected from the following.

[0044]

Chem.

[0045] In the sixth embodiment of the first aspect, W is

[0046]

Chem.

[0047] .

[0048] In the seventh embodiment of the first aspect, Z is a peptide that can be cleaved enzymatically. In the eighth embodiment, Z is cathepsin-cleavable. In the ninth embodiment, Z is a 2-amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe. In the tenth embodiment of the first aspect, Z is Val-Ala or Ala-Val.

[0049] In the eleventh embodiment of the first aspect, U and U' are independently absent, or

[0050]

Chem.

[0051] selected from,

[0052] wherein in the formula,

[0053]

Chem.

[0054] is the point of attachment to Z;

[0055]

Chem.

[0056] is the point of attachment to Q;

[0057] p is an integer of 1 to 6;

[0058] q is an integer of 1 to 20;

[0059] X is O or -CH2-;

[0060] each r is independently 0 or 1.

[0061] In the twelfth embodiment of the first aspect, U' is absent and U is

[0062]

Chem.

[0063] .

[0064] In the thirteenth embodiment of the first aspect, Q is a heterobifunctional group capable of binding to Ab via a chemical bond or an enzyme-mediated bond. In the fourteenth embodiment of the first aspect, Q is

[0065]

Chem.

[0066] selected from,

[0067] wherein,

[0068] [ka]

[0069] It is a connection point to U, and if U does not exist, it is a connection point to Z;

[0070] [ka]

[0071] This is a connection point to U', and if U' does not exist, it is a connection point to Ab.

[0072] In a 15th embodiment of the first aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is

[0073] [ka]

[0074] There, t is 1; W is absent or a self-sacrificing group; Z is absent or a 2-amino acid peptide.

[0075] In the sixteenth embodiment of the first aspect, the disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, where R 2 is -CH3 or -(CH2CH2O)3-CH3, and s is an integer between 1 and 10.

[0076] In the 17th embodiment of the first aspect, the disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D-NH2 is an amino-substituted compound that modulates STING activity. In the 18th embodiment of the first aspect, the amino-substituted compound that modulates STING activity includes a guanine derivative or an adenine derivative.

[0077] In the 19th embodiment, the amino-substituted compound that modulates STING activity is of Formula (II):

[0078]

Chemical Formula

[0079] or a pharmaceutically acceptable salt thereof,

[0080] wherein,

[0081] X 10 is SH or OH;

[0082] X 20 is SH or OH;

[0083] Y a is O, S, or CH2;

[0084] Y b is O, S, NH, or NR a , wherein R a is C1-C4 alkyl ;

[0085] R 10 is hydrogen, fluoro, OH, NH2, OR b , or NHR b ;

[0086] R 20 is hydrogen or fluoro;

[0087] R 30 is hydrogen; R 40 is hydrogen, fluoro, OH, NH2, OR b , or NHR b , or R 30 and R 40 together form CH2O;

[0088] R 50 is hydrogen or fluoro;

[0089] R b These are C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl;

[0090] Ring A 10 is an optionally substituted 5-membered or 6-membered monocyclic heteroaryl ring containing 1 to 4 heteroatoms selected from N, O, or S, or an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 1 to 5 heteroatoms selected from N, O, or S, in which case ring A 10 It contains at least one N atom in the ring, Y b is ring A 10 It is bonded to the carbon atom;

[0091] Ring B 10 This is an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O, or S, in which case ring B 10 It contains at least two N atoms in the ring;

[0092] However, any ring A 10 or ring B 10 This is conditional on the carbonyl group of formula (I) being bonded via an -NH- group.

[0093] In the 20th embodiment, the amino-substituted compound that modulates STING activity is:

[0094] [ka]

[0095] Alternatively, a pharmaceutically acceptable salt thereof,

[0096] During the ceremony,

[0097] [ka]

[0098] is the bonding site to the carbonyl group in formula (I).

[0099] In the 21st embodiment of the first aspect, the amino-substituted compound that modulates STING activity is of the formula (III):

[0100] [ka]

[0101] A compound of or a pharmaceutically acceptable salt thereof, During the ceremony,

[0102] X 10 is either SH or OH;

[0103] X 20 is either SH or OH;

[0104] Y c is O, S, or CH2;

[0105] Y d is O, S, or CH2;

[0106] B 100 is equation (B 1 -A) or formula (B 1 The base represented by -B) is:

[0107] [ka]

[0108] R 13 , R 14 , R 15 , R 16 , and R 17 Each is independently a hydrogen atom or a substituent;

[0109] R 1000 is a hydrogen atom, or a bond to the carbonyl group of formula (I);

[0110] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 Each of these can be independently N or CR 1a And in that case, R 1a is a hydrogen atom or substituent;

[0111] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 Each is independently either N or C;

[0112] R 105 is a hydrogen atom or substituent;

[0113] B 200 is equation (B 2 -A) or formula (B 2 The base represented by -B);

[0114] [ka]

[0115] R 23 , R 24 , R 25 , R 26 , and R 27 Each of these is independently a hydrogen atom or a substituent.

[0116] R 100’ is a bond to hydrogen or the carbonyl group of formula (I);

[0117] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each of these can be independently N or CR 2a And in that case, R2a is hydrogen or a substituent;

[0118] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

[0119] R 205 is a hydrogen atom or a substituent, wherein R 105 and R 205 are each independently bonded to the 2-position or 3-position of the 5-membered ring to which they are bonded;

[0120] provided that,

[0121] B 100 or B 200 one of them is bonded to the carbonyl group of formula (I) via a -NH- group.

[0122] In the twenty-second embodiment of the first aspect, the amino-substituted compound that modulates STING activity is a compound of formula (IIIa):

[0123] ##STR##

[0124] or a pharmaceutically acceptable salt thereof, wherein,

[0125] B 100 is a group represented by formula (B 1 -A) or formula (B 1 -B);

[0126] ##STR##

[0127] R 13 , R 14 , R15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent;

[0128] R 1000 is hydrogen or a bond to the carbonyl group of formula (I);

[0129] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 are each independently N or CR 1a , where R 1a is hydrogen or a substituent;

[0130] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C;

[0131] R 105 is a hydrogen atom or a substituent;

[0132] B 200 is a group represented by formula (B 2 -A) or formula (B 2 -B);

[0133]

Chemical formula

[0134] R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent,

[0135] R 100’ is hydrogen or a bond to the carbonyl group of formula (I);

[0136] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each is independently N or CR 2a And in that case, R 2a is a hydrogen atom or substituent;

[0137] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 Each is independently either N or C;

[0138] R 205 R is a hydrogen atom or substituent, in which case 105 and R 205 Each of them is independently bonded to the 2nd or 3rd position of the 5-membered ring to which they are joined;

[0139] however,

[0140] B 100 or B 200 One of them is,

[0141] [ka]

[0142] And,

[0143] During the ceremony,

[0144] R 18 is hydrogen or C 1-6 It is alkyl;

[0145] R 19 It is a halogen atom;

[0146] Furthermore, the other is bonded to the carbonyl group of formula (I) via an -NH- group.

[0147] In the 23rd embodiment of the first aspect, the amino-substituted compound that modulates STING activity is

[0148] [ka]

[0149] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0150] [ka]

[0151] is the bonding site to the carbonyl group in formula (I).

[0152] In a second aspect, the present disclosure is formula (IV):

[0153] [ka]

[0154] Provides compounds or pharmaceutically acceptable salts thereof, During the ceremony,

[0155] a is an integer between 1 and 20;

[0156] b is an integer between 1 and 20;

[0157] k is 0, 1, 2, or 3;

[0158] m is 0, 1, 2, 3, or 4;

[0159] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0160] R 2 H, C1-C4 alkyl and -(CH2CH2O) s-Selected from CH3, where s is an integer between 1 and 10;

[0161] R 4 The side chains are selected from hydrogen and any naturally occurring amino acid;

[0162] R 5 The alkyl group is selected from C1-C4 alkyl and O-C1-C4 alkyl groups;

[0163] L is a severable linker;

[0164] Ab stands for antibody, antibody fragment, or antigen-binding fragment.

[0165] In the first embodiment of the second aspect, L is

[0166] [ka]

[0167] And,

[0168] During the ceremony:

[0169] [ka]

[0170] This is a bonding site to the carbonyl group;

[0171] [ka]

[0172] is a connection point to Ab;

[0173] W is a self-sacrificing element;

[0174] Z is either nonexistent or a peptide of 2-5 amino acids;

[0175] U and U' do not exist independently, or they are spacers;

[0176] Q is a heterodifunctional group.

[0177] In a third aspect, the present disclosure is given by formula (V):

[0178] [ka]

[0179] Provides compounds or pharmaceutically acceptable salts thereof, During the ceremony,

[0180] D-NH- is

[0181] [ka]

[0182] and;

[0183] X is either O or CH2;

[0184] f is an integer between 1 and 10;

[0185] g is an integer between 1 and 20.

[0186] U and U' do not exist independently, or they are spacers;

[0187] Q is a heterodifunctional group;

[0188] Ab stands for antibody, and Ab can stand for antibody, antibody fragment, or antigen-binding fragment.

[0189] In the first embodiment of the third aspect, X is O.

[0190] In a fourth aspect, the present disclosure is given by formula (VI):

[0191] [ka]

[0192] We provide compounds or pharmaceutically acceptable salts thereof. During the ceremony,

[0193] a is an integer between 1 and 20.

[0194] m is 0, 1, 2, 3, or 4;

[0195] n is either 0 or 1;

[0196] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0197] Each R 1 These are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogen;

[0198] R 2 C1-C4 alkyl and -(CH2CH2O) s -Selected from CH3; in this case, s is an integer between 1 and 10;

[0199] R 3 and R 3’ Each is independently selected from hydrogen and C1-C3 alkyl;

[0200] L is a severable linker;

[0201] LP is a lipid.

[0202] In the first embodiment of the fourth aspect, the lipid is cholesterol or a phospholipid. In the second embodiment of the fourth aspect, the lipid is a phospholipid. In the third embodiment of the fourth aspect, the phospholipid is selected from phosphatidylcholine, phosphatidyldicerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipids, and egg yolk phospholipids. In the fifth embodiment of the fourth aspect, the phospholipid is phosphatidylethanolamine, in which case the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine.

[0203] In a sixth embodiment of the fourth aspect, the disclosure provides a lipid complex comprising a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In a seventh embodiment, the lipid complex is in the form of a liposome.

[0204] In the eighth embodiment of the fourth aspect, the lipid complex further comprises one or more phospholipids. In the ninth embodiment, the one or more phospholipids are selected from phosphatidylcholine, phosphatidyldicerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipids, and egg yolk phospholipids. In the tenth embodiment, the phospholipid is phosphatidylcholine, and the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0205] In the eleventh embodiment of the fourth aspect, the lipid complex further comprises fatty alcohols and / or cholesterol.

[0206] In the twelfth embodiment of the fourth aspect, the lipid complex further comprises at least one PEGylated lipid. In the thirteenth embodiment, the PEGylated lipid is selected from PEGylated phospholipids and PEGylated diacylglycerols. In the fourteenth embodiment, the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0207] In a fifth aspect, the disclosure provides a pharmaceutical composition comprising a compound or complex described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0208] In a sixth aspect, the Disclosure provides a method for treating cancer in a subject requiring treatment for cancer, the method comprising administering a pharmaceutically acceptable amount of a compound or complex described herein to the subject.

[0209] In a seventh aspect, the Disclosure provides a method for stimulating an immune response in a subject requiring such stimulation, the method comprising administering a pharmaceutically acceptable amount of the compound or complex described herein to the subject. [Brief explanation of the drawing]

[0210] [Figure 1] This figure shows the preparation of the Ab-STING agonist complex via stochastic cysteine ​​conjugation. [Figure 2] This figure shows the preparation of the Ab-STING agonist complex via transglutaminase conjugation. [Figure 3] This figure shows the preparation of the Ab-STING agonist complex via transglutaminase conjugation. [Figure 4] This figure shows the preparation of the Ab-STING agonist complex via saltase conjugation. (SEQ ID NOs: 3 and 4) [Figure 5] This figure shows the payload release of ADC-1 in a tritosome assay. [Figure 6] This figure shows the payload release of ADC-3 in a tritosome assay. [Figure 7] This figure shows the payload release of ADC-9 in a tritosome assay. [Figure 8] This figure shows the plasma PK (payload dose of 0.1 mg / kg) of ADC-1 in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 9]This figure shows the plasma PK of ADC-8 (payload dose of 0.055 mg / kg, released CDN BLQ) in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 10] This figure shows the plasma PK of ADC-9 (payload dose of 0.05 mg / kg, released CDN BLQ) in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 11] This figure shows the efficacy (payload dose) of ADC-1 in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 12] This figure shows the efficacy (payload dose) of ADC-5 in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 13] This figure shows the efficacy (payload dose) of ADC-8 in CT26 tumor-bearing Balb / C mice expressing GCC. [Figure 14] This figure shows the efficacy (payload dose) of ADC-9 in CT26 tumor-bearing Balb / C mice expressing GCC. [Modes for carrying out the invention]

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. All publications and patents referenced herein are incorporated in their entirety by reference.

[0212] Unless otherwise specified by the context, the singular forms "a," "an," and "the" refer to multiple objects.

[0213] As used herein, the term “or” is logical OR (i.e., AND / OR) and does not indicate exclusive OR unless explicitly indicated, such as the terms “either,” “otherwise,” “alternatively,” and words of similar effect.

[0214] As used herein, the term "approximately" refers to a range of ±10%.

[0215] Antibody-drug conjugates In some embodiments, the present disclosure is formula (I)

[0216] [ka]

[0217] Provides compounds or pharmaceutically acceptable salts thereof,

[0218] During the ceremony,

[0219] a is an integer between 1 and 20;

[0220] b is an integer between 1 and 20;

[0221] m is 0, 1, 2, 3, or 4;

[0222] n is either 0 or 1;

[0223] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0224] Each R 1 These are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogen;

[0225] R 2 C1-C4 alkyl and -(CH2CH2O) s -Selected from CH3, where s is an integer between 1 and 10;

[0226] R 3 and R 3’ Each is independently selected from hydrogen and C1-C3 alkyl;

[0227] L is a severable linker;

[0228] Ab stands for antibody, antibody fragment, or antigen-binding fragment.

[0229] In some embodiments, the present disclosure is based on formula (IV),

[0230] [ka]

[0231] Provides compounds or pharmaceutically acceptable salts thereof,

[0232] During the ceremony,

[0233] a is an integer between 1 and 20;

[0234] b is an integer between 1 and 20;

[0235] k is 0, 1, 2, or 3.

[0236] m is 0, 1, 2, 3, or 4;

[0237] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0238] R 2 H, C1-C4 alkyl and -(CH2CH2O) s -Selected from CH3, where s is an integer between 1 and 10;

[0239] R 4 These are selected from hydrogen and any naturally occurring amino acid side chains;

[0240] R 5 The alkyl group is selected from C1-C4 alkyl and O-C1-C4 alkyl groups;

[0241] L is a severable linker;

[0242] Ab stands for antibody, antibody fragment, or antigen-binding fragment.

[0243] In some embodiments, the present disclosure is formula (V):

[0244] [ka]

[0245] Provides compounds or pharmaceutically acceptable salts thereof,

[0246] During the ceremony,

[0247] D-NH is

[0248] [ka]

[0249] and;

[0250] X is either O or CH2;

[0251] f is an integer between 1 and 10;

[0252] g is an integer between 1 and 20.

[0253] U and U' do not exist independently, or they are spacers;

[0254] Q is a heterodifunctional group;

[0255] Ab stands for antibody, antibody fragment, or antigen-binding fragment.

[0256] In the compounds of formulas (I) and (IV), the amino-substituted compound D-NH2 can be any amino-containing drug. In certain embodiments, D-NH2 is an immunomodulator. As used herein, the term “immunomodulator” refers to a compound that elicits an immune response in a target that requires it. Examples of immunomodulators include IDO inhibitors, MEK inhibitors, and STING inhibitors. Examples include, but are not limited to, diurators, CCR4 antagonists, PD-1 / PD-L1 inhibitors, and TLR7 / 8 agonists. In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 comprises a guanine derivative or an adenine derivative. In some embodiments, the STING modulator is of the formula:

[0257] [ka]

[0258] These are cyclic dinucleotides or cyclic dinucleotide-like compounds (CDNs, respectively), In the formula, A' and A'' are independently nucleosides or their synthetic analogs; Q, Q', Q 2 , and Q 2’ Each of them is independently oxygen or sulfur. In some embodiments, Q ’ and Q 2’ is sulfur. Examples of CDNs containing cyclic dinucleotides and cyclic dinucleotide-like compounds within the scope of this disclosure include US9,724,408, US10,106,574, US9,549,944, US9,695,212, US9,718,848, US9,994,607, US10,047,115, US20180230178, US20180064745, and US20180281. Examples of compounds such as those disclosed in 32, US20180002369, US20180186828, US20190016750, US20180162899, US20180369268, US2018273578, US20180092937, WO2017 / 027646, and WO2018 / 100558, as well as compounds such as c-di-GMP, 2'3'-cGAMP, and SB 11285 (Spring Bank Pharmaceuticals).

[0259] In a particular embodiment, the STING modulator is given by equation (II):

[0260] [ka]

[0261] A compound of or a pharmaceutically acceptable salt thereof,

[0262] During the ceremony,

[0263] X 10 is -SH or -OH;

[0264] X 20 is -SH or -OH;

[0265] Y a is -O-, -S-, or -CH2-;

[0266] Y b -O-, -S-, -NH-, or -NR a - and in that case R a It is a C1-C4 alkyl group;

[0267] R 10 These are hydrogen, fluoro, -OH, -NH2, -OR b , or -NHR b and;

[0268] R 20 is hydrogen or fluorine;

[0269] R 30 is hydrogen, R 40 These are hydrogen, fluoro, -OH, -NH2, -OR b , or -NHR b is, or R 30 and R 40 They come together to form -CH2O-;

[0270] R 50 is hydrogen or fluorine;

[0271] R bThese are C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl;

[0272] Ring A 10 is an optionally substituted 5-membered or 6-membered monocyclic heteroaryl ring containing 1 to 4 heteroatoms selected from N, O, or S, or an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 1 to 5 heteroatoms selected from N, O, or S, in which case ring A 10 It contains at least one N atom in the ring, Y b is ring A 10 It is bonded to the carbon atom;

[0273] Ring B 10 This is an optionally substituted 9-membered or 10-membered bicyclic heteroaryl ring containing 2 to 5 heteroatoms selected from N, O, or S, in which case ring B 10 It contains at least two N atoms within the ring.

[0274] As described herein, Ring A 10 and ring B 10 Since it can contain one or more substituents, it can be optionally substituted. Preferred substituents on the unsaturated carbon atom of the heteroaryl group include -halo, -NO2, -CN, and -R + , -C(R + )=C(R + )2, -C≡CR + , -OR + , -SR゜, -S(O)R゜, -SO2R゜, -SO3R + , -SO2N(R + )2, -N(R + )2, -NR + C(O)R + , -NR + C(S)R + , -NR + C(O)N(R + )2, -NR + C(S)N(R + )2, -N(R + )C(=NR + )-N(R +)2, -N(R + )C(=NR + )-R°,-NR + CO2R + , -NR + SO2R°, -NR + SO2N(R + )2, -OC(O)R + , -O-CO2R + ,-OC(O)N(R + )2, -C(O)R + -C(S)R°, -CO2R + -C(O)-C(O)R + ,-C(O)N(R + )2, -C(S)N(R + ) 2、 -C(O)N(R + )-OR + ,-C(O)N(R + )C(=NR + )-N(R + )2, -N(R + )C(=NR + )-N(R + )-C(O)R + -C(=NR + )-N(R + )2, -C(=NR + )-OR + , -N(R + )-N(R + )2, -C(=NR + )-N(R + )-OR + -C(R°)=N-OR + ,-P(O)(R + )2, -P(O)(OR + )2, -OP(O)-OR + and -P(O)(NR + )-N(R + )2 is one example, and is generally selected from among them, in which case R + is independently a hydrogen or optionally substituted aliphatic aryl group, heteroaryl group, or heterocyclyl group, or R + These two independent entities, together with the intervening atom(s), form a 5-7 membered aryl ring that can be optionally substituted. It forms a heteroaryl, alicyclic, or heterocycline. In some embodiments, R + Hydrogen and C are independent of each other. 1-6 Aliphatic, or C 3-6 It is an alicyclic compound. Each R° is an independently and optionally substituted aliphatic group, aryl group, heteroaryl group, alicyclic group, or heterocyclyl group.

[0275] As detailed above, in some embodiments, R + Two independent entities of (or any other variable elements similarly defined herein and in the claims) together with the atoms(s) interposed therein, together with the atoms(s) of a 3- to 13-membered alicyclic ring, a 3- to 12-membered heterocycline ring having 1- to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 6- to 10-membered aryl ring, or a 5- to 10-membered heteroaryl ring having 1- to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, form a monocyclic or bicyclic ring.

[0276] In some embodiments, the STING modulator is given by equation (IIA),

[0277] [ka]

[0278] A compound of or a pharmaceutically acceptable salt thereof,

[0279] In the formula, R 10 and R 40は Each is independently hydrogen, fluoro, -OH, or -OCH2CF3, and ring A 10 and B 10 The definition is as defined for the compound of formula (II), except for ring A 10 or ring B 10 The condition is that one of them is bonded to the carbonyl group of the parent molecule of compound (I), (IV), or (V) via an -NH- group.

[0280] In some embodiments, ring A 10 This is an optionally substituted six-membered monocyclic heteroaryl ring containing one, two, or three nitrogen atoms.

[0281] In some embodiments, ring B 10 The following is true:

[0282] [ka]

[0283] During the ceremony:

[0284] Z 10 , Z 20 , Z 30 and Z 40 Each is independently N or CR 200 and;

[0285] R 210 is hydrogen, or C1-C6 alkyl, halo(C1-C6)alkyl, or C It is a 3-C6 cycloalkyl group;

[0286] R 230 is hydrogen or -NH2;

[0287] R 200 , R 220 , and R 240 Each of these is independently hydrogen, halogen, -OH, -NH2, -CN, C1-C6 alkyl, halo(C1-C6)alkyl, or C3-C6 cycloalkyl.

[0288] In some embodiments, the STING modulator is:

[0289] [ka]

[0290] or a pharmaceutically acceptable salt thereof,

[0291] During the ceremony,

[0292] [ka]

[0293] This is the bond point to the carbonyl group of the parent molecule.

[0294] In a particular embodiment, the STING modulator is given by equation (X):

[0295] [ka]

[0296] A cyclic dinucleotide or a pharmaceutically acceptable salt thereof, During the ceremony,

[0297] Formula (A-1):

[0298] [ka]

[0299] The substructure represented by is the substructure represented by equation (ZA), or equation (ZB), i.e.,

[0300] [ka]

[0301] And,

[0302] R 105 and R 205 Each of these is independently a hydroxyl group or a halogen atom;

[0303] B 100 is equation (B 1 -A) or formula (B 1 -B):

[0304] [ka]

[0305] It is a base represented by,

[0306] R 13 , R 14 , R 15 , R 16 , and R 17 Each of these is independently a hydrogen atom or a substituent;

[0307] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 Each is independently N or CR 1a And,

[0308] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 Each is independently either N or C;

[0309] R 1a is a hydrogen atom or substituent;

[0310] B 200 is equation (B 2 -A) or formula (B 2 -B):

[0311] [ka]

[0312] It is a base represented by,

[0313] R 23 , R 24 , R 25 , R 26 , and R 27Each is independently a hydrogen atom or a substituent;

[0314] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each is independently N or CR 2a and;

[0315] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 Each is independently either N or C;

[0316] R 2a is a hydrogen atom or substituent;

[0317] X 1 and X 2 Each of these is independently either an oxygen atom or a sulfur atom;

[0318] Q 1a Q 2a , Q3a, and Q 4a Each of these is independently either an oxygen atom or a sulfur atom;

[0319] B 100 or B 200 One of the components is bonded to the five-membered ring of the parent structure via an -NH- group.

[0320] In another embodiment, B 100 or B 200 One of them is:

[0321] [ka]

[0322] And,

[0323] During the ceremony,

[0324] R 18 is hydrogen or C 1-6 It is alkyl; and the other is bonded to the five-membered ring of the parent structure via an -NH- group;

[0325] R 19 One is a halogen atom; the other is bonded to the carbonyl group of formula (I) via an -NH- group.

[0326] In another embodiment, the present disclosure provides an antibody-drug conjugate as described herein.

[0327] As described herein, a particular R group is selected from hydrogen and substituents. Examples of "substituents" include halogen atoms, cyano groups, nitro groups, optionally substituted hydrocarbon groups, optionally substituted heterocyclic groups, acyl groups, optionally substituted amino groups, optionally substituted carbamoyl groups, optionally substituted thiocarbamoyl groups, optionally substituted sulfamoyl groups, optionally substituted hydroxyl groups, optionally substituted sulfanyl (SH) groups, and optionally substituted silyl groups.

[0328] When used herein, examples of "hydrocarbon group" (including "hydrocarbon group" of "optionally substituted hydrocarbon group") include C 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 2-6 Alkynyl group, C 3-10 Cycloalkyl groups, C 3-10 Cycloalkenyl group, C 6-14 Aryl group and C 7-16 An example is the aralkyl group.

[0329] In this specification, examples of "optionally substituted hydrocarbon groups" include hydrocarbon groups having optionally one substituent selected from the following substituent group A, which include:

[0330] (1) Halogen atom,

[0331] (2) Nitro group,

[0332] (3) Cyano group,

[0333] (4) Oxo group,

[0334] (5) Hydroxyl group,

[0335] (6) C which is optionally halogenated 1-6 Alkoxy group,

[0336] (7)C 6-14 Aryloxy groups (e.g., phenoxy, naphthoxy),

[0337] (8)C 7-16 Aralkyloxy group (e.g., benzyloxy),

[0338] (9) Aromatic heterocyclyloxy groups of 5-14 member rings (e.g., pyridyloxy),

[0339] (10) Non-aromatic heterocyclyloxy groups of 3-14 member rings (e.g., morpholinyloxy, piperidinyloxy),

[0340] (11)C 1-6 Alkyl-carbonyloxy groups (e.g., acetoxy, propanoyloxy),

[0341] (12)C 6-14 Aryl-carbonyloxy groups (e.g., benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy),

[0342] (13)C 1-6 Alkoxy-carbonyloxy groups (e.g., methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy),

[0343] (14) Mono- or di-C 1-6 Alkyl-carbamoyloxy groups (e.g., methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy),

[0344] (15)C 6-14 Aryl-carbamoyloxy groups (e.g., phenylcarbamoyloxy, naphthylcarbamoyloxy),

[0345] (16) Aromatic heterocyclylcarbonyloxy groups of 5-14 member rings (e.g., nicotinoyloxy),

[0346] (17) Non-aromatic heterocyclylcarbonyloxy groups of 3-14 member rings (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy),

[0347] (18) C which is optionally halogenated 1-6 Alkyl sulfonyloxy groups (e.g., methyl sulfonyloxy, trifluoromethyl sulfonyloxy),

[0348] (19)C 1-6 C optionally substituted with an alkyl group 6-14 Aryl sulfonyloxy groups (e.g., phenylsulfonyloxy, toluenesulfonyloxy),

[0349] (20) C which is optionally halogenated 1-6 Alkylthio group,

[0350] (21) Aromatic heterocyclic groups of 5-14 members,

[0351] (22) Non-aromatic heterocyclic groups of 3-14 member rings,

[0352] (23) Formyl group,

[0353] (24) Carboxy group,

[0354] (25) C which is optionally halogenated 1-6 Alkyl-carbonyl group,

[0355] (26)C 6-14 Aryl-carbonyl group,

[0356] (27) Aromatic heterocyclylcarbonyl groups of 5-14 member rings,

[0357] (28) Non-aromatic heterocyclylcarbonyl groups of 3-14 member rings,

[0358] (29)C 1-6 Alkoxy-carbonyl group,

[0359] (30)C 6-14 Aryloxycarbonyl groups (e.g., phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),

[0360] (31)C 7-16 Aralkyloxycarbonyl group (e.g., benzyloxycarbonyl, phenethyloxycarbonyl),

[0361] (32) Carbamoyl group,

[0362] (33) Thiocarbamoyl group,

[0363] (34) Mono- or di-C 1-6 Alkyl-carbamoyl group,

[0364] (35)C 6-14 Aryl-carbamoyl group (e.g., phenylcarbamoyl),

[0365] (36) Aromatic heterocyclylcarbamoyl groups of 5-14 member rings (e.g., pyridylcarbamoyl, thienylcarbamoyl),

[0366] (37) Non-aromatic heterocyclylcarbamoyl groups of 3-14 member rings (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),

[0367] (38) C which is optionally halogenated 1-6 Alkyl sulfonyl group,

[0368] (39)C 6-14Aryl sulfonyl group,

[0369] (40) Aromatic heterocyclylsulfonyl groups of 5-14 member rings (e.g., pyridylsulfonyl, thienylsulfonyl),

[0370] (41) C which is optionally halogenated 1-6 Alkyl sulfinyl group,

[0371] (42)C 6-14 Aryl sulfinyl groups (e.g., phenyl sulfinyl, 1-naphthyl sulfinyl, 2-naphthyl sulfinyl),

[0372] (43) Aromatic heterocyclylsulfinyl groups of 5-14 member rings (e.g., pyridylsulfinyl, thienylsulfinyl),

[0373] (44) Amino group,

[0374] (45) Mono- or di-C 1-6 Alkylamino groups (e.g., methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, N-ethyl-N-methylamino),

[0375] (46) Mono- or di-C 6-14 Arylamino group (e.g., phenylamino),

[0376] (47) Aromatic heterocyclylamino groups of 5-14 member rings (e.g., pyridylamino),

[0377] (48)C 7-16 Aralkylamino group (e.g., benzylamino),

[0378] (49) Formylamino group,

[0379] (50)C 1-6Alkyl-carbonylamino groups (e.g., acetylamino, propanoylamino, butanoylamino),

[0380] (51)(C 1-6 Alkyl)(C 1-6 Alkyl-carbonyl)amino group (e.g., N-acetyl-N-methylamino),

[0381] (52)C 6-14 Aryl-carbonylamino groups (e.g., phenylcarbonylamino, naphthylcarbonylamino),

[0382] (53)C 1-6 Alkoxycarbonylamino groups (e.g., methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino),

[0383] (54)C 7-16 Aralkyloxycarbonylamino group (e.g., benzyloxycarbonylamino),

[0384] (55)C 1-6 Alkylsulfonylamino group (e.g., methylsulfonylamino, ethylsulfonylamino),

[0385] (56)C 1-6 C optionally substituted with an alkyl group 6-14 Aryl sulfonylamino group (e.g., phenylsulfonylamino, toluenesulfonylamino),

[0386] (57) C which can be optionally halogenated 1-6 alkyl group,

[0387] (58)C 2-6 Alkenyl group,

[0388] (59)C 2-6 Alkynyl group,

[0389] (60)C 3-10Cycloalkyl groups,

[0390] (61) C 3-10 Cycloalkenyl group, and

[0391] (62)C 6-14 An example is the aryl group.

[0392] The number of substituents in the "optionally substituted hydrocarbon group" is, for example, 1 to 5, usually 1 to 3. If there are two or more substituents, each substituent may be the same or different.

[0393] In this specification, examples of "heterocyclic groups" (including "heterocyclic groups" in "optionally substituted heterocyclic groups") include (i) aromatic heterocyclic groups, (ii) non-aromatic heterocyclic groups, and (iii) 7- to 10-membered bridging heterocyclic groups, each containing 1 to 4 heteroatoms selected from nitrogen, sulfur, and oxygen atoms as ring constituent atoms other than carbon atoms.

[0394] In this specification, examples of "aromatic heterocyclic groups" (including "aromatic heterocyclic groups of 5 to 14 members") include aromatic heterocyclic groups of 5 to 14 members (usually 5 to 10 members) that contain 1 to 4 heteroatoms selected from nitrogen, sulfur, and oxygen atoms as ring constituent atoms other than carbon atoms.

[0395] Suitable examples of the “aromatic heterocyclic group” include monocyclic aromatic heterocyclic groups of 5 or 6 members such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, and triazinyl; and

[0396] Benzothiophenyl, benzofuranil, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, phlopyridinyl, pyrrolopyridinyl, pyraz Examples include 8-14 member condensed polycyclic (usually bicyclic or tricyclic) aromatic heterocyclic groups such as lopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, imidazopyrimidinyl, thienopyrimidinyl, phlopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, sinnolinyl, carbazolyl, β-carbolinyl, phenanthiinyl, acridinyl, phenadinyl, phenothiazinyl, and phenosadinyl.

[0397] In this specification, examples of "non-aromatic heterocyclic groups" (including "non-aromatic heterocyclic groups of 3 to 14 members") include non-aromatic heterocyclic groups of 3 to 14 members (usually 4 to 10 members) that contain 1 to 4 heteroatoms selected from nitrogen, sulfur, and oxygen atoms as ring constituent atoms other than carbon atoms.

[0398] Suitable examples of the "non-aromatic heterocyclic group" include azilidinyl, oxyranil, thyranil, azetidinil, oxetanil, thietanil, tetrahydrothienyl, tetrahydrofuranil, pyrrolinil, pyrrolidinyl, imidazolinil, imidazolidinyl, oxazolinil, oxazolidinil, pyrazolinil, pyrazolidinil, thiazolinil, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydro monocyclic non-aromatic heterocyclic groups of 3 to 8 members, such as sooxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranil, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl, and diazocanyl; and

[0399] Dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzoisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolidinyl, indolinyl, isoindolinyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenantridinyl, hexa Examples include 9- to 14-membered condensed polycyclic (usually bicyclic or tricyclic) non-aromatic heterocyclic groups such as hydrophenothiazinyl, hexahydrophenoxadinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrosinnolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl, and octahydroisoquinolyl.

[0400] In this specification, preferred examples of “7- to 10-membered ring crosslinked heterocyclic groups” include quinuclidinyl and 7-azabicyclo[2.2.1]heptanyl.

[0401] In this specification, examples of "nitrogen-containing heterocyclic groups" include "heterocyclic groups" that contain at least one nitrogen atom as a ring constituent atom.

[0402] In this specification, examples of "optionally substituted heterocyclic groups" include heterocyclic groups having any substituent(s) selected from the substituent group A described above.

[0403] The number of substituents in the "optionally substituted heterocyclic group" is, for example, 1 to 3. If there are two or more substituents, each substituent may be the same or different.

[0404] In this specification, examples of "acyl groups" include formyl groups, carboxyl groups, carbamoyl groups, thiocarbamoyl groups, sulfino groups, sulfo groups, sulfamoyl groups, and phosphono groups, each of which is optional. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 3-10 Cycloalkenyl group, C 6-14 Aryl group, C 7-16 The group has an aralkyl group, one or two substituents selected from a 5-14 membered aromatic heterocyclic group and a 3-14 membered non-aromatic heterocyclic group, each of which is a halogen atom, optionally halogenated C 1-6 It optionally has 1 to 3 substituents selected from alkoxy, hydroxyl, nitro, cyano, amino, and carbamoyl groups.

[0405] Other examples of "acyl groups" include hydrocarbon-sulfonyl groups, heterocyclylsulfonyl groups, hydrocarbon-sulfinyl groups, and heterocyclylsulfinyl groups.

[0406] Here, a hydrocarbon-sulfonyl group refers to a sulfonyl group to which a hydrocarbon group is attached, a heterocyclylsulfonyl group refers to a sulfonyl group to which a heterocyclic group is attached, a hydrocarbon-sulfinyl group refers to a sulfinyl group to which a hydrocarbon group is attached, and a heterocyclylsulfinyl group refers to a sulfinyl group to which a heterocyclic group is attached.

[0407] Suitable examples of "acyl groups" include formyl groups, carboxyl groups, and C 1-6 Alkyl-carbonyl group, C 2-6 Alkenyl-carbonyl group (e.g., crotonoyl), C 3-10 Cycloalkyl-carbonyl group (e.g., cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), C 3-10 Cycloalkenyl-carbonyl group (e.g., 2-cyclohexenecarbonyl), C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, C 6-14 Aryloxycarbonyl group (e.g., phenyloxycarbonyl, naphthyloxycarbonyl), C 7-16 Aralkyloxycarbonyl groups (e.g., benzyloxycarbonyl, phenethyloxycarbonyl), carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 2-6 Alkenyl-carbamoyl group (e.g., diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl group (e.g., cyclopropylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl group (e.g., phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl group, 5-14 membered aromatic heterocyclylcarbamoyl group (e.g., pyridylcarbamoyl), thiocarbamoyl group, mono- or di-C 1-6 Alkyl-thiocarbamoyl groups (e.g., methylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), mono- or di-C 2-6 Alkenyl-thiocarbamoyl group (e.g., diallylthiocarbamoyl), mono- or di-C 3-10Cycloalkyl-thiocarbamoyl groups (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14 Aryl-thiocarbamoyl group (e.g., phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl groups (e.g., benzylthiocarbamoyl, phenethylthiocarbamoyl), 5-14 membered aromatic heterocyclylthiocarbamoyl groups (e.g., pyridylthiocarbamoyl), sulfino groups, C 1-6 Alkyl sulfinyl group (e.g., methyl sulfinyl, ethyl sulfinyl), sulfo group, C 1-6 Alkyl sulfonyl group, C 6-14 Aryl sulfonyl groups, phosphono groups, and mono- or di-C groups 1~6 Examples include alkylphosphono groups (e.g., dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

[0408] In this specification, examples of "optionally substituted amino groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C 7-16 Aralkyl group, C1 -6 Alkyl-carbonyl group, C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl groups, 5-14 membered aromatic heterocyclic groups, carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 7-16 Aralkyl-carbamoyl group, C 1-6 Alkyl sulfonyl group and C 6-14 Examples include amino groups having one or two substituents selected from arylsulfonyl groups.

[0409] Preferred examples of optionally substituted amino groups include amino groups, mono- or di-(optionally halogenated C) 1-6 Alkyl)amino group (e.g., methylamino, trifluoromethylamino, dimethylamino, ethylamino, diethylamino, propylamino, dibutylamino), mono- or di-C 2-6 Alkenylamino group (e.g., diallylamino), mono- or di-C 3-10 Cycloalkylamino groups (e.g., cyclopropylamino, cyclohexylamino), mono- or di-C 6-14 Arylamino group (e.g., phenylamino), mono- or di-C 7-16 Aralkylamino group (e.g., benzylamino, dibenzylamino), mono- or di-(optionally halogenated C) 1-6 Alkyl)-carbonylamino group (e.g., acetylamino, propionylamino), mono- or di-C 6-14 Aryl-carbonylamino group (e.g., benzoylamino), mono- or di-C 7-16 Aalkyl-carbonylamino groups (e.g., benzylcarbonylamino), mono- or di-5- to 14-membered aromatic heterocyclylcarbonylamino groups (e.g., nicotinoylamino, isonicotinoylamino), mono- or di-3- to 14-membered non-aromatic heterocyclylcarbonylamino groups (e.g., piperidinylcarbonylamino), mono- or di-C 1-6 Alkoxy-carbonylamino groups (e.g., tert-butoxycarbonylamino), 5-14 membered aromatic heterocyclylamino groups (e.g., pyridylamino), carbamoylamino groups, (mono- or di-C) 1-6 Alkyl-carbamoyl)amino group (e.g., methylcarbamoylamino), (mono- or di-C) 7-16 Aalkyl-carbamoyl)amino group (e.g., benzylcarbamoylamino), C 1-6 Alkylsulfonylamino group (e.g., methylsulfonylamino, ethylsulfonylamino), C 6-14 Aryl sulfonylamino group (e.g., phenylsulfonylamino), (C 1-6 Alkyl)(C1-6 Alkyl-carbonyl)amino group (e.g., N-acetyl-N-methylamino) and (C 1-6 Alkyl)(C 6-14 Examples include aryl-carbonyl)amino groups (for example, N-benzoyl-N-methylamino).

[0410] In this specification, examples of "optionally substituted carbamoyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C 7-16 Aralkyl group, C 1-6 Alkyl-carbonyl group, C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl groups, 5-14 membered aromatic heterocyclic groups, carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl groups and mono- or di-C 7-16 Examples include a carbamoyl group having one or two substituents selected from the aralkyl-carbamoyl group.

[0411] Preferred examples of optionally substituted carbamoyl groups include carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 2-6 Alkenyl-carbamoyl group (e.g., diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl groups (e.g., cyclopropylcarbamoyl, cyclohexylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl group (e.g., phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl group, mono- or di-C 1-6 Alkyl-carbonyl-carbamoyl group (e.g., acetylcarbamoyl, propio Nilcarbamoyl), mono- or di-C 6-14 Examples include aryl-carbonyl-carbamoyl groups (e.g., benzoylcarbamoyl) and aromatic heterocyclylcarbamoyl groups with 5 to 14 member rings (e.g., pyridylcarbamoyl).

[0412] In this specification, examples of "optionally substituted thiocarbamoyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C 7-16 Aralkyl group, C 1-6 Alkyl-carbonyl group, C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl groups, 5-14 membered aromatic heterocyclic groups, carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl groups and mono- or di-C 7-16 Examples include thiocarbamoyl groups having one or two substituents selected from aralkyl-carbamoyl groups.

[0413] Preferred examples of optionally substituted thiocarbamoyl groups include thiocarbamoyl groups, mono- or di-C 1-6 Alkyl-thiocarbamoyl groups (e.g., methylthiocarbamoyl, ethylthiocarbamoyl, dimethylthiocarbamoyl, diethylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), mono- or di-C 2-6 Alkenyl-thiocarbamoyl group (e.g., diallylthiocarbamoyl), mono- or di-C 3-10 Cycloalkyl-thiocarbamoyl groups (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14Aryl-thiocarbamoyl group (e.g., phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl group (e.g., benzylthiocarbamoyl, phenethylthiocarbamoyl), mono- or di-C 1-6 Alkyl-carbonyl-thiocarbamoyl groups (e.g., acetylthiocarbamoyl, propionylthiocarbamoyl), mono- or di-C 6-14 Examples include aryl-carbonyl-thiocarbamoyl groups (e.g., benzoylthiocarbamoyl) and aromatic heterocyclylthiocarbamoyl groups with 5 to 14 member rings (e.g., pyridylthiocarbamoyl).

[0414] In this specification, examples of "optionally substituted sulfamoyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C 7-16 Aralkyl group, C 1-6 Alkyl-carbonyl group, C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl groups, 5-14 membered aromatic heterocyclic groups, carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl groups and mono- or di-C 7-16 Examples include sulfamoyl groups having one or two substituents selected from aralkyl-carbamoyl groups.

[0415] Suitable examples of optionally substituted sulfamoyl groups include sulfamoyl groups, mono- or di-C 1-6 Alkyl-sulfamoyl groups (e.g., methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, N-ethyl-N-methylsulfamoyl), mono- or di-C 2-6Alkenyl sulfamoyl group (e.g., diallyl sulfamoyl), mono- or di-C 3-10 Cycloalkyl-sulfamoyl groups (e.g., cyclopropylsulfamoyl, cyclohexylsulfamoyl), mono- or di-C 6-14 Aryl-sulfamoyl group (e.g., phenylsulfamoyl), mono- or di-C 7-16 Aralkyl sulfamoyl groups (e.g., benzyl sulfamoyl, phenethyl sulfamoyl), mono- or di-C 1-6 Alkyl-carbonyl-sulfamoyl groups (e.g., acetylsulfamoyl, propionylsulfamoyl), mono- or di-C 6-14 Aryl-carbonyl-sulfamoyl group (for example) Examples include benzoyl sulfamoyl and aromatic heterocyclyl sulfamoyl groups of 5-14 member rings (e.g., pyridyl sulfamoyl).

[0416] In this specification, examples of "optionally substituted hydroxyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C 7-16 Aralkyl group, C 1-6 Alkyl-carbonyl group, C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5-14 membered aromatic heterocyclylcarbonyl group, 3-14 membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl groups, 5-14 membered aromatic heterocyclic groups, carbamoyl groups, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 7-16 Aralkyl-carbamoyl group, C 1-6 Alkyl sulfonyl group and C 6-14 Examples include hydroxyl groups having substituents selected from arylsulfonyl groups.

[0417] Preferred examples of optionally substituted hydroxyl groups include hydroxyl groups and C 1-6 Alkoxy group, C 2-6 Alkenyloxy groups (e.g., allyloxy, 2-butenyloxy, 2-pentenyloxy, 3-hexenyloxy), C 3-10 Cycloalkyloxy group (e.g., cyclohexyloxy), C 6-14 Aryloxy group (e.g., phenoxy, naphthyloxy), C 7-16 Aralkyloxy group (e.g., benzyloxy, phenethyloxy), C 1-6 Alkyl-carbonyloxy groups (e.g., acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pivaloyloxy), C 6-14 Aryl-carbonyloxy group (e.g., benzoyloxy), C 7-16 Aalkyl-carbonyloxy group (e.g., benzylcarbonyloxy), 5-14 membered aromatic heterocyclylcarbonyloxy group (e.g., nicotinoyloxy), 3-14 membered non-aromatic heterocyclylcarbonyloxy group (e.g., piperidinylcarbonyloxy), C 1-6 Alkoxy-carbonyloxy groups (e.g., tert-butoxycarbonyloxy), 5-14 membered aromatic heterocyclyloxy groups (e.g., pyridyloxy), carbamoyloxy groups, C 1-6 Alkyl-carbamoyloxy group (e.g., methylcarbamoyloxy), C 7-16 Aralkyl-carbamoyloxy group (e.g., benzylcarbamoyloxy), C 1-6 Alkyl sulfonyloxy groups (e.g., methyl sulfonyloxy, ethyl sulfonyloxy) and C 6-14 Examples include aryl sulfonyloxy groups (e.g., phenylsulfonyloxy).

[0418] In this specification, examples of "optionally substituted sulfanyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group, C7-16 Aralkyl group, C 1-6 Alkyl-carbonyl group, C 6-14 Examples include sulfanyl groups and halogenated sulfanyl groups having substituents optionally selected from aryl-carbonyl groups and aromatic heterocyclic groups of 5 to 14 members.

[0419] Preferred examples of optionally substituted sulfanyl groups include sulfanyl(-SH) groups and C 1-6 Alkylthio group, C 2-6 Alkenylthio groups (e.g., allylthio, 2-butenylthio, 2-pentenylthio, 3-hexenylthio), C 3-10 Cycloalkylthio group (e.g., cyclohexylthio), C 6-14 Arylthio group (e.g., phenylthio, naphthylthio), C 7-16 Aralkylthio group (e.g., benzylthio, phenethylthio), C 1-6 Alkyl-carbonylthio group (e.g., acetylthio, propionylthio, butyrylthio, isobutylylthio, pivaloylthio), C 6-14 Examples include aryl-carbonylthio groups (e.g., benzoylthio), aromatic heterocyclylthio groups with 5 to 14 member rings (e.g., pyridylthio), and halogenated thio groups (e.g., pentafluorothio).

[0420] In this specification, examples of "optionally substituted silyl groups" include C, each having 1 to 3 substituents selected from substituent group A. 1-6 Alkyl alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl group and C 7~16 Examples include silyl groups having 1 to 3 substituents selected from aralkyl groups. An example of a optionally substituted silyl group is a tri-C group. 1-6 Examples include alkylsilyl groups (e.g., trimethylsilyl, tert-butyl(dimethyl)silyl).

[0421] In some embodiments, the STING modulator is given by equation (III):

[0422] [ka]

[0423] A compound of or a pharmaceutically acceptable salt thereof,

[0424] During the ceremony,

[0425] R 105 and R 205 Each of these is independently a hydroxyl group or a halogen atom;

[0426] B 100 is equation (B 1 -A) or formula (B 1 The base represented by -B);

[0427] [ka]

[0428] R 13 , R 14 , R 15 , R 16 , and R 17 Each is independently a hydrogen atom or a substituent;

[0429] R 1000 is a hydrogen atom, or a bond to the carbonyl group of formula (I);

[0430] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 Each is independently N or CR 1a and;

[0431] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16Each is independently either N or C;

[0432] R 105 and R 205 Each of these is independently a hydroxyl group or a halogen atom;

[0433] R 1a is a hydrogen atom or substituent;

[0434] B 200 is, equation (B 2 -A) or formula (B 2 The base represented by -B) is:

[0435] [ka]

[0436] R 23 , R 24 , R 25 , R 26 , and R 27 Each is independently a hydrogen atom or a substituent;

[0437] R 100’ is a bond to hydrogen or the carbonyl group of formula (I);

[0438] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 Each is independently N or CR 2a and;

[0439] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 Each is independently either N or C;

[0440] R 2a is a hydrogen atom or substituent;

[0441] X 1 and X 2 Each of these is independently either an oxygen atom or a sulfur atom;

[0442] Q 1 Q 2 , Q3, and Q 4 Each is independently a hydrogen atom or a sulfur atom;

[0443] however,

[0444] B 100 or B 200 One of them is

[0445] [ka]

[0446] And,

[0447] During the ceremony,

[0448] R 18 is hydrogen or C 1-6 It is alkyl;

[0449] R 19 is a halogen atom, and B 100 or B 200 The other side is bound to the five-membered ring of the parent structure via an -NH- group.

[0450] In some embodiments, the STING modulator is a compound of formula (III), or a pharmaceutically acceptable salt thereof, where R 205 F is B 100 teeth

[0451] [ka]

[0452] That is the case.

[0453] In some embodiments, the cyclic dinucleotide is:

[0454] [ka]

[0455] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0456] [ka]

[0457] This is the bond point to the carbonyl group of the parent molecule.

[0458] The "L" group is a cleavable linker. As used herein, the term "linker" refers to any chemical moiety that can connect an antibody, antibody fragment, or antigen-binding fragment (Ab) to a drug-containing moiety in the compounds of formulas (I) and (IV). The linker can be branched and can be substituted with 1 to 20 drug-containing moieties. In some embodiments, the linker can be substituted with 1 to 10 drug-containing moieties. In some embodiments, the linker can be substituted with 1 to 5 drug-containing moieties. In some embodiments, the linker can be substituted with 1 or 2 drug-containing moieties. In some embodiments, the linker can be substituted with 1 drug-containing moiety.

[0459] The linker "L" is cleavable. In certain embodiments, the linker may be susceptible to acid-induced cleavage, photo-induced cleavage, enzymatic cleavage, etc., under conditions in which the drug and / or antibody can maintain its active state. In some embodiments, the cleavable linker can be enzymatically cleaved. In some embodiments, the cleavable linker can be cleaved by a protease, peptidase, esterase, glycosidase, phosphodiesterase, phosphatase, or lipase. In some embodiments The cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B and VAGP tetrapeptide. In certain embodiments, the linker may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated into the whole therein by reference.

[0460] In a particular embodiment, "L" is the formula:

[0461] [ka]

[0462] It has,

[0463] During the ceremony:

[0464] [ka]

[0465] This is the bonding point to the nitrogen atom;

[0466] [ka]

[0467] This is the connection point to Ab.

[0468] In some embodiments, "L" is the formula:

[0469] [ka]

[0470] It has,

[0471] During the ceremony:

[0472] [ka]

[0473] This is the bonding point to the nitrogen atom;

[0474] [ka]

[0475] This is the binding site for the antibody.

[0476] The "W" group is either absent or a self-sacrificing group. As used herein, the term "self-sacrificing" refers to a group that undergoes an electron cascade resulting in the release of the group to which it is bonded. In some embodiments, the self-sacrificing group includes one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cyclization-elimination, 1,5-cyclization-elimination, 1,3-cyclization-elimination, intramolecular 5-exo-trig cyclization, and / or 6-exo-trig cyclization. In certain embodiments, the self-sacrificing group may be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated in whole by reference.

[0477] The "Z" group is absent or the peptide consists of 2-5 amino acids. In certain embodiments, the peptide is the linker cleavage site, thereby promoting drug release upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003), Nat. Biotechnol. 21:778-784). Examples of peptides with two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolynise (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides with three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The above amino acid combinations can also exist in the reverse order (i.e., cit-val).

[0478] The peptides of this disclosure may contain amino acid residues that are naturally occurring and / or not naturally occurring. The term “naturally occurring amino acids” refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. “Non-natural amino acids” (i.e., amino acids that are not naturally occurring) include, without limitation, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalene, ornithine ("Orn"), and others. The peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0479] Amino acids include both natural and unnatural D-form amino acids. "D-" indicates an amino acid with a "D" (dextrorotatory) configuration, as opposed to the configuration found in naturally occurring ("L-") amino acids. Natural and unnatural amino acids can be commercially purchased (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0480] The "U" and "U'" groups are either absent independently or are spacers. As used herein, the term "spacer" refers to a chemical moiety that functions as a connecting group. In this disclosure, a spacer can connect an antibody, antibody fragment, or antigen fragment to a heterodifunctional group, and / or connect a heterodifunctional group to a peptide "Z", or, if "Z" is absent, to a "W" group. Non-limiting exemplary spacers include: -NH-, -S-, -O-, -NHC(=O)CH2CH2-, -S(=O)2-CH2CH2-, -C(=O)NHNH-, -C(=O)O-, -C(=O )NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2=CH2-, -C≡C-, -CH=NO-, polyethylene glycol (PEG),

[0481] [ka]

[0482] In the compounds of this disclosure, if "U" is present, it may be a branched group substituted with 1 to 10 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 to 5 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 or 2 "-C(O)-WZ-" groups. In some embodiments, "U" is substituted with 1 "-C(O)-WZ-" group. In certain embodiments, the spacer may be one of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated in their entirety by reference.

[0483] The "Q" group is a heterodifunctional group. In this disclosure, the term "heterodifunctional group" refers to a chemical moiety that connects the linker of which it is part to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO2017 / 191579. A heterodifunctional group is characterized by having different reactive groups at both ends of the chemical moiety. The heterodifunctional group may be directly bonded to "Ab" or, instead, connected via the linker "U'". Bonding to "Ab" can be achieved via chemical or enzymatic bonding, or a combination of both. Chemical bonding involves a controlled reaction with a reaction handle at "Q" or "U'" of an accessible amino acid residue on the surface of the antibody. Examples of chemical bonding include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via a non-natural amino acid incorporated by genetic engineering, in which a non-natural amino acid residue with a desired reaction handle is introduced to "Ab". In enzymatic bonding, an enzyme intervenes in the coupling of the linker with an accessible amino acid residue on the antibody, antibody fragment, or antigen-binding fragment. Examples of enzymatic linkages include, but are not limited to, peptide transfers using saltase, peptide transfers using microbial transglutaminase, and N-glycan manipulations. Chemical and enzymatic linkages can also be used sequentially. For example, enzymatic linkages can also be used to introduce a unique reaction handle to "Ab" which is used in subsequent chemical linkages. In certain embodiments, heterodifunctional groups are incorporated as a whole by reference (PCT Publication WO2018 / 20081). 2. It may be any of those disclosed in WO2018 / 100558.

[0484] In some embodiments, "Q" is

[0485] [ka]

[0486] Selected from,

[0487] During the ceremony,

[0488] [ka]

[0489] It is a connection point to U, and if U does not exist, it is a connection point to Z;

[0490] [ka]

[0491] This is a connection point to U', and if U' does not exist, it is a connection point to Ab.

[0492] In some embodiments, the present disclosure is derived from formula (XX):

[0493] [ka]

[0494] Provide compounds of or pharmaceutically acceptable salts thereof; In the formula, n, m, a, t, D-NH-, R 1 , R 2 , R 3 , R 3’ W, Z, and U are as described herein, and Q * Q is a reactive functional group that can bind to an antibody, antibody fragment, or antigen-binding fragment. *Examples of groups include, but are not limited to, activated carboxylic acid groups such as acid chlorides-C(O)-Cl and acid anhydrides, haloacetamides, maleimides, alkynes, cycloalkynes, such as cyclooctin, oxanoboradienes, norbornene, azides, diaryltetrazine, monoaryltetrazine, aldehydes, ketones, hydroxylamines, vinyl sulfones, and aziridines. In certain embodiments, the reactive functional group may be any of those disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, which are incorporated in their entirety by reference.

[0495] The "Ab" group is an antibody, antibody fragment, or antigen-binding fragment. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. Target antigens generally have numerous binding sites, also called epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have one corresponding antibody. The term "antibody" as used herein is used in its broadest sense and specifically includes monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity. Antibodies may be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001), Immuno Biology, 5th Ed., Garland Publishing, New York)

[0496] When used herein, the term “antibody” also refers to a full-length immunoglobulin molecule, or a molecule containing an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immune-specifically binds to the antigen or a portion of the target of interest, such targets include, but are not limited to, cancer cells (one or more) that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. The immunoglobulins may originate from any species. However, in one embodiment, the immunoglobulins may be of human, mouse, or rabbit origin.

[0497] The term "single-domain antibody," also known as a nanobody, refers to an antibody fragment consisting of a single monomeric variable antibody domain with a molecular weight of approximately 12 kDa to 15 kDa. Single antibodies can be based on either a heavy-chain variable domain or a light-chain. An example of a single-domain antibody is V H H fragment and V NAR Examples include, but are not limited to, Harmsen MM et al. Applied Microbiology and Biotechnology, 77(1):13-22.

[0498] An "antibody fragment" is a portion of an intact antibody, generally including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab'), sub.2, and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity-determining regions), and epitope-binding fragments, single-chain antibody molecules, and any of the above that bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens; as well as multispecific antibodies formed from antibody fragments.

[0499] An "intact antibody" contains a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3, similar to the antigen-binding variable region. The constant domains may be the constant domains of the native sequence (e.g., the constant domains of the human native sequence) or amino acid sequence variants thereof.

[0500] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain various antibodies directed against various determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with this disclosure may be produced by the hybridoma method first described by Kohler et al, (1975), Nature, 256:495, or by the recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol.Biol.222:581-597 (1991).

[0501] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies in which, to the extent that they exhibit the desired biological activity, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (U.S. Patent No. 4,816,567 and Morrison). et al, (1984), Proc. Natl. Acad. Sci. USA, 81:6851-6855). In this specification, the chimeric antibodies in question include "primatized" antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.) and a human constant region sequence.

[0502] Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to cloned cell lines that produce a single type of antibody, uses cells from various species, including mice, hamsters, rats, and humans. Another method for preparing MAbs uses genetic engineering, including recombinant DNA technology. Monoclonal antibodies produced from these technologies include, among others, chimeric antibodies and humanized antibodies. Chimeric antibodies combine DNA coding regions from one or more different species. For example, a chimeric antibody may have a variable region derived from mouse and a constant region derived from human. Humanized antibodies contain non-human portions but are primarily human-derived. Like chimeric antibodies, humanized antibodies may contain an entirely human constant region. However, unlike chimeric antibodies, the variable region may be partially human-derived. The non-human synthetic portion of humanized antibodies is often derived from the CDR of the mouse antibody. In any case, these regions are important for the antibody to recognize and bind to a specific antigen. While useful for diagnosis and short-term treatment, mouse antibodies cannot be administered to humans long-term without increasing the risk of adverse immunogenic reactions. This reaction, called human anti-mouse antibody (HAMA), occurs when the human immune system recognizes mouse antibodies as foreign substances and attacks them. The HAMA reaction can lead to toxic shock and even death.

[0503] Chimeric and humanized antibodies reduce the likelihood of HAMA reactions by minimizing the non-human portion of the administered antibody. Furthermore, chimeric and humanized antibodies may have the additional advantage of activating secondary human immune responses, such as antibody-dependent cell-mediated cytotoxicity.

[0504] Intact antibodies may have one or more "effector functions," which refer to biological activities that may be caused by the antibody's Fc region (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors; BCRs).

[0505] Intact antibodies can be assigned to various "classes" depending on the amino acid sequence of the heavy chain constant domain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0506] Useful non-immune-reactive proteins, polypeptides, or peptide antibodies include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF") such as TGF-alpha and TGF-beta, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, lectins, and apolipoproteins derived from low-density lipoproteins.

[0507] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the serum of immunized animals. Various procedures known in the art may be used to produce polyclonal antibodies against a target antigen. For example, to produce polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injection with the target antigen or a derivative thereof. Various adjuvants, including but not limited to Freund's (complete and incomplete) adjuvants, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and pluronic polyols, polyanions, peptides, oil emulsions, water oyster hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum, may be used to enhance the immune response depending on the host species. Such adjuvants are also well known in the art.

[0508] Useful monoclonal antibodies are a homogeneous population of antibodies against specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be prepared by using any technique known in the art that provides the production of antibody molecules by serially passaged cell lines in culture. These include the hybridoma technique first described by Kohler and Milstein (1975, Nature, 256, 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Lis). Examples include, but are not limited to, s, Inc., pp. 77-96. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas that produce the mAbs used in this disclosure may be cultured in vitro or in vivo.

[0509] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be produced by any of the many techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today, 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

[0510] Antibodies can also be bispecific antibodies. Methods for producing bispecific antibodies are known in the field. Conventional production of full-length bispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy-light chain pairs, in which these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed using affinity chromatography, is rather cumbersome and results in low product yields. Similar procedures are described in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0511] According to different approaches, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion may be with an immunoglobulin heavy chain constant domain including at least a portion of the hinge region, CH2 region, and CH3 region. The first heavy chain constant region (CH1) may contain a site necessary for light chain binding, which is present in at least one of the fusions. The immunoglobulin heavy chain fusion, and optionally a nucleic acid having a sequence encoding an immunoglobulin light chain, is inserted into a separate expression vector and simultaneously translocated into a suitable host organism. This provides excellent flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where an unequal ratio of the three polypeptide chains used in construction yields the optimal yield. However, if a high yield is obtained by expressing at least two polypeptide chains in equal ratios, or if their ratios are not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0512] A bispecific antibody may have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure allows for easy separation because immunoglobulin light chains are present on only half of the bispecific molecules, thus facilitating the separation of desired bispecific compounds from unwanted immunoglobulin chain combinations (WO94 / 04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology, 151:6954-6961; Carter et al., 1992, Bio / Technology, 10:163-167; Carter et al., 1995, J. of Hematotherapy, 4:463-470; Merchant et al., 1998, Nature Biotechnology, 16:677-681). Using such techniques, as defined herein Bispecific antibodies can be prepared for conjugation as ADCs in the treatment or prevention of diseases such as those mentioned above.

[0513] Hybrid antibodies or bifunctional antibodies can be derived biologically, i.e., by cell fusion techniques, or chemically, particularly by crosslinking agents or disulfide crosslinking reagents, and may include whole antibodies or fragments thereof (EP105360;WO83 / 03679;EP217577).

[0514] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens, or to other antibodies bound to tumor cells or the matrix. In this sense, "functionally active" means that the fragment, derivative, or analog can induce an anti-idiotype antibody that recognizes the same antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary embodiments, the antigenicity of an idiotype of an immunoglobulin molecule can be enhanced by a framework that is the C-terminus of a CDR sequence that specifically recognizes the antigen, and by deletion of the CDR sequence. To determine which CDR sequence binds to the antigen, a synthetic peptide containing a CDR sequence can be used in a binding assay with the antigen by any binding assay method known in the art (e.g., BIA core assay) (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. of Immunology 125(3):961-969).

[0515] Other useful antibody fragments include, but are not limited to, the F(ab')2 fragment, which contains a variable region, a light chain constant region, and the CH1 domain of the heavy chain and can be produced by pepsin digestion of the antibody molecule, and the Fab fragment, which can be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. Other useful antibodies are the heavy and light chain dimers of the antibody, or any minimal fragment thereof such as Fv or single-chain antibodies (SCA) (e.g., as described in U.S. Patent No. 4,946,778; Bird, 1988, Science, 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; and Ward et al., (1989), Nature, 334:544-54), or any other molecule with the same specificity as the antibody.

[0516] Furthermore, recombinant antibodies such as chimeric monoclonal antibodies and humanized monoclonal antibodies, which contain both human and non-human portions and can be produced using standard recombinant DNA technology, are useful antibodies. Chimeric antibodies are molecules in which different portions originate from different animal species, such as those having a variable region derived from mouse monoclonal and a human immunoglobulin constant region. (See, for example, Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397). Humanized antibodies are non-human antibody molecules that have one or more complementarity-determining regions (CDRs) derived from non-human species and a framework region derived from a human immunoglobulin molecule. (See, for example, Queen, U.S. Patent No. 5,585,089) Such chimeric monoclonal antibodies and humanized monoclonal antibodies are, for example, WO87 / 02671;EP184,187;EP171496;EP173494;WO86 / 01533;U.S. Patent No. 4,816,567;EP12023;Berter et al., 1988, Science, 240:1041-1043;Liu et al., 1987, Proc. Natl. Acad. Sci. USA, 84:3439-3443;Liu et al., 1987, J. Immunol. 139:3521-3526;Sun et al., 1987, Proc. Natl. Acad. Sci. USA, 84:214-218; Nishimura et al., 1987, Cancer Res. 47: 999-1005; Wood et al., 1985, Nature, 314: 446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559;Morrison,1985,Science,229:1202-1207;Oi et al.,1986,BioTechniques,4:214;US Patent No. 5,225,539;Jones et al.,1986,Nature,321:552-525;Verhoeyan et al. al. (1988), Science, 239:1534; and Beidler et al. It can be produced using recombinant DNA techniques known in the art, using the method described in al., 1988, J.Immunol. 141:4053-4060.

[0517] Complete human antibodies cannot express the genes for endogenous immunoglobulin heavy and light chains, but they can be produced using transgenic mice that can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or part of the polypeptides of this disclosure. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma techniques. The human immunoglobulin transgenes in the transgenic mice are rearranged during B cell differentiation, and subsequently undergo class switching and somatic mutation. Therefore, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies using such techniques. For an overview of this technique for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, and of protocols for producing such antibodies, see, for example, U.S. Patents 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Other human antibodies are commercially available, for example, from Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0518] Complete human antibodies that recognize selected epitopes can be produced using a technique called "guided selection." In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of complete human antibodies that recognize the same epitope (Jespers et al. (1994), Biotechnology, 12:899-903). Human antibodies can also be prepared using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991), Marks et al., J.Mol.Biol., 222:581 (1991)).

[0519] The antibody may be an antibody fusion protein or a functionally active fragment thereof, for example, in which case the antibody is fused to the amino acid sequence of another protein (or a portion thereof, e.g., at least 10, 20, or 50 amino acids of a protein) via a covalent bond (e.g., a peptide bond) at either the N-terminus or C-terminus. The antibody or fragment may be covalently bonded to the other protein at the N-terminus of a constant domain.

[0520] Antibodies include analogues and derivatives that are modified by such covalent bonds in any type of molecule, insofar as the covalent bond allows the antibody to retain its antigen-binding immunospecificity. For example, but not limited to, derivatives and analogues of antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular antibody units or other proteins, etc. Therefore, further modifications are included. Numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis in the presence of tunicamycin. Furthermore, analogs or derivatives may contain one or more non-natural amino acids.

[0521] Antibodies in antibody-drug conjugates include antibodies with modifications (e.g., substitution, deletion, or addition) to amino acid residues that interact with the Fc receptor. In particular, antibodies include those with modifications to amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, for example, WO97 / 34631). Antibodies immune-specific to cancer cell antigens can be commercially available, for example, from Genentech (San Francisco, Calif.), or can be produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immune-specific to cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.

[0522] The antibody in the ADC may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody may also be an antibody fragment, such as a Fab fragment.

[0523] Known antibodies for the treatment or prevention of cancer can be conjugated as ADCs. Immunospecific antibodies against cancer cell antigens are commercially available or can be produced by any method known to those skilled in the art, such as recombinant expression technology. Nucleotide sequences encoding immunospecific antibodies against cancer cell antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.Examples of antibodies available for cancer treatment include: a humanized anti-HER2 monoclonal antibody for the treatment of metastatic breast cancer; RITUXAN.RTM. (Rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for the treatment of non-Hodgkin lymphoma; OvaRex (AltaRex Corporation, MA), a mouse antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), a mouse IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY), an anti-EGFRIgG chimeric antibody for the treatment of epidermal growth factor-positive cancers such as head and neck cancer; Vitaxin (MedImmune, Inc., MD), a humanized antibody for the treatment of sarcoma; Campath I / H (Leukosite, MA), a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); and Smart MI95 (Protein Design), a humanized anti-CD33IgG antibody for the treatment of acute myeloid leukemia (AML). LymphoCide (Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin lymphoma; Smart ID10 (Protein Design Labs, Inc., CA), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin lymphoma; Oncolym (Techniclone, Inc., CA), a radiolabeled mouse anti-HLA-Dr10 antibody for the treatment of non-Hodgkin lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin lymphoma; Avastin (Genentech, Inc., CA), a humanized anti-VEGF antibody for the treatment of lung cancer and colorectal cancer; Epratuzamab (Immunomedics, Inc., NJ and Examples include, but are not limited to, Amgen (CA) and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0524] Other antibodies useful in cancer treatment include the following antigens: CA125 (ovary), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colorectal), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (black Antibodies against chromoma, MUC1-KLH (breast cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu tumor gene product (carcinoma) are examples, but are not limited to these. Some specific and useful antibodies include, but are not limited to, BR96mAb (Trail, PA, et al. Science, (1993):261,212-215), BR64 (Trail, PA, et al. Cancer Research, (1997):57,100-105), mAbs against the CD40 antigen such as S2C6mAb (Francisco, JA, et al. Cancer Res. (2000):60:3225-3231), mAbs against the CD70 antigen such as 1F6mAb, and mAbs against the CD30 antigen such as AC10 (Bowen, MA, et al. (1993), J.Immunol.,151:5896-5906; Wahl et al.,2002, Cancer Res.62(13):3736-42).Many other internally distributed antibodies that bind to tumor-associated antigens can be used, and these have been outlined (Franke, AE, et al. Cancer Biother Radiopharm. (2000), 15:459-76; Murray, JL, (2000), Semin. Oncol., 27:64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

[0525] Antibodies that bind to antigens associated with antigen-presenting cells, such as CD40, OX40L, Endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, Dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2, can also be conjugated as ADCs.

[0526] Known antibodies for the treatment or prevention of autoimmune diseases may be conjugated as ADCs. Autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren's syndrome, immune thrombocytopenia, and multiple sclerosis. Immunospecific antibodies against cellular antigens involved in the production of autoimmune antibodies can be obtained by any method known to those skilled in the art, such as chemosynthesis or recombinant expression techniques. SLE is characterized by the overexpression of the interferon-alpha (IFN-alpha) cytokine gene (Bennett et al. (2003), Jour. Exp. Med. 197:711-723). Type 1 interferons (IFN-alpha / beta) play an important role in the pathogenesis of lupus (Santiago-Raber, (2003), Jour. Exp. Med. 197:777-788). Knockout mice (-IFN-alpha / beta) showed significantly reduced anti-erythrocyte autoantibodies, pure red blood cell erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, renal disease, and mortality. These results suggest that type 1 IFN mediates lupus in mice, and that reducing its activity with human counterparts may be beneficial. Anti-IFN Ab, conjugated to the bis-1,8-naphthalimide drug moiety, may be an effective therapeutic agent for SLE and other autoimmune diseases.

[0527] In another embodiment, useful antibodies in ADCs are immune-specific for the treatment of autoimmune diseases and include, but are not limited to, antinuclear antibodies, anti-dsDNA; anti-ssDNA; anti-cardiolipin antibodies IgM, IgG; antiphospholipid antibodies IgM, IgG; anti-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies; thyroglobulin antibodies; anti-SCL-70; anti-Jo; anti-U1RNP; anti-La / SSB; anti-SSA; anti-SSB; anti-peripheral cell antibodies; anti-histones; anti-RNP; C-ANCA; P-ANCA; anti-centromere; anti-fibrillarin; and anti-GBM antibodies.

[0528] Antibodies of ADCs can bind to either receptors or receptor complexes expressed on activated lymphocytes. These receptors or complexes may include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement regulatory proteins. Non-limiting examples of preferred immunoglobulin superfamily members include CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152 / CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable TNF receptor superfamily members include CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins include CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins include type C, type S, and type I lectins.

[0529] As used herein, the term “viral antigen” includes, but is not limited to, peptides, polypeptide proteins of any virus that can elicit an immune response (e.g., HIVgp120, HIVnef, RSVF glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLVtax, herpes simplex virus glycoproteins (e.g., Gb, Gc, Gd, and Ge) and hepatitis B surface antigens). As used herein, the term “microbial antigen” includes, but is not limited to, peptides, polypeptides, proteins, sugars, polysaccharides, or lipid molecules of any microorganism that can elicit an immune response (e.g., polypeptides of bacteria, fungi, pathogenic protozoa, or yeast containing LPS and capsular polysaccharides 5 / 8).

[0530] Antibodies immune to viral or microbial antigens can be commercially available, for example, from BD Biosciences (San Francisco, Calif.), Chemicon International, Inc. (Temecula, Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.), or can be produced by any method known to those skilled in the art, such as chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immune to viral or microbial antigens can be obtained, for example, from the GenBank database or similar databases, literature publications, or by routine cloning and sequencing.

[0531] In some embodiments, useful antibodies in current ADCs treat or prevent viral or microbial infections according to the methods disclosed herein. Examples of available antibodies useful for treating viral or microbial infections include SYNAGIS (MedImmune, Inc., MD), a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for treating patients with RSV infection; and C, which is useful for treating HIV infection. Examples of antibodies that can be used include, but are not limited to, PRO542 (Progenics), a D4 fusion antibody; OSTAVIR (Protein Design Labs, Inc., CA), a human antibody useful for treating hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA), a humanized IgG antibody useful for treating cytomegalovirus (CMV); and anti-LPS antibodies.

[0532] The ADC of the strain of the strain was determined by the strains of Streptococcus pyogenes, Streptococcus pneumoniae, and Neisseria gonorrheae、Neisseria meningitidis、Corynebacterium diphtheriae、Clostridium botulinum、Clostridium perfringens、Clostridium tetani、Haemophilus influenzae、Klebsiella pneumoniae、Klebsiella ozaenas、Klebsiella rhinoscleromotis、Staphylococcus aureus、Vibrio colerae、Escherichia coli、Pseudomonas aeruginosa、Campylobacter(Vibrio) fetus、Aeromonas hydrophila、Bacillus cereus、Edwardsiella tarda、Yersinia enterocolitica、Yersinia pestis、Yersinia pseudotuberculosis、Shigella dysenteriae、Shigella flexneri、Shigella sonnei、Salmonella typhimurium、Treponema pallidum、Treponema pertenue、Treponema carateneum、Borrelia vincentii、Borrelia burgdorferi、Leptospira icterohemorrhagiae、Mycobacterium tuberculosis、Pneumocystis carinii、Francisella tularensis、Brucella abortus、Brucella suis、Brucella melitensis、Mycoplasma spp.、Rickettsia prowazeki、Rickettsia tsutsugumushi、Chlamydia spp.); Pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); Protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryanosoma). Antibodies against antigens derived from gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms) are included, but are not limited to these.

[0533] Other antibodies useful in ADCs for treating viral diseases include Poxviridae, Herpesviridae, herpes simplex virus 1, herpes simplex virus 2, Examples of antibodies against antigens of pathogenic viruses include, but are not limited to, those of Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A / non-B hepatitis viruses, Rhinoviridae, Coronaviridae, Rotoviridae, and human immunodeficiency virus.

[0534] The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from that of the native polypeptide. Typically, an amino acid sequence variant has at least about 70% sequence identity with at least one receptor-binding domain of the native antibody or at least one ligand-binding domain of the native receptor, and usually the sequence will be at least about 80%, and more often at least about 90%, homologous to such receptor or ligand-binding domains. An amino acid sequence variant has substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence. Amino acids are represented by their conventional names, one-letter codes, and three-letter codes.

[0535] "Sequence identity" is defined as the percentage of amino acid sequence variant residues that are identical after aligning sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. Methods and computer programs for sequence comparison are well known in the art. One such computer program is "Align 2," developed by Genentech, Inc., which was filed with the United States Copyright Office, Washington, DC 20559, on December 10, 1991, along with user documentation.

[0536] The terms “Fc receptor” or “FcR” are used to describe receptors that bind to the Fc region of an antibody. An exemplary FcR is a native human FcR. Furthermore, FcRs may include receptors that bind to IgG antibodies (gamma receptors) and encompass the RIII subclass, which includes allele variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine system activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine system inhibitory motif (ITIM) in its cytoplasmic domain. (See Overview M in Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are outlined in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those identified later, are encompassed in the term “FcR” as used herein. This term also encompasses FcRn, a neonatal receptor involved in the transfer of maternal IgG to the fetus (Guyer et al.). al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0537] "Complement-dependent cell injury" or "CDC" refers to the ability of molecules to lyse targets in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to molecules (e.g., antibodies) that have formed a complex with an alloantigen. To assess complement activation, For example, a CDC assay like the one described in Gazzano-Santoro et al., J.Immunol.Methods, 202:163 (1996) may be performed.

[0538] "Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain.

[0539] The term "variable" refers to the fact that certain parts of the variable domain differ significantly in sequence between antibodies, and that these differences are used to determine the binding affinity and specificity of each antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The native heavy chain and light chain variable domains each contain four FRs that primarily employ a beta-sheet configuration, connected by three hypervariable regions that connect parts of the beta-sheet structure, and in some cases form loops that make up parts of it. The hypervariable regions in each chain are very close together by the FRs and, together with the hypervariable region of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0540] As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody involved in antigen binding. The hypervariable region generally contains amino acid residues from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. (see above)) and / or residues from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J.Mol.Biol., 196:901-917). Residues in the "framework region" or "FR" are variable domain residues other than hypervariable region residues as defined herein.

[0541] Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to easily crystallize. Pepsin treatment yields the F(ab')2 fragment, which still has two antigen-binding sites and can crosslink to the antigen.

[0542] "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen binding site. This region consists of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. It consists of a domain dimer. This configuration is responsible for the interaction of the three hypervariable regions within each variable domain, which define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific hypervariable regions) possesses the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0543] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is a notation used herein for Fab' in which the cysteine ​​residue(s) of the constant domain have at least one free thiol group. The F(Ab')2 antibody fragment was originally created as a pair with the Fab' fragment, which has a hinge cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0544] The "light chains" of antibodies derived from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0545] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, with these domains present on a single polypeptide chain. The Fv polypeptide further contains a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form a structure desired for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). scFv fragments of anti-ErbB2 antibodies are described in WO93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458.

[0546] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment containing a variable heavy domain (VH) attached to a variable light domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are paired with a complementary domain on another chain, creating two antigen-binding sites. Diabodies are described more fully, for example, in EP404,097;WO93 / 11161; and Hollinger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448.

[0547] The "humanization" of non-human (e.g., rodent) antibodies is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. Humanization is a method for transferring mouse antigen-binding information into non-immunogenic human antibody acceptors, resulting in numerous therapeutically useful drugs. The humanization method generally begins by transferring all six mouse complementarity-determining regions (CDRs) into the human antibody framework (Jones et al, (1986) Nature 321:522-525). Antibodies grafted with these CDRs generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues also need to be incorporated to maintain proper CDR configuration (Chothia et al, (1989) Nature 342:877). Transplantation of key mouse framework residues supporting the structural arrangement of grafted CDRs into human acceptors has been shown to restore antigen binding and affinity (Riechmann et al (1992)J). (Mol.Methods 157:4986-4995, and Presta et al (2001) Thromb.Haemost.85:379-389). For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and ability. In some cases, residues in the framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of at least one, usually two, variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies are optional and will include at least a portion of the constant region (Fc) of immunoglobulins, and usually at least a portion of the constant region (Fc) of human immunoglobulins. For further details, see U.S. Patent No. 6407213, Jones et al. (1986), Nature, 321:522-525, Riechmann et al. (1988), Nature, 332:323-329, and Presta, (1992) Curr. Struct. Biol., 2:593-596.

[0548] A "parent antibody" is an antibody containing an amino acid sequence derived from a sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. The parent antibody may contain either a native or wild-type sequence. The parent antibody may have modifications (such as additions, deletions, and / or substitutions) to existing amino acid sequences relative to other native, wild-type, or modified forms of the antibody. The parent antibody is directed towards the target antigen. Antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also intended.

[0549] An “isolated” antibody is one that has been identified, separated, and / or recovered from components of its natural environment. These environmental contaminants are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody would be purified to (1) exceed 95% or 99% by weight as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by gas-phase protein sequencing, or (3) to be homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Since an isolated antibody is free from at least one component of the antibody’s natural environment, it contains the antibody in situ within recombinant cells. However, typically, isolated antibodies would be prepared by at least one purification step.

[0550] An antibody that "binds" to a target molecular or antigen is one that can bind to that antigen with sufficient affinity so that it is useful for targeting cells expressing the antigen.

[0551] The terms “to treat” or “treatment” refer to both therapeutic and preventive or protective measures aimed at preventing or slowing (mitigating) undesirable physiological changes or impairments, such as the onset or transmission of cancer. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom reduction, attenuation of disease severity, a stable (i.e., non-worsening) disease state, delayed or slowed disease progression, relief or remission of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” can also mean extending survival compared to the survival expected if no treatment were administered. Yes, it is possible. Those who require treatment include individuals who already have a medical condition or disability, those who are prone to developing such a condition or disability, or those who need to prevent a medical condition or disability.

[0552] "Phage display" is a technique in which mutant polypeptides are presented as fusion proteins with the coat protein on the surface of phages, such as filamentous phage particles. One of the advantages of phage display is that it allows for the rapid and efficient screening of large libraries of random protein mutants for sequences that bind to target molecules with high affinity. Display of peptide and protein libraries on phages is used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods are typically used to display small random peptides and small proteins via fusion to either PIII or PVIII of filamentous phages. (Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992) and references cited therein.) In monovalent phage display, a library of proteins or peptides is fused to the phage coat protein or a portion thereof and expressed at low levels in the presence of wild-type proteins. The binding effect is reduced for multivalent phages so that selection is based on endogenous ligand affinity, and phagemide vectors are used, thereby simplifying DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991). Phage display includes techniques for producing antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, pp. 627-628).

[0553] A "phagemide" is a plasmid vector containing a bacterial replication origin, e.g., Co1E1, and a copy of the intergenetic region of a bacteriophage. Phagemids can be used with any known bacteriophage, including filamentous bacteriophages and lambda bacteriophages. Plasmids will also generally contain selective markers for antibiotic resistance. Segments of DNA cloned into these vectors can be grown as plasmids. When cells containing these vectors are provided with all the genes necessary for the generation of phage particles, the plasmid replication mode changes to rolling circle replication, producing a copy of one strand of plasmid DNA and packaged phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene or a fragment thereof, conjugated as a gene fusion to a heterologous polypeptide gene, so that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0554] In this disclosure, the “Ab” group (i.e., antibody, antibody fragment, and / or antigen fragment) can be conjugated to more than one drug-containing moiety. In some embodiments, “Ab” can be conjugated to 1 to 20 drug-containing moieties. In some embodiments, “Ab” can be conjugated to 1 to 10 drug-containing moieties. In some embodiments, “Ab” can be conjugated to 1 to 5 drug-containing moieties. In some embodiments, “Ab” can be conjugated to 1 to 2 drug-containing moieties. In some embodiments, “Ab” can be conjugated to 1 It can be attached to one of the drug-containing portions.

[0555] The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For a review of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

[0556] Suitable examples of acid addition salts include acetate, adipine, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivaphosphate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

[0557] Suitable examples of base addition salts include alkali metal salts such as ammonium salts, sodium salts, and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; salts with organic bases such as dicyclohexylamine salts and N-methyl-D-glucamine; and salts with amino acids such as arginine and lysine.

[0558] For example, Berge lists the following FDA-approved commercially available salts: anionic acetates, besilates (benzenesulfonates), benzoates, bicarbonates, tartarates, bromides, calcium edetate (ethylenediaminetetraacetate), camphor sulfonates, carbonates, chlorides, citrates, dihydrochlorides, edetate (ethylenediaminetetraacetate), edisylates (1,2-ethanedisulfonates). Estrous acid salts (lauryl sulfate), ethyl acid salts (ethanesulfonate), fumarate, gluceptate salts (glucoheptone), gluconate, glutamate, glycolyl arsanilate (glycolamide phenyl arsonate), hexyl resorcinate, hydravamin (N,N'-di(dehydroabiethyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-Hydro (Loxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate (2-naphthalenesulfonate), nitrate, pamoate (embolate), pantothenate, phosphate / diphosphate, polygalactulose, salicylate, stearate, basic acetate, succinate, sulfate, tannate, tartrate, theoclate (8-chlorotheophylline), and triethiozide; organic cations such as benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0559] Berge further lists the following commercially sold (outside the US) salts that are not FDA approved: anions such as adipines, alginates, aminosalicylates, anhydromethylene citrates, arecoline, aspartates, bisulfates, and butyl bromide. , camphor salts, digluconates, hydrobromides, disuccinates, glycerophosphates, hemisulfates, hydrofluorides, hydroiodides, methylenebis(salicylate), napadisylates (1,5-naphthalenedisulfonate), oxalates, pectates, persulfates, phenylethyl barbiturates, picrates, propions, thiocyans, tosylates, and undecanoic acids; organic cations such as benetamine (N-benzylphenethylamine), cremisole (1-p-chlorobenzyl-2-pyrroridine-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and metal cations such as barium and bismuth.

[0560] The compounds described herein may also include suitable carriers, excipients, and adjuvants, which may vary depending on the mode of administration.

[0561] In some embodiments, the pharmaceutical composition can be formulated as a suitable parenteral dosage form. The formulation can be prepared by various methods known in the art. The pharmaceutical composition can be administered into the bloodstream, into the muscle, or directly into an organ. Suitable means of parenteral administration include intravenous, intraarterial, intraperitoneal, subarachnoid, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needled syringes, needleless syringes, and injection techniques.

[0562] Parenteral compositions are typically aqueous solutions that may contain salts, carbohydrates, and excipients such as buffers. However, compositions may also be formulated as sterile, non-aqueous solutions or as dry forms used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

[0563] For example, the preparation of parenteral compositions under sterile conditions by freeze-drying can be easily achieved using standard techniques well known to those skilled in the art.

[0564] Compositions for parenteral administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Accordingly, compositions can be formulated as solids, semi-solids, or thixotropic liquids for administration as implantable depots resulting in modified release of the activator.

[0565] Parenteral formulations can be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

[0566] In another embodiment, the pharmaceutical composition can be formulated into a suitable oral dosage form, such as tablets, capsules, powders, pellets, suspensions, solutions, emulsions, etc. Other suitable carriers may be present, such as disintegrants, diluents, chelating agents, binders, flow enhancers, lubricants, fillers, bulking agents, and anti-adhesion agents.

[0567] Oral medication formulations may also contain other suitable pharmaceutical excipients, such as sweeteners, vehicles / humectants, colorants, flavorings, preservatives, viscosity enhancers / thickeners, etc.

[0568] The dosage of the pharmaceutical composition disclosed herein can be adjusted to suit the individual patient.

[0569] lipid complex In certain embodiments, this disclosure relates to formula (VI),

[0570] [ka]

[0571] Provides compounds or pharmaceutically acceptable salts thereof, During the ceremony,

[0572] a is an integer between 1 and 20.

[0573] m is 0, 1, 2, 3, or 4;

[0574] n is either 0 or 1;

[0575] D-NH- is a component of amino-substituted compounds, and amino-substituted compounds have the formula D-NH2;

[0576] Each R 1 These are independently selected from C1-C4 alkyl, O-C1-C4 alkyl, and halogen;

[0577] R 2 C1-C4 alkyl and -(CH2CH2O) s -Selected from CH3, where s is an integer between 1 and 10;

[0578] R 3 and R 3’ Each is independently selected from hydrogen and C1-C3 alkyl;

[0579] L is a severable linker;

[0580] LP is a lipid.

[0581] In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 is a cyclic dinucleotide (CDN) or a CDN-like compound. In some embodiments, D-NH2 is one of the formulas described herein.

[0582] In certain embodiments, the present disclosure provides compounds of formula (VI), where L is a compound of formula (VI).

[0583] [ka]

[0584] It has, In the formula, W, Z, t, U, Q, and U' are as described herein.

[0585] In some embodiments, the compound of formula (VI) is related to a lipid complex. The term "lipid complex" is a term recognized in the art in the preparation of pharmaceutical compounds. The complex is characterized by a non-covalent bond between the lipid and the compound of formula (VI). Without being constrained by any particular theory, the compound of formula (VI) can associate with the lipid complex via electrostatic interactions, hydrophilic interactions, hydrophobic interactions, or any combination thereof.

[0586] In some embodiments, the lipid complexes described herein take the form of lipid particles. In one embodiment, the complex is in the form of lipid nanoparticles. In one embodiment, the complex is in the form of liposomes. In some embodiments, the liposomes of this disclosure comprise a lipid monolayer or a lipid multilayer. In some embodiments, the liposome comprises a lipid bilayer and an aqueous core. In some embodiments, the average particle size of the liposomes is about 5 nm to about 1 μm. In some embodiments, the average particle size of the lipid nanoparticles or liposomes is about 5 nm to about 500 nm, about 10 nm to about 150 nm, and about 30 nm to about 100 nm. In some embodiments, the compound of formula (V) is present in the aqueous core, the lipid bilayer, or a combination thereof.

[0587] In certain embodiments, LP can be cholesterol or a phospholipid. In some embodiments, LP is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidyldicerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, soybean phospholipids, and egg yolk phospholipids. In some embodiments, the phospholipid is phosphatidylethanolamine. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine.

[0588] In some embodiments, the disclosure provides lipid complexes comprising a compound of formula (VI). In some embodiments, the lipid complex may be a liposome.

[0589] In some embodiments, the lipid complex contains about 0.5 to about 40 mole percent of the compound of formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex contains about 1 to about 35 mole percent of the compound of formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex contains about 1 to about 30 mole percent of the compound of formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex contains about 1 to about 20 mole percent of the compound of formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex contains about 1 to about 15 mole percent of the compound of formula (VI), or a pharmaceutically acceptable salt thereof.

[0590] In some embodiments, the lipid complex further comprises one or more additional phospholipids. In some embodiments, the phospholipids are selected from phosphatidylcholine, phosphatidyl diserol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipids, and egg yolk phospholipids.

[0591] In some embodiments, the lipid complex contains one or more phospholipids in an amount of about 30 to about 90 mole percent. In some embodiments, the lipid complex contains one or more phospholipids in an amount of about 40 to about 85 mole percent. In some embodiments, the lipid complex contains one or more phospholipids in an amount of about 50 to about 80 mole percent.

[0592] In some embodiments, the lipid complex further contains fatty alcohols or cholesterol. In some embodiments, the lipid complex contains about 5 to about 35 mole percent of cholesterol. In some embodiments, the lipid complex contains about 5 to about 30 mole percent of cholesterol. In some embodiments, the lipid complex contains about 5 to about 25 mole percent of cholesterol.

[0593] In some embodiments, the lipid complex further comprises at least one PEGylated phospholipid. In some embodiments, the at least one PEGylated lipid is a PEGylated phospholipid. In some embodiments, the PEGylated phospholipid is N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine The phosphoethanolamine is selected from N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, N-(carbonyl-methoxypolyethylene glycol 2000)-distearoyl-rac-glycerol, and N-(carbonyl-methoxypolyethylene glycol 5000)-distearoyl-rac-glycerol, pharmaceutically acceptable salts thereof, or mixtures thereof. In some embodiments, the pegylated phospholipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0594] In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 0 to about 6 mole percent. In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 0.5 to about 5 mole percent. In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 1 to about 5 mole percent. In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 3 mole percent. In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 4 mole percent. In some embodiments, the lipid complex contains one or more pegylated phospholipids in an amount of about 5 mole percent. Methods of using compounds and compositions

[0595] The specific compounds described herein are STING agonists and are therefore useful in stimulating an immune response in their target. The compositions can be used in the treatment of viruses.

[0596] The compounds of this disclosure exhibit regulatory / agonist activity of STING. The particular compounds of this disclosure may be superior in terms of efficacy, pharmacokinetics (e.g., absorption, distribution, metabolism, excretion), solubility (e.g., water solubility), interactions with other drugs (e.g., drug-metabolizing enzyme inhibitory activity), safety (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central nervous system toxicity), and / or stability (e.g., chemical stability, enzyme stability), and may be useful as pharmaceuticals.

[0597] The compounds disclosed herein can be used to enhance STING activity in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, and humans).

[0598] The compounds disclosed herein are used to treat diseases that may be affected by STING (hereinafter referred to as "STING-related diseases"), such as cancer, such as colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumors), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma), duodenal cancer, small intestine cancer, and breast cancer (e.g., invasive ductal carcinoma, non-invasive **Cancers:** Ductal carcinoma, inflammatory breast cancer; ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor); testicular tumors; prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, castration-resistant prostate cancer); liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic cholangiocarcinoma); thyroid cancer (e.g., medullary thyroid carcinoma); kidney cancer (e.g., renal cell carcinoma (e.g., clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter); uterine cancer (e.g., cervical cancer, endometrial cancer, uterine sarcoma); gestational trophoblastoma; brain tumors (e.g., medulloblastoma, glioma, pineal astrocytic tumor, pilocytic cell tumor) It can be used as a drug such as an agent for the prevention or treatment of cancers of unknown primary origin (e.g., astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell tumor, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, hematological cancer (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), cancer of unknown primary origin; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; and agents for the treatment of precancerous lesions (e.g., myelodysplastic syndrome).

[0599] In certain embodiments, the compounds of the present disclosure can be used as drugs for colorectal cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

[0600] Furthermore, the compounds or combination agents of the disclosed herein can be used concurrently with non-pharmacological therapies. More precisely, the compounds or combination agents of the disclosed herein can be used in combination with non-pharmacological therapies such as (1) surgery, (2) antihypertensive chemotherapy using angiotensin II, etc., (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiotherapy.

[0601] For example, by using the compounds disclosed herein before or after the aforementioned surgeries, or before or after combination therapy of two or three of these treatments, effects such as prevention of resistance development, extension of disease-free survival, suppression of cancer metastasis or recurrence, and life extension may be obtained.

[0602] In addition, treatment with the compounds of this disclosure or the combination agents of this disclosure can be combined with supportive therapies such as: (i) administration of antibiotics (e.g., β-lactam antibiotics such as Pansporin, macrolide antibiotics such as Clarithromycin) for complications of various infections; (ii) administration of high-calorie intravenous fluids, amino acid preparations, or multivitamins to improve malnutrition; (iii) administration of morphine for pain relief; (iv) administration of pharmaceutical agents to improve side effects such as nausea, vomiting, loss of appetite, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, alopecia, liver damage, kidney damage, DIC, fever, etc.; and (v) administration of pharmaceutical agents to suppress multidrug resistance such as cancer. [Examples]

[0603] General synthesis methods and intermediates The compounds of this disclosure can be prepared by those skilled in the art based on this disclosure and knowledge of the art, and / or by referring to the schemes and synthetic examples shown below. Exemplary synthetic routes are described in the following schemes and examples. It should be understood that variable elements appearing in the following schemes and examples, such as the "R" group, should be interpreted independently of those appearing elsewhere in this application. Those skilled in the art will readily understand how the schemes and examples shown below illustrate the preparation of the compounds described herein.

[0604] Scheme 1: General routes for converting carboxylic acids to their corresponding acid chlorides or (isobutyl carbonate)propionic anhydride.

[0605] [ka]

[0606] Scheme 1 shows a general pathway for activating carboxylic acid i to the corresponding acid chloride or ii such as (isobutyl carbonate)propionic anhydride, where Yc is an alkyl or aromatic chain, and R 6 is a methyl or polyether-containing alkyl chain. Preparation of ii, where E is OCO2CH2CH(CH3)2, can be achieved by treating i with isobutyl chloroformate in the presence of a base. When E is Cl, the formation of ii is achieved by treating i with oxalyl chloride or Ghosez reagent (α-chloroenamine, a reactive intermediate in synthesis: 1-chloro-N,N,2-trimethylpropenylamine; see Haveaux B., et al. Org. Synth. 1980, 59, 26).

[0607] Scheme 2: A general route for attaching a spacer to the amino group of the drug moiety using a stepwise approach

[0608] [ka]

[0609] Scheme 2 shows a general method for attaching a spacer like ii to the amino group of a drug moiety like iii. Compound iii is subjected to overall silyl protection by treatment with trimethylsilyl chloride and pyridine, and then reacted with activated spacer ii. Removal of the silyl group with NEt3-3HF yields iv. Deprotection of the amine of iv is achieved by treatment with an acid such as TFA or HCl, and vi is obtained by treating the resulting amine salt with activated dipeptide carbonate v.

[0610] Scheme 3: A general route for attaching a spacer to the amino group of the drug moiety using a stepwise approach

[0611] [ka]

[0612] Scheme 3 shows a general method for attaching a spacer like ii to the amino group of a drug moiety like iii-a, in which case X 1 and X 2 is oxygen, sulfur, or CH2, and X 3 is nitrogen or CH, and R 3 R is hydrogen or fluoro, 4 R is hydroxyl, hydrogen, fluoro, or R 3 Together with R, they form OCH2, c is OH or SH, R d The group is methyl or (CH2)3NHC(O)NH2. Compound iii-a is subjected to overall silyl protection by treatment with trimethylsilyl chloride and pyridine, and then reacted with the activation spacer ii. Removal of the silyl group with NEt3-3HF yields iv-a. Deprotection of the amine of iv-a is achieved by treatment with an acid such as TFA or HCl, and vi-a is obtained by treating the resulting amine salt with the activated dipeptide carbonate v.

[0613] Scheme 4: A general route for attaching a spacer to the amino group of a drug moiety using a stepwise approach.

[0614] [ka]

[0615] Scheme 4 shows a general method for attaching a spacer group like ii to an amino group of a drug moiety like iii-b, following the general method described in Scheme 3.

[0616] Scheme 5: General pathway for producing spacer peptide acid chlorides

[0617] [ka]

[0618] Scheme 5 shows a general method for preparing acid chloride viii. Deprotection of Boc-amine i with an acid such as TFA or HCl is followed by treatment with activated carbonate v to obtain carboxylic acid vii. Conversion of vii to acid chloride viii can be achieved by treatment with oxalyl chloride or sulfonyl chloride.

[0619] Scheme 6: Alternative common pathways for attaching a spacer to the amino group of the drug moiety

[0620] [ka]

[0621] Scheme 6 shows an alternative common method for directly converting payload iii to compound ix. After overall silyl protection with trimethylsilyl chloride, iii is treated with acid chloride vii. Removal of the silyl group yields ix.

[0622] Scheme 7: Alternative common pathways for attaching spacers to the amino group of the drug moiety

[0623] [ka]

[0624] Scheme 7 presents an alternative general method for directly converting payload iii-a to compound ix-a according to the general method described in Scheme 6.

[0625] Scheme 8: Alternative common pathways for attaching spacers to the amino group of the drug moiety

[0626] [ka]

[0627] Scheme 8 shows an alternative general method for attaching a spacer to the amino group of a drug moiety, such as iii-b, according to the general method described in Scheme 6.

[0628] Scheme 9: General route for preparing the linker payload

[0629] [ka]

[0630] Scheme 9 shows a general method for preparing the linker payload xi from vi, where Yd is a linear or branched alkyl or polyether chain and Z is a binding handle such as maleimide, BCN, or DBCO. The amine of vi is deprotected with an acid such as TFA or HCl, followed by a reaction with the activated ester x to obtain the linker payload xi.

[0631] Scheme 10: A general route for preparing the linker payload.

[0632] [ka]

[0633] Scheme 10 shows a general method for preparing the linker payload xi-a from vi-a according to the general method described in Scheme 9.

[0634] Scheme 11: A general route for preparing the linker payload.

[0635] [ka]

[0636] Scheme 11 shows a general method for attaching a binding handle to a compound like vi-b, following the general method described in Scheme 9.

[0637] Scheme 12: A general route for preparing the linker payload.

[0638] [ka]

[0639] Scheme 12 shows an alternative method for preparing the linker payload xi from iv. Deprotection of the amine in iv with an acid such as TFA or HCl is followed by reaction xii to obtain xi.

[0640] Scheme 13: General route for preparing the linker payload

[0641] [ka]

[0642] Scheme 13 shows an alternative method for preparing linker payload xi-a from iv-a according to the general method described in Scheme 12.

[0643] Scheme 14: General route for preparing the linker payload

[0644] [ka]

[0645] Scheme 14 shows a general method for preparing a glucuronidation-containing linker payload xvi. Deprotection of the amine of iv with an acid such as TFA or HCl is followed by reaction with an activated carbonate xiii to obtain xiv. Overall acetic acid hydrolysis and removal of the FMOC group of xiv with a base such as lithium hydroxide yield xv. Treatment of xv with an activated binding handle x yields a glucuronidation-containing linker payload xvi.

[0646] Scheme 15: General route for preparing the linker payload

[0647] [ka]

[0648] Scheme 15 shows a general method for preparing the glucuronidation-containing linker payload xvi-a according to the general method described in Scheme 14.

[0649] Scheme 16: General route for preparing the linker payload

[0650] [ka]

[0651] Scheme 16 shows a general method for preparing a terminal amine-containing linker payload xviii, where Ye is a linear alkyl chain or a polyether-containing alkyl chain. The reaction of the activated binding handle xvii with an acid such as TFA or HCl follows the deprotection of the amine vi. The terminal amine-containing linker payload xviii is obtained by removal of the terminal protecting group with an acid such as TFA.

[0652] Scheme 17: General route for preparing the linker payload

[0653] [ka]

[0654] Scheme 17 shows a general method for preparing the terminal amine-containing linker payload xviii-a according to the general method described in Scheme 16.

[0655] Scheme 18: General route for preparing the linker payload

[0656] [ka]

[0657] Scheme 18 shows a general method for preparing a linker payload like xxii that incorporates a dipeptide spacer, in which R 7 The moiety is hydrogen, alkyl, or aromatic. Compound iii is protected overall by treatment with trimethylsilyl chloride and pyridine, then treated with Boc-proline succinimide ester. xix is ​​obtained by removing the silyl group with a fluoride source such as NEt3-3HF. Deprotection of the amine of xix with an acid such as TFA or HCl is followed by reaction with amino acid xx activated with succinimide ester to obtain xxi. Deprotection of the amine of xxi with an acid such as TFA or HCl is followed by introduction of a maleimide handle and linker payload x xii is obtained.

[0658] Scheme 19: General route for preparing the linker payload

[0659] [ka]

[0660] Scheme 19 shows a general method for preparing a linker payload like xxii-a according to the general method described in Scheme 18.

[0661] Preparation of exemplary ADCs and intermediates definition AA: LC-MS method using ammonium acetate

[0662] Ac: Acetate

[0663] ACN: Acetonitrile

[0664] atm: atmosphere

[0665] aq: aqueous solution

[0666] BCN: Bicyclo[6.1.0]nonine

[0667] BLQ: Below the limit of quantification

[0668] Bn: Benzyl

[0669] Boc:tert-butoxycarbonyl

[0670] tBu:tert-butyl

[0671] Bz: Benzoyl

[0672] C: Celsius

[0673] calcd: Calculated value

[0674] Cbz: Benzyloxycarbonyl

[0675] DAR: Drug / Antibody Ratio

[0676] DBCO: Dibenzocyclooctin

[0677] DCC:N,N'-Dicyclohexylcarbodiimide

[0678] DCM: Dichloromethane

[0679] DIPEA: N,N-diisopropylethylamine

[0680] DMAP: 4-dimethylaminopyridine

[0681] DMA: N,N-dimethylacetamide

[0682] DMB: 2,4-dimethoxybenzyl

[0683] DMF: N,N-dimethylformamide

[0684] DMSO: Dimethyl sulfoxide

[0685] DTT: Dithiothreitol

[0686] ε: Absorption coefficient

[0687] E0.1%: Absorption coefficient of 0.1% solution

[0688] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

[0689] EDTA: Ethylenediaminetetraacetic acid

[0690] Et: Ethyl

[0691] EtOH: Ethanol

[0692] æ:ethyl acetate

[0693] FA: LC-MS method using formic acid

[0694] Fmoc: Fluorenylmethyloxycarbonyl

[0695] h: time

[0696] HATU: Hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide

[0697] HIC: Hydrophobic Interaction chromatography

[0698] HOAt: 1-Hydroxy-7-Azabenzotriazole

[0699] HPLC: High-Pressure Liquid Chromatography

[0700] HRMS: High resolution mass spectrometry

[0701] I C 50 : 50% inhibitory concentration

[0702] LC: Liquid Chromatography

[0703] LCMS: Liquid Chromatography Mass Spectrometry

[0704] m / z: mass-to-charge ratio

[0705] MHz: Megahertz

[0706] Me: Methyl

[0707] MeOH: methanol

[0708] μM: micron

[0709] min: minutes

[0710] mL: milliliter

[0711] MS: Mass Spectrum

[0712] MWCO: Molecular weight cutoff

[0713] NMR: nuclear magnetic resonance

[0714] PBS: Phosphate-buffered saline

[0715] PE: Petroleum ether

[0716] Ph: Phenyl

[0717] psi: pounds per square inch

[0718] psig: pounds / square inch gauge

[0719] Pyr: Pyridine

[0720] QTOF: Quadrupole time of flight

[0721] rt: room temperature

[0722] SEC: Size exclusion chromatography:

[0723] SFC: Supercritical Fluid Chromatography

[0724] TBS: tert-butyldimethylsilyl

[0725] TCEP: (Tris(2-carboxyethyl)phosphine)

[0726] TEA: Triethylamine

[0727] TFA: Trifluoroacetic acid

[0728] THF: Tetrahydrofuran

[0729] TIPS: Triisopropylsilyl

[0730] TMS: Trimethylsilyl

[0731] Tris: Tris(hydroxymethyl)aminomethane

[0732] UPLC: Ultra-high-performance liquid chromatography

[0733] Analysis method NMR conditions: 1 Unless otherwise specified, 1H NMR spectra are obtained using a 400MHz Bruker spectrometer. I obtained it. 31 The PN MR spectrum was obtained using a 400 MHz Bruker spectrometer at 162 Record in mHz, unless otherwise specified. 1 It was obtained using H decoupling. 19 F NMR Unless otherwise specified, the vectors were recorded at 376 mHz using a 400 MHz Bruker spectrometer.

[0734] Requirements for HRMS: HRMS spectra were obtained using an Agilent 1260 Infinity II bioinert multisampler, bioinert column compartment, bioinert quaternary pump, DAD multiwavelength detector, and OsakaSoda CapcelLPAK. The results were obtained using an Agilent 6545LC-QTOF mass spectrometer equipped with a C1 UG120 (5 μm, 2.0 mm inner diameter × 35 mm length) reversed-phase column. The solvent system for LC consisted of a 0.1% formic acid aqueous solution for mobile phase A, and a 0.1% formic acid in acetonitrile, or an equivalent solvent system, for mobile phase B. Typically, LC elution was initiated with a 95% / 5% mobile phase A / B ratio. After a section run at the fixed composition, usually after 0.5 minutes, the mobile phase The voltage was varied from 100% B over a linear gradient of 1.5 minutes. The flow rate was 0.7 mL / min for 5 minutes.

[0735] Typically, 2 μL of a 1 mM solution in DMSO was injected into the mass spectrometer. QTOF settings: gas temperature 35°C, dry gas flow rate = 10 L / min, nebulizer pressure 55 psig, sheath gas temperature and flow rate 35°C and 12 L / min, respectively. The following voltage settings were applied: capillary voltage 4500V, nozzle voltage 2000V, and fragmenter voltage 75V.

[0736] The data were analyzed using Agilent Mass Hunter Qualitative Analysis software version 8.07.00SP2. The mass error [ppm] was 1 × 10⁻⁶. 6 The difference between the precise mass and the observed mass was expressed by dividing by the precise mass obtained by multiplying by [a certain factor].

[0737] LCMS requirements: LC-MS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 series LC system connected to a Micromass mass spectrometer using a reversed-phase C18 column. Various gradients and analysis times were selected to optimize the characterization of the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained either 0.1% formic acid (as indicated by FA) or 10 mM ammonium acetate (as indicated by AA). One example of a solvent gradient used was from 100% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 1 mL / min over a 16.5 minute run.

[0738] In some cases, LC-MS spectra were recorded using a reversed-phase C18 column on an Agilent 1290 Infinity UPLC system connected to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system connected to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 series HPLC system connected to a Waters Micromass ZQ mass spectrometer. Various gradients and analysis times were selected to optimize the characterization of the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained either 0.1% formic acid (as indicated by FA) or 10 mM ammonium acetate (as indicated by AA). One example of a solvent gradient used was from 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid), with a flow rate of 0.5 mL / min over a 5-minute run.

[0739] Preparative HPLC: Separation by preparative HPLC was performed using a Gilson instrument powered by a 322 pump, with an 18×150 mm Sunfire C-18 column eluting via a water-ACN gradient. Fractionation was performed using a UV / visible light 155 detector with the wavelength set between 200 nm and 400 nm. Mass-gated fractionation was performed using an Agilent 1100 LC / MSD instrument.

[0740] Those skilled in the art will recognize that the gradient, column length, and flow rate can be changed, and that, depending on the chemical species being analyzed, some conditions may be more favorable than others for characterizing the compound.

[0741] Preparative SFC: Preparative SFCs are eluted with appropriate proportions of supercritical carbon dioxide and alcohol in 10, 20, or 30 mm × 250 mm ChiraLPak columns containing 0.3% diethylamine, 0.3% TEA, and 0.3% formic acid, or without acid or base additives. Typically, the procedure was performed using IA, IB, IC, ID, IE, and IF) 10 or 20 mm × 250 mm Phenomenex Lux Cellulose-4 or 2-ethylpyridine columns. Typical conditions included constant composition with flow rates in the range of 10–100 mL / min and a column temperature of 40°C. Preparative SFC was performed using a Jasco SFC preparative purification system with UV / visible light fractionation set to 200 nm–400 nm and back pressure adjusted to 10 MPa.

[0742] Those skilled in the art will recognize that the gradient, column length, and flow rate can be changed, and that, depending on the chemical species being analyzed, some conditions may be more favorable than others for characterizing the compound.

[0743] Conditions for analysis SEC: SEC spectra were recorded at 280 nm using a Hewlett Packard HP1100 or Agilent 1100 series LC system equipped with a diode array detector using an SEC column (typically TosoH Biosep TSK Gel, G3000SWxl; P / N8541; 250A; 5 μm; 7.8 mm × 300 mm). The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8, 10% acetonitrile (v / v) or 1x PBS. Typically, the mixture was run at a flow rate of 1 mL / min for 20 minutes at a constant composition.

[0744] Conditions for HIC analysis: HIC spectra were recorded at 280 nm using a Hewlett Packard HP1100 or Agilent 1100 series LC system equipped with a diode array detector using an HIC column (typically Tosoh Butyl-NPR, 4.6 × 35 mm, 2.5 μm, P / N: 14947). Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7, and mobile phase B was 75% 25 mM sodium phosphate, pH 7, 25% isopropanol. Typically, the flow is run for 20 minutes, but a 12-minute linear gradient from 95% / 5% A / B to 100% B is used between the first and last sections of the constant composition flow.

[0745] LC-QTOF requirements: LC-MS spectra were recorded using a reversed-phase column (typically Agilent, PLRP-S, 5 μm, 1000 angstroms, 2.1 mm × 50 mm) heated to 80°C on an Agilent 1260 Bioinert series LC system connected to an Agilent 6545 QTOF mass spectrometer. Various gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water gradient and contained 0.1% formic acid. One example of a solvent gradient used was from 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) with the conditions shown in Table 1.

[0746] [Table 1]

[0747] Samples were intact or reduced (20 μL of 1–5 mg / mL ADC solution treated with 4 μL of 0.5 M DTT solution at 37°C for 30 minutes). Raw data was deconvolved within an appropriate mass range using Agilent BioConfirm software to obtain the molecular weight(s) of the protein, and DAR was calculated using the Agilent DAR calculator.

[0748] LC / MS / MS conditions: LC / MS / MS analysis was performed using a Shimadzu UFLC LC-20ADXR binary pump, a SIL-30ACMP autosampler system, and an AB SCIEX Triple Quad 4500ESI mass spectrometer.

[0749] Typically, after passing the sample through a Waters Xselect C18 CSH 3.5u, 2.1 mm ID × 30 mm column, a 5 μL sample aliquot was injected into the LC / MS / MS. Mobile phase A contained 0.1% aqueous formic acid, and mobile phase B was 5% aqueous 0.1% formic acid with 95% acetonitrile. The total run time was 3 minutes at 1.5 mL / min with a linear gradient from 100% A to 100% B over a flow rate of 1.5 minutes. First, the instrument was run with 100% aqueous mobile phase solvent for 0.5 minutes, and then with 100% organic solvent for the next 1.5 minutes.

[0750] Preparative SEC: Preparative SEC purification was performed using a Gilson preparative HPLC system equipped with a UV detector and an SEC column (typically GE Superdex 200 Increase 10 / 300GL). The mobile phase was 1×PBS (pH 7.4). A typical run was a constant composition at a flow rate of 1 mL / min for 30 minutes. Fractionation was initiated based on UV thresholds (214 and 280 nm).

[0751] ADC concentration: The ADC concentration was calculated from the UV absorbance at 280 nm, measured using the NanoDrop (2000c; Fisher Scientific) coefficient, after subtracting the UV absorbance from the corresponding linker payload structure.

[0752] Example 1 N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate tert-butyl (intermediate 1)

[0753] [ka]

[0754] Step 1: 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid 2-[(methylamino)methyl]benzoic acid (7.50 g, 44 mmol) was added to 1,4-dioxane (136 mL) and 1N sodium hydroxide aqueous solution (182 mL) with di-tert-butyl dicarbonate (19.2 g, 88 mmol). The reaction mixture was stirred at room temperature for 2 hours. Next, the reaction mixture was concentrated to remove the organic solvent, and the pH was adjusted to pH 3 using 1N HCl. The product was extracted with SiO(3×). The combined organic phase was washed with brine, dried with (Na2SO4), filtered, and evaporated. The crude compound was purified by silica gel chromatography (0-50% SiO / DCM) to obtain 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (8.88 g, 75%). LCMS(AA): m / z = 264.1 (MH). Step 2: N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate tert-butyl (intermediate 1)

[0755] 1-Chloro-N,N,2-trimethylpropenylamine (0.52 mL, 4.0 mmol) was slowly added at 0°C to a solution of 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (525 mg, 2.0 mmol) in DCM (16 mL). The reaction mixture was then warmed to room temperature and stirred for 1 hour. The reaction mixture was then concentrated to dryness to obtain crude N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate tert-butyl (intermediate 1.562 mg, 100%).

[0756] Example 2 N-[[2-(2-chloro-2-oxo-ethyl)phenyl]methyl]-N-methylcarbamate tert-butyl (intermediate 2)

[0757] [ka]

[0758] Step 1: 2-[2-[[tert-butoxycarbonyl(`methyl)amino]methyl]phenyl]acetic acid 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid (1.50 g, 5.7 mmol) was dissolved in THF (20 mL) and iodomethane (2.8 mL, 45.2 mmol) was added at room temperature. The reaction mixture was cooled to 0°C and sodium hydride (mineral) was added. A 60% dispersion in oil (905 mg, 22.6 mmol) was added in three portions. The reaction mixture was then stirred at room temperature for 16 hours. The reaction was stopped with water, and the organic solvent was removed by concentration. The aqueous phase was acidified to pH 3 using 1N HCl, and the crude product was extracted with Et2O(2×). The combined organic phase was washed with brine, dried, filtered, and evaporated to obtain 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (1.36 g, 86%). LCMS(AA): m / z = 280.2(M+H).

[0759] Step 2: N-[[2-(2-chloro-2-oxo-ethyl)phenyl]methyl]-N-methylcarbamate tert-butyl, intermediate 2 Starting with 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (650 mg, 2.33 mmol), the title compound was prepared according to the procedure described in Step 2 of Example 1 to obtain crude intermediate 2 (693 mg, 100%).

[0760] Example 3 4-[tert-butoxycarbonyl(methyl)amino]butanoate isobutoxycarbonyl (intermediate 3)

[0761] [ka]

[0762] Step 1: 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid The title compound was prepared using 4-(tert-butoxycarbonylamino)butanoic acid (2.92 g, 14.4 mmol) instead of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid, following the procedure described in Step 1 of Example 2, to obtain 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (2.89 g, 93%). LCMS(AA): m / z = 216.1 (MH).

[0763] Step 2: 4-[tert-butoxycarbonyl(methyl)amino]butanoate isobutoxycarbonyl (intermediate 3) Isobutyl chloroformate (0.22 mL, 1.73 mmol) was added dropwise to a solution of 4-((tert-butyl-butoxycarbonyl)(methyl)amino)butanoic acid (360 mg, 1.57 mmol) dissolved in DCM (7 mL) and cooled to 0°C, and triethylamine (0.24 mL, 1.73 mmol). The reaction mixture was stirred at room temperature for 2 hours until complete addition. The mixture was then concentrated to dryness to obtain crude 4-[tert-butoxycarbonyl(methyl)amino]butanoic acid isobutoxycarbonyl (intermediate 3, 499 mg, 100%).

[0764] Example 4 The compounds listed below were prepared as described in Example 3, using the starting materials shown in Table 2 instead of 4-(tert-butoxycarbonylamino)butanoic acid.

[0765] [Table 2]

[0766] Example 5 N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}-2-[(methylamino)methyl]benzamide (intermediate 6)

[0767] [ka]

[0768] Step 1: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4- Iloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate tert-butyl 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahylo-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-1,9-dihydro-6H-purin-6-one (compound I-5c, see PCT / IB2018 / 058846, 160 mg, 0.19 mmol) as an N,N-diethylethaneamine salt was dissolved in anhydrous pyridine, concentrated to dryness (3 × 2 mL), and then placed under vacuum for 15 minutes. The residue was dissolved in pyridine (3 mL) under an argon atmosphere, and chlorotrimethylsilane (0.15 mL, 1.13 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. Next, intermediate 1 (800 mg, 2.82 mmol) dissolved in pyridine (3 mL) was added via syringe. The reaction mixture was stirred under an argon atmosphere at room temperature for 16 hours. Next, the reaction mixture was concentrated to dryness, and MeOH (10 mL) and ammonium hydroxide (28-30% aqueous solution, 10 mL) were added, and the mixture was stirred for 30 minutes. The reaction mixture was concentrated to dryness, and the residue was dissolved in MeOH (15 mL). Triethylamine trifluoride (0.12 mL, 0.75 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated to dryness, the crude residue was adsorbed onto Celite, and purified by reverse-phase flash column chromatography (0-50% ACN / triethylammonium acetate aqueous solution (10 mM)) to obtain [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate tert-butyl (120 mg, 58%) as an N,N-diethylethaneamine salt. LCMS(AA): m / z = 897.3(M+H). 1 HN MR(400MHz,CD3OD)δ8.70(s,1H),8.49(s,1H),8.41(d,J=6.0Hz,1H),7.70(d,J=7.5Hz,1H),7.58-7.52( m,1H),7.44-7.39(m,1H),7.33(d,J=7.8Hz,1H),6.85(d,J=6.0Hz,1H),6.17(d,J=8.3Hz,1H),5.64-5.5 8(m,1H),5.49-5.44(m,1H),5.08-5.02(m,1H),4.88-4.78(m,1H),4.76-4.67(m,2H),4.37-4.21(m,3H) ,4.06-4.00(m,1H),3.83-3.74(m,1H),2.82(s,3H),2.60-2.32(m,4H),1.54-1.46(m,1H),1.42(s,9H). 31 PNMR(162MHz,CD3OD)δ55.19(s,1P),53.20(s,1P).

[0769] Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}-2-[(methylamino)methyl]benzamide, intermediate 6 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-p 50 mg, 0.045 mmol of tert-butyl phosphate-2-yl carbamoyl benzyl methylcarbamate was added to a round-bottom flask and cooled to 0°C. A solution of trifluoroacetic acid (0.24 mL, 3.2 mmol) and DCM (0.58 mL) was added via syringe, and the reaction mixture was stirred at 0°C for 30 minutes. Next, the reaction mixture was concentrated to dryness and left under vacuum for 2 hours to obtain intermediate 6 (41 mg, 100%) as 2,2,2-trifluoroacetate. LC-MS (AA): m / z = 797.1 (M + H).

[0770] Example 6 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7- [6-6-oxo-6,9-dihydro-1H-purine-2-yl]carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl, compound 1(C-1)

[0771] [ka]

[0772] Step 1: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl, intermediate 7 To a solution of tert-butyloxycarbonyl-valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate (58 mg, 0.10 mmol) and 4-dimethylaminopyridine (12 mg, 0.10 mmol) in DMF (0.38 mL) and triethylamine (0.055 mL, 0.40 mmol), a solution of intermediate 6 (45 mg, 0.05 mmol) in DMF (1.5 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 15 minutes. Celite was added, and the mixture was concentrated to dryness. The crude residue absorbed by Celite was purified by reverse-phase flash column chromatography (0-40% ACN / triethylammonium acetate aqueous solution (10 mM)) and obtained as the N,N-diethylethaneamine salt [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8 -Methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (intermediate 7, 10 mg, 16%) were obtained. LCMS(AA): m / z = 1216.3 (M+H). 1H NMR(400MHz,CD3OD)δ8.70(s,1H),8.43( brs,1H),8.40(d,J=5.9Hz,1H),7.70(d,J=7.5Hz,1H),7.56-7.46(m,3H),7.42-7.38(m,1H),7.32-7.19(m,3H),6.84(d,J=5.5 Hz,1H),6.15(d,J=7.3Hz,1H),5.62-5.56(m,1H),5.54-5.48(m,1H),5.08-5.02(m,1H),5.04(s,2H),4.88-4.83(m,1H),4.81-4 .76(m,2H),4.53-4.47(m,1H),4.37-4.22(m,3H),4.08-4.03(m,1H),3.92-3.89(m,1H),3.82-3.74(m,1H),2.88(s,3H),2.58-2 .30(m,4H),2.10-2.03(m,1H),1.54-1.47(m,1H),1.46-1.44(m,3H),1.44(s,9H),0.98(d,J=6.8Hz,3H),0.93(d,J=6.8Hz,3H). 31 P NMR (162 MHz) z,CD3OD)δ55.01(s,1P),53.03(s,1P).

[0773] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl, intermediate 8

[0774] [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate tert-butyl was used instead of intermediate 7 (10 mg, 0.008 mmol) to prepare the title compound according to the procedure described in Step 2 of Example 5, and stirred at room temperature for 10 minutes to 2,2,2- [2-({9-[(2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-{[(2NS)-2-amino-3-methylbutanoyl]amino}propanoyl]aminobenzyl (intermediate 8, 9 mg, 100%) was obtained as trifluoroacetate. LCMS(AA): m / z = 1116.3 (M + H). Step 3: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15 aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl, compound C-1

[0775] Intermediate 8 (8 mg, 0.007 mmol) and N-succinimidyl 6-maleimidohexanoate (3.1 mg, 0.010 mmol) were dissolved in THF (0.20 mL) and DMF (0.10 mL), respectively, to which N,N-diisopropylethylamine (2.5 μL, 0.014 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0-100% ACN / ammonium acetate aqueous solution (10 mM)) to obtain the ammonium salt [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1, [3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl (C-1, 4.2 mg, 45%) was obtained. LCMS(AA):m / z=1309.4(M+H).HRMS(m / z):[M+H]+C 55 H 66 N 12 O 18 The calculated value for P2S2 is 1309.3607, and the measured value is 1309.3639.

[0776] Example 7 Carbono chloride 2-[tert-butoxycarbonyl(methyl)amino]ethyl (intermediate 9)

[0777] [ka]

[0778] Step 1: Carbono chloride 2-[tert-butoxycarbonyl(methyl)amino]ethyl, intermediate 9 To a solution of tert-butyl N-(2-hydroxyethyl)-N-methylcarbamate (296 mg, 1.69 mmol) cooled to 0°C in THF (4.2 mL) and N,N-diisopropylethylamine (1 mL, 5.9 mmol), triphosgene (752 mg, 2.53 mmol) was slowly added. The reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was filtered, the solids were washed with THF, and the filtrate was concentrated to dryness to obtain crude 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonochloride (intermediate 9, 402 mg, 100%).

[0779] Example 8 N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (intermediate 10)

[0780] [ka]

[0781] Step 1: 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid The title compound was prepared according to the procedure described in Example 6, Step 1, starting with 2-[(methylamino)methyl]benzoate HCl (150 mg, 0.72 mmol) instead of intermediate 6. Following purification by silica gel chromatography (0-25% MeOH / DCM), 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (306 mg, 67%) was obtained. LCMS (AA): m / z = 583.4 (MH).

[0782] Step 2: N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl, intermediate 10 To a solution of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (295 mg, 0.50 mmol) cooled to 0°C and mixed with THF (1.5 mL), oxalyl chloride (2.0 M solution in DCM, 0.25 mL, 0.50 mmol) was added, followed by the addition of 3 drops of DMF. The reaction mixture was stirred at 0°C for 45 minutes. The mixture was then concentrated to dryness to obtain crude intermediate 10 (304 mg, 100%).

[0783] Example 8A (4-Chloro-4-oxobutyl)methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl (intermediate 34)

[0784] [ka]

[0785] Step 1: 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]methyl butanoate The title compound was prepared according to the procedure described in Example 6, Step 1, starting with methyl HCl 4-(methylamino)butanoate (1.44 g, 8.16 mmol) instead of intermediate 6. Following purification by silica gel chromatography (0-70% HCl / DCM), methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 92%) was obtained. LCMS (AA): m / z = 549.3 (MH).

[0786] Step 2: 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]methylbutanoate (3.45 g, 6.27 mmol) was added to a solution of THF (40.7 mL) and water (20.3 mL) at 0°C with lithium hydroxide monohydrate (657 mg, 15.7 mmol). The homogeneous mixture was then warmed to room temperature and stirred for 2 hours. The reaction mixture was cooled to 0°C and hydrochloric acid (1.0 mol / L aqueous solution, 1.83 mL). The mixture was adjusted to pH < 4 by adding an additional 1N HCl. This solution was warmed to room temperature and diluted with HCl and water. The aqueous phase was further extracted with HCl. The combined organic phase was washed with brine and dried on magnesium sulfate. After filtration and solvent removal, the residue was purified by silica gel chromatography (0-10% MeOH / SiO). Upon solvent removal, 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (3.17 g, 94%) was obtained as a white solid. LCMS (AA): m / z = 535.3 (MH).

[0787] Step 3: (4-chloro-4-oxobutyl)methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbony Starting with 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (387 mg, 0.721 mmol) instead of 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (387 mg, 0.721 mmol), the title compound was prepared according to the procedure described in Step 2 of Example 8, and crude intermediate 34 (400 mg, 100%) was obtained during the concentration of the reaction mixture.

[0788] Example 9 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl (intermediate 7)

[0789] [ka]

[0790] [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl Ru, intermediate 7 The title compound was prepared according to the procedure described in Example 5, Step 1, using intermediate 10 instead of N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate tert-butyl (intermediate 1). The crude product was purified by reverse-phase flash column chromatography (0-50% ACN / ammonium bicarbonate (5 mM) aqueous solution) to obtain intermediate 7 as an ammonium salt. Next, this substance was dissolved in MeOH (2 mL) and triethylamine (5 mL) was added. The mixture was shaken for 1 minute, the solvent was removed, and it was freeze-dried overnight to obtain the N,N-diethylethaneamine salt [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9, [11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (147 mg, 48%) was obtained. LCMS(AA): m / z = 1216.3 (M + H). 1 1H NMR Example 6, Process Same as 1.

[0791] Example 10 [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecane-13-ylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (intermediate 11)

[0792] [ka]

[0793] Step 1: 2-(2,5,8,11-tetraoxa-14-azapentadecane-15-yl)benzoic acid To a solution of 2-carboxybenzaldehyde (1.10 g, 7.3 mmol) dissolved in MeOH (15 mL), 2,5,8,11-tetraoxatridecane-13-amine (1.82 g, 8.8 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Boron hydride Sodium (139 mg, 3.7 mmol) was slowly added gradually. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was stopped by the addition of acetone (0.65 mL), and then concentrated to dryness. The crude residue was purified by silica gel chromatography (0-25% MeOH / DCM) to obtain 2-(2,5,8,11-tetraoxa-14-azapentadecane-15-yl)benzoic acid (2.25 g, 87%). LCMS (AA): m / z = 342.3 (M + H).

[0794] Step 2: 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecane-15-yl)benzoic acid The title compound was prepared using DCM as the solvent, starting with 2-(2,5,8,11-tetraoxa-14-azapentadecane-15-yl)benzoic acid (764 mg, 2.24 mmol) instead of intermediate 6, following the procedure described in Example 6, Step 1, and stirring for 16 hours. Following purification by silica gel chromatography (0-25% MeOH / DCM), 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecane-15-yl)benzoic acid (855 mg, 47%). LCMS (AA): m / z = 759.4 (MH).

[0795] Step 3: [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecane-13-ylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl, intermediate 11 Starting with 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonylamino)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (591 mg, 0.777 mmol) as the basis, the title compound was prepared according to the procedure described in Step 2 of Example 8, and crude intermediate 11 (605 mg, 100%) was obtained during the concentration of the reaction mixture.

[0796] Example 11 The compounds listed in Table 3 below were prepared as described in Example 5, using the starting materials shown in the table instead of N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate tert-butyl (intermediate 1).

[0797] [Table 3-1]

[0798] [Table 3-2]

[0799] Example 12 N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}-4-(methylamino)butanamide (intermediate 17)

[0800] [ka]

[0801] Step 1: [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl] tert-butyl methylcarbamate The title compounds are, respectively, 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-1,9-dihydro-6H-purine-6-one and 7-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6- Amino-9H-purine-9-yl)-15-fluoro-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one (compound number 14, see WO2018100558A2, 200 mg, 0.265 mmol) and intermediate 3 (503 mg, 1.59 mmol) were used to prepare the compound according to the procedure described in Example 5, Step 1. Purification by reverse-phase flash column chromatography (0-50% ACN / ammonium bicarbonate (5mM) aqueous solution) yielded the ammonium salt [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate tert-butyl (148 mg, 59%). LCMS(AA): m / z = 910. 5(M+H). 1 H NMR(400MHz,D2O)δ8.70(s,1H),8.3 4(s,1H),8.01(s,1H),7.28(s,1H),6.56(d,J=16.1Hz,1H),6.43(d,J=8.4Hz, 1H),5.67(dd,J=51.0,3.9Hz,1H),5.18-4.99(m,2H),4.82-4.77(m,1H),4.63 -4.58(m,1H),4.49-4.38(m,3H),4.31-4.25(m,1H),4.12-4.06(m,1H),3.43- 3.36(m,2H),2.88(s,3H),2.73-2.68(m,2H),2.04-1.97(m,2H),1.34(s,9H). 31 P-NMR(162MHz,D2O)δ55.47(s,1P),52.07(s,1P). 19 F-NMR(376MHz,CD3OD)δ-165.50(m,1F),-203.45(m,1F).

[0802] Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}-4-(methylamino)butanamide, intermediate 17 The title compound is [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate tert-butyl as an ammonium salt instead of [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R, 15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate tert-butyl (42 mg, 0.045 mmol) was used and prepared by following the procedure described in Step 2 of Example 5 and stirring at room temperature for 30 minutes. N-{9-[(2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15R, 15aR, 16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide (intermediate 17, 41 mg, 100%) was obtained as 2,2,2-trifluoroacetate. LCMS(AA): m / z = 810.2 (M + H).

[0803] Example 13 The compounds listed below in Table 5 were prepared as described in Example 12, using the starting materials shown in Table 4 instead of 4-[tert-butoxycarbonyl(methyl)amino]butanoic acid isobutoxycarbonyl (intermediate 3).

[0804] [Table 4]

[0805] [Table 5]

[0806] Example 14 The compounds listed in Table 7 were prepared as described in Example 6, using the starting materials shown in Table 6 instead of intermediate 6.

[0807] [Table 6]

[0808] [Table 7]

[0809] Example 15 The compounds listed in Table 8 (Intermediate 19) were prepared as described in Example 6, Step 1, using the starting materials shown in the table instead of tert-butyloxycarbonyl-valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl) carbonate.

[0810] [Table 8]

[0811] Example 16 The compounds listed in Table 10 were prepared as described in steps 2 and 3 of Example 6, using the starting materials shown in Table 9 instead of intermediate 7.

[0812] [Table 9]

[0813] [Table 10]

[0814] Example 17 The compounds listed in Table 12 are N-succinimidyl 6-maleimidohexanoate. Alternatively, the preparation was carried out using the starting materials shown in Table 11, as described in Example 6, Step 3.

[0815] [Table 11-1]

[0816] [Table 11-2]

[0817] [Table 11-3]

[0818] [Table 12-1]

[0819] [Table 12-2]

[0820] [Table 12-3]

[0821] Example 18 The compounds listed in Table 14 were prepared as described in Example 6, Steps 2 and 3, using the starting materials shown in Table 13 instead of intermediates 7 and 6-maleimidohexanoate N-succinimidyl.

[0822] [Table 13-1]

[0823] [Table 13-2]

[0824] [Table 13-3]

[0825] [Table 13-4]

[0826] [Table 13-5]

[0827] [Table 14-1]

[0828] [Table 14-2]

[0829] [Table 14-3]

[0830] [Table 14-4]

[0831] [Table 14-5]

[0832] [Table 14-6]

[0833] Example 19 [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo- 6,9-Dihydro-1H-Prin-2-yl}amino)benzyl]-2,2-dimethyl-4-oxobutyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-Dioxo-2,5-Dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl, Compound 20(C-20)

[0834] [ka]

[0835] Step 1: [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6 -Oxo-6,9-dihydro-1H-purine-2-yl}amino)benzyl]-2,2-dimethyl-4-oxobutyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl, compound 20 (4-nitrophenyl)carbonate [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) are dissolved in DMF (0.60 mL) and N,N-diisopropylethylamine (26 μL, 0.15 mmol) at room temperature as N-{9-[(2R,5 A solution of R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}-3,3-dimethyl-4-(methylamino)butanamide (intermediate 14, 20 mg, 0.02 mmol) in DMF (0.54 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. Celite was added, and the mixture was concentrated to dryness. The crude residue absorbed by Celite was purified by reverse-phase flash column chromatography (0-100% ACN / ammonium acetate aqueous solution (10 mM)) and obtained as an ammonium salt [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][ [1,3,6,9,11,2,10]Pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl (C-20, 4 mg, 17%) was obtained. LCMS(AA): m / z = 1287.4 (MH).

[0836] Example 20 The compounds listed in Table 16 are processed using starting materials A and B shown in Table 15 instead of intermediate 14 and (4-nitrophenyl)carbonate [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl, respectively. It was prepared as described in Example 19.

[0837] [Table 15-1]

[0838] [Table 15-2]

[0839] [Table 16]

[0840] Example 21 N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14-pentaoxaheptadecane-17-amide (intermediate 20)

[0841] [ka]

[0842] Step 1: (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-euic acid To a solution of 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione (4.0 g, 10.5 mmol) in anhydrous DCM (10 mL), (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoate (3.4 g, 12.9 mmol) dissolved in DMF (40 mL) was added, followed by the addition of N,N-diisopropylethylamine (6.9 mL, 42 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered and concentrated to dryness. The crude residue was purified by preparative HPLC to obtain (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-euic acid (1.26 g, 24%). LC-MS (AA): m / z = 489.3 (M + H).

[0843] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14-pentaoxaheptadecane-17-amide, intermediate 20 (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-euic acid (65 mg, 0.133 mmol) and N,N'-dicyclohexylcarbodiimide (30 mg, 0.145 mmol) were dissolved in 1,4-dioxane (0.68 mL) and N-hydroxysuccinimide (15.3 mg, 0.133 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. Celite was added, the mixture was filtered, rinsed with 1,4-dioxane, and concentrated to dryness. The crude residue absorbed by Celite was purified by silica gel chromatography (0-10% MeOH / DCM) to obtain intermediate 20 (14.6 mg, 19%). LCMS(AA): m / z = 586.3(M+H).

[0844] Example 22 The compounds listed in Table 17 were prepared as described in Example 9, using the starting materials shown in the table instead of intermediate 10.

[0845] [Table 17]

[0846] Example 23 (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotet Radicin-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, compound 24(C24)

[0847] [ka]

[0848] Step 1: (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-ylo Xy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate methyl Using N,N-diisopropylethylamine as the base, the title compound was prepared starting with intermediate 6 (104 mg, 0.094 mmol) and (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoylamino]-4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylate methyl (150 mg, 0.16 mmol) according to the procedure described in Example 6, Step 1 (for synthesis, see Jeffrey, SC et al. Bioconjugate Chem. 2006, 17, 831-840), and as the ammonium salt (2S,3S,4S, 5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfa Nyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate (71 mg, 48%) was obtained. LCMS(AA): m / z = 1570.9 (M + H).

[0849] Step 2: (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, intermediate 22 (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8- To a solution of methyl tetrahydro-2H-pyran-2-carboxylate (66 mg, 0.042 mmol) of metanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate (66 mg, 0.042 mmol), LiOH (0.5 M aqueous solution, 1.25 mL, 0.625 mmol) was added at room temperature, and the reaction mixture was stirred for 2.5 hours. The reaction mixture was neutralized to pH 7 with 1 M HCl, and the mixture was concentrated to dryness. The crude residue was purified by reverse-phase flash column chromatography (0-50% ACN / ammonium bicarbonate (5 mM) aqueous solution) to obtain intermediate 22 (26 mg, 50%) as an ammonium salt. Next, this substance was dissolved in MeOH (5 mL), and triethylamine (1 mL) was added. The mixture was shaken for 1 minute. The solvent was removed, and the resulting residue was freeze-dried overnight to obtain the N,N-diethylethaneamine salt (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldeca Hydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (intermediates 22, 30 mg, and 100%) were obtained.LCMS(AA): m / z = 1209.0 (M + H).

[0850] Step 3: (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14 -(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, compound 24 The title compound was prepared using intermediate 22 (24 mg, 0.02 mmol) instead of intermediate 8, following the procedure described in Step 3 of Example 6, and the ammonium salt was (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10 -Dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (C-24, 17 mg, 71%) was obtained. LCMS (AA): m / z = 1402.4 (M+H). HRMS (m / z): [M+H]+, C 56 H 65 N 11 O 24Calculated value for P2S2: 1402.3193; Measured value: 1402.3232.

[0851] Example 24 The compounds listed in Table 18 (Compound 25) were each prepared as described in Example 23, Step 3, using the starting materials shown in the table instead of N-succinimidyl 6-maleimidohexanoate.

[0852] [Table 18]

[0853] [ka]

[0854] Example 25 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo -6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl}carbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl, compound 26(C26)

[0855] [ka]

[0856] Step 1: 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid To a solution of glycine (24 mg, 0.32 mmol) in triethylamine (0.10 mL, 0.72 mmol), DMF (0.50 mL), and DMSO (0.50 mL), (4-nitrophenyl)carbonate [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (220 mg, 0.34 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then cooled to 0°C, water (1 mL) was added, and the solution was acidified to pH 4 by adding formic acid. The mixture was concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0-100% ACN / 0.1% formic acid aqueous solution) to obtain 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid (87 mg, 44%). LCMS (AA): m / z = 586.3 (MH).

[0857] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]- 6-Oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl}carbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl, compound 26 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrole-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid (40 mg, 0.068 mmol) and triethylamine (35 μL, 0.25 mmol) in DMF (1 mL) were mixed with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (46 mg, 0.12 mmol) at room temperature, and the reaction mixture was stirred for 10 minutes. Next, a solution of (2R)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide (intermediate 15, 50 mg, 0.047 mmol) in DMF (0.60 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. Next, the mixture was cooled to 0°C, water (1 mL) was added, and the solution was acidified to pH 4 by adding formic acid. The reaction mixture was concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0-100% ACN / ammonium acetate aqueous solution (10 mM)). [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9 ,11,2,10]Pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl}carbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl(C-2 6, 32 mg, and 50% were obtained. LCMS(AA): m / z = 1316.4(M+H).HRMS(m / z): [M+H]+, C 53 H 67 N 13 O 19 Calculated value for P2S2: 1316.3666; Measured value: 1316.3705.

[0858] Example 26 (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminoacetyl)-N-(9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2, 10-Dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide, compound 27 (C-27)

[0859] [ka]

[0860] Step 1: {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl} tert-butyl carbamate In DMF (1.6 mL), a mixture of intermediate 15 (60 mg, 0.05 mmol) and (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl (19 mg, 0.07 mmol) was added to triethylamine (50 μL, 0.355 mmol), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was stopped with water, Celite was added, and the reaction mixture was concentrated to dryness. The crude residue absorbed by Celite was purified by reverse-phase column chromatography (0-100% ACN / ammonium bicarbonate aqueous solution (5 mM)). The product was prepared. Triethylamine (14 μL, 0.1 mmol) was added to an aqueous solution of the product. By freeze-drying overnight, the N,N-diethylethaneamine salt {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl}carbamate tert-butyl (49 mg, 87%) was obtained. LCMS(AA): m / z = 904.4(M+H).

[0861] Step 2: (2R)-1-(aminoacetyl)-N-{9-[(2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxy as an N,N-diethylethaneamine salt To a solution of tert-butyl sadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)pyrrolidine-1-yl]-2-oxoethyl}carbamate (37 mg, 0.033 mmol) in MeOH (1 mL), hydrochloric acid (4 M solution in dioxane, 1 mL, 4 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was concentrated to dryness and left under vacuum for 2 hours to obtain (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide (37 mg, 100%) as the hydrochloride salt. LCMS(AA): m / z = 804.2 (M + H).

[0862] Step 3: (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-(9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydro Xy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-ethanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide, compound 27 (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7- Example 26 was developed starting with [yl]-6-oxo-6,9-dihydro-1H-purine-2-yl]pyrrolidine-2-carboxamide (30 mg, 0.027 mmol) and (2S)-2-[[(2NS)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoic acid (2,5-dioxopyrrolidine-1-yl) (24 mg, 0.04 mmol). The title compound was prepared according to the procedure described in Step 1 (see US20180015176A1 for synthesis). Purification by reverse-phase flash column chromatography (0-100% ACN / formic acid aqueous solution (0.1%)) was performed on (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R ,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}pyrrolidine-2-carboxamide (C-27, 20 mg, 64%) was obtained. LCMS(AA): m / z = 1167.1 (M+H). HRMS(m / z): [M+H]+, C 45 H 60 N 12 O 17 Calculated value for P2S2: 1167.3189; Measured value: 1167.3206.

[0863] Example 27 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl, compound 28(C-28)

[0864] [ka]

[0865] Step 1: (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10 ]Pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropane-2-yl]amino}-3-methyl-1-oxobutan-2-yl]amino}-2-oxoethyl)tert-butyl carbamate The title compound was prepared according to the procedure described in Step 3 of Example 6, starting with intermediate 8 (23 mg, 0.016 mmol) and (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl) instead of 6-maleimidohexanoate N-succinimidyl. The reaction mixture was stirred at room temperature for 30 minutes. The N,N-diethylethaneamine salt was purified by reverse-phase flash column chromatography (0-100% ACN / triethylammonium acetate aqueous solution (10 mM)) to obtain (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrim Zin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropane-2-yl]amino}-3 13 mg, 57% tert-butyl methyl-1-oxobutan-2-yl]amino}-2-oxoethyl)carbamate was obtained. LC-MS (AA): m / z = 1273.1 (M + H).

[0866] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl, compound 28 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine- (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R The title compound was prepared starting with tert-butyl (6.6 mg, 0.005 mmol) of )-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]amino}-2-oxoethyl)carbamate, following the procedure described in Step 2 of Example 5. The reaction mixture was stirred at room temperature for 5 minutes to obtain [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (C-28, 5.6 mg, 91%) as 2,2,2-trifluoroacetate. LCMS(AA): m / z = 1173.3(M+H). Example 28 The compounds listed in Table 20 were prepared as described in Example 27, using the starting materials shown in Table 19 instead of (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl.

[0867] [Table 19]

[0868] [Table 20]

[0869] Example 29 {14-[(2,5-dioxopyrrolidine-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate tert-butyl (intermediate 23)

[0870] [ka]

[0871] Step 1: {14-[(2,5-dioxopyrrolidine-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate tert-butyl, intermediate 23 N-hydroxysuccinimide (46 mg, 0.39 mmol) was added to a solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-euic acid (120 mg, 0.32 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (78 mg, 0.49 mmol) in DCM (6.4 mL) at room temperature. The reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was stopped with water and extracted with DCM (×3). The combined organic phases were washed with brine, dried with (Na2SO4), filtered, and evaporated to obtain crude {14-[(2,5-dioxopyrrolidine-1-yl)oxy]-14-oxo-3,6,9,12-tetraoxatetradec-1-yl}carbamate tert-butyl (intermediate 23, 146 mg, 100%). LCMS(AA): m / z = 447.2 (MH).

[0872] Example 30 N-[(1S)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]-1-methyl-2-oxo-ethyl]carbamate tert-butyl (intermediate 24)

[0873] [ka]

[0874] Step 1: 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid Starting with 2-carboxybenzaldehyde (6.0 g, 40 mmol) and 2,4-dimethoxybenzylamine (12 mL, 80 mmol), the title compound was prepared according to the procedure described in Step 1 of Example 10 to obtain 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (7.6 g, 61%). LCMS(AA): m / z=3 02.2(M+H).

[0875] Step 2: 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (380 mg, 1.26 mmol) and triethylamine (0.5 mL, 3.55 mmol) were dissolved in DMF (6 mL) to (tert-butoxycarbonyl)-L-alaniate 2,5-dioxopyrrolidine-1-yl (640 mg, 2.2 mmol). Water (2 mL) was added to help solubilize the reaction mixture, and the mixture was stirred at room temperature for 1 day. The reaction mixture was then concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0-100%) ACN / formic acid (0.1%) aqueous solution to obtain 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (322 mg, 51%). LCMS(AA): m / z = 473.3(M+H). Step 3: N-[(1S)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]-1-methyl-2-oxo-ethyl]carbamate tert-butyl (intermediate 24)

[0876] The title compound was prepared using 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (270 mg, 0.57 mmol) instead of 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid according to the procedure described in Step 2 of Example 1 to obtain crude intermediate 24 (280 mg, 100%).

[0877] Example 31 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-(9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}benzamide, compound 31(C-31)

[0878] [ka]

[0879] Step 1: [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](amino)-1-oxopropane-2-yl]amino}-3-methyl-1-oxobutan-2-yl]tert-butyl carbamate The title compound was prepared according to the procedure described in Step 1 of Example 26, using intermediate 16 (45 mg, 0.53 mmol) and (tert-butoxycarbonyl)-L-valate 2,5-dioxopyrrolidine-1-yl) instead of intermediate 15 and (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl, respectively. The ammonium salt was obtained by purification by reverse-phase flash column chromatography (0-100% ACN / ammonium acetate (10 mM) aqueous solution) and [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfani Rudecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate tert-butyl (24 mg, 44%) was obtained. LCMS(AA): m / z = 1053.0 (M + H).

[0880] Step 2: 2-{[(2S)-2-({(2S)-2-amino]-3-methylbutanoyl]ami}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}benzamide [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate tert-buty Instead of ammonium salt, use [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine- Starting with tert-butyl 2-yl carbamoyl benzyl amino-1-oxopropan-2-yl amino-3-methyl-1-oxobutan-2-yl carbamate (24 mg, 0.023 mmol), the title compound was prepared according to the procedure described in Step 2 of Example 5, and 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[((2R,5R, 7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}benzamide (24 mg, 97%) was obtained. LCMS(AA): m / z = 953.1 (M + H).

[0881] Step 3: 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-(9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}benzamide, compound 31 Starting with 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide (24 mg, 0.022 mmol) as 2,2,2-trifluoroacetate instead of intermediate 8, the title compound was produced according to the procedure described in Example 6, Step 3. Prepare and use the ammonium salt 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydrox C-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}benzamide (C-31, 6.2 mg, 23%) was obtained. LCMS (AA): m / z = 1146.1 (M + H). HRMS (m / z): [M + H]+, C 46 H 57 N 11 O 16 Calculated value for P2S2: 1146.2974; Measured value: 1146.2998.

[0882] Example 32 4-Nitrophenyl Carbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl (intermediate 25)

[0883] [ka]

[0884] Step 1: 6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexaneamide To a solution of (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide (240 mg, 0.82 mmol) dissolved in DMF (2 mL) and THF (5.5 mL), N,N-diisopropylethylamine (0.39 mL, 2.24 mmol) was added. The reaction mixture was cooled to 0°C, and 1-{[6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione (320 mg, 0.75 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 30 minutes. The reaction mixture was diluted with water and extracted with ELISA (3×). The combined organic phases were washed with saturated sodium bicarbonate aqueous solution and brine, dried (Na2SO4), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (50-100% SiO2 / DCM) to obtain 6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide (347 mg, 76%). LCMS(AA): m / z = 609.4(M+H).

[0885] Step 2: 4-nitrophenyl carbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl, intermediate 25 THF (4.2 mL) and N,N-diisopropylethylamine (0.37 mL, 2.1 mL) of 6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide (257 mg, 0.42 mmol) cooled to 0°C. 4-nitrophenyl chloroformate (362 mg, 1.69 mmol) was added all at once to a mol(1) solution. The reaction mixture was heated to room temperature and stirred for 3 hours. The reaction mixture was diluted with water and extracted by DCM (3×). The combined organic phase was washed with saturated sodium bicarbonate solution and brine, dried (Na₂SO₄), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-100% siRNA / DCM) to obtain 4-nitrophenyl carbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]aminobenzyl (intermediate 25, 137 mg, 42%). LCMS (AA): m / z = 774.4 (M + H).

[0886] Example 33 The compounds listed in Table 21 (Intermediate 26) were prepared as described in Example 32, using the starting materials shown in the table instead of 1-{[6-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione.

[0887] [Table 21]

[0888] Example 34 (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidine-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide (intermediate 27) and (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (intermediate 27A)

[0889] [ka]

[0890] Step 1: (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate The title compound was prepared by following the procedure described in Step 1 of Example 26, starting with (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidine-1-yl)oxy]-4-oxobutanoate tert-butyl (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol), respectively, instead of intermediate 15 and (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl. Purification by silica gel chromatography (0-10% MeOH / DCM) yielded (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (3.8g, 100%).

[0891] Process 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,2 0-Heptaoxa-23-Azaheptacosan-27-Tert-Butyl Eucate (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added, and the mixture was stirred under hydrogen (15 psi) for 3 hours. The reaction mixture was filtered, washed with methanol, and the filtrate was evaporated to dryness to obtain (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (2.56 g, 85%).

[0892] Step 3: (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (25S)-25amino-24-oxo2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euicate tert-butyl (3.32 g, 6.5 mmol), 4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoic acid (1.5 g, 4.91 mmol), and 1-hydroxy-7-azabenzotriazole (884 mg, 6.57 mmol) were mixed in a DCM (50 mL) solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.25 g, 6.56 mmol) and triethylamine (1.26 mL, 9.1 mmol) were added at 0°C. The reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture was diluted with ethyl acetate, and potassium carbonate (20 mL, 4.0 M aqueous solution) was added. The mixture was stirred for 15 minutes, the organic phase was isolated, washed with saturated aqueous solution of sodium bicarbonate and brine, dried to (Na2SO4), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-10% MeOH / DCM) to obtain (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (2.27 g, 44%).

[0893] Step 4: (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl] The title compound was prepared by starting with (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-eurycate tert-butyl (278 mg, 0.35 mmol) instead of tert-butyl tylcarbamate and following the procedure described in step 2 of Example 5. Purification by silica gel chromatography (0-20% MeOH / DCM) yielded (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (intermediate 27A, 180 mg, 70%). LC-MS (AA): m / z = 742.4 (M + H).

[0894] Step 5: (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidine-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide, intermediate 27 The title compound was prepared by following the procedure described in Example 29, starting with (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (60 mg, 0.08 mmol) instead of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-euic acid. The reaction mixture was stirred at room temperature for 30 minutes to obtain (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidine-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide (intermediate 27, 68 mg, 100%). LCMS(AA): m / z = 839.4(M+H).

[0895] Example 35 The compounds listed in Table 23 were prepared as described in Example 6, Step 3, using the starting materials shown in Table 22 instead of N-succinimidyl 6-maleimidohexanoate.

[0896] [Table 22]

[0897] [Table 23]

[0898] Example 36 {(29S)-30-[(2,5-dioxopyrrolidine-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl} tert-butyl carbamate (intermediate 30)

[0899] [ka]

[0900] Step 1: (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontane-30-euic acid The title compound was prepared by stirring at room temperature for 2 hours using DMF as the solvent, starting with (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (238 mg, 1.17 mmol) and 1-[(26-oxo-2,5,8,11,14-pentaoxaheptadecane-17-yl)oxy]pyrrolidine-2,5-dione (300 mg, 0.58 mmol) instead of (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoic acid and 1-[(17-oxo-2,5,8,11,17,20,23-octaoxahexacosan-26-yl)oxy]pyrrolidine-2,5-dione (300 mg, 0.58 mmol) as the procedure described in Step 1 of Example 21. (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontane-30-euic acid (344 mg, 99%) was obtained by purification using reverse-phase flash column chromatography (0-100% ACN / triethylammonium acetate (10 mM) aqueous solution). LC-MS (AA): m / z = 599.4 (M + H).

[0901] Step 2: {(29S)-30-[(2,5-dioxopyrrolidine-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}carbamate tert-butyl, intermediate 30 The title compound was prepared by starting with (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontane-30-euic acid (100 mg, 0.166 mmol) instead of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-euic acid, and following the procedure described in Step 2 of Example 21, using DCM as the solvent, and stirring at room temperature for 1 hour. The reaction mixture was filtered and concentrated to obtain crude {(29S)-30-[(2,5-dioxopyrrolidine-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}carbamate tert-butyl (intermediate 30, 116 mg, 100%).

[0902] Example 37 N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid[4-[[(2S)-2-[[(2S)-2-(tert-butoxymethyl Bonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (intermediate 32)

[0903] [ka]

[0904] Step 1: N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl 2-Amino-9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-1H-purin-6-one (1.62 g, 3.27 mmol) (see Prakash, TPJ Med. Chem. 2005, 48, 1199-1210 for synthesis) was dissolved in anhydrous pyridine, concentrated to dry (3 × 3 mL), and then placed under vacuum for 15 minutes. The residue was dissolved in pyridine (43 mL) under an argon atmosphere, and a solution of intermediate 10 (5.91 g, 9.80 mmol) in pyridine (32 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. MeOH (20 mL) was added, and the reaction mixture was concentrated to dry. MeOH (100 mL) and ammonium hydroxide (28-30% aqueous solution, 50 mL) were added, and the resulting mixture was stirred for 60 minutes. The reaction mixture was concentrated to dryness, the crude residue was adsorbed onto Celite, and purified by silica gel chromatography (0-100% SiO₂ / hexane) to obtain N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (2.17 g, 63%). LCMS(AA): m / z = 1062.0(M+H).

[0905] Step 2: N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (2.17 g, 2.04 mmol) dissolved in THF (27 mL) was added to a polypropylene test tube. Triethylamine (2.26 mL, 16.1 mmol) was added, followed by triethylamine trifluoride (1.19 mL, 7.33 mmol), and the reaction mixture was stirred at room temperature for 16 hours. The reaction of the reaction mixture was slowly stopped by adding a saturated aqueous solution of sodium bicarbonate, and then diluted with ethyl acetate. The organic phase was separated, washed with brine, dried with (Na2SO4), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-20% MeOH / DCM) to obtain N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (1.18 g, 69%). LCMS(AA): m / z = 834.4 (M+H).

[0906] Step 3: N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl, intermediate 32 N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (1.13 g, 1.36 mmol) was dissolved in anhydrous pyridine and concentrated to dryness (3 × 15 mL). The residue was dissolved in pyridine (13.3 mL), and 4,4'-dimethoxytrityl chloride (650 mg, 1.9 mmol) was added, followed by 4-dimethylaminopyridine (8 mg, 0.068 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness, adsorbed onto Celite, and purified by silica gel chromatography (0-5% MeOH / DCM, with 0.5% NET3) to obtain N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (intermediate 32, 1.12 g, 73%). LCMS(AA): m / z = 1136.5 (M+H).

[0907] Example 38 [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl or [ 2-({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (intermediate 33)

[0908] [ka]

[0909] Step 1: N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purine-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]N-methylcarbamate[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl] -6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (intermediate 32, 1.1g, 0.97mmol) and N-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro- A mixture of 5-purine-9-yltetrahydrofuran-2-yl]methyl]sulfamic acid (4-nitrophenyl) (667 mg, 1.17 mmol) (for synthesis, see PCT application number PCT / IB2019 / 052364), 4-dimethylaminopyridine (122 mg, 0.97 mmol), and a 4-angstrom molecular sieve (1.74 g) in anhydrous DCM (13.6 mL) was stirred at room temperature under argon for 10 minutes. Then triethylamine (0.60 mL, 4.3 mmol) was added, and the reaction mixture was stirred at 40°C for 16 hours. The reaction mixture was filtered and concentrated, and the resulting residue was purified by reverse-phase column chromatography (10-100% ACN / ammonium bicarbonate aqueous solution (5mM)) to obtain N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-{[tert-butyl(dimethyl)silyl]oxy}-4-fluoro-5-purine [-9-yl-tetrahydrofuran-2-yl]methyl}sulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (0.94g, 62%) was obtained. LCMS(AA): m / z = 1565.0 (M + H).

[0910] Step 2: N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purine-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]N-methylcarbamic acid[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl In a polypropylene test tube, N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]cal To a solution of [bamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (939 g, 0.60 mmol) in THF (40 mL), triethylamine trifluoride (0.52 mL, 3.17 mmol) and triethylamine (0.98 mL, 6.95 mmol) were added. The reaction solution was stirred overnight at room temperature, then diluted with dichloromethane and sequentially washed with saturated aqueous solution of NaHCO3 and brine. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography (10-100% ACN / ammonium bicarbonate aqueous solution (5 mM)) to obtain N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purine-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamic acid[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl( 795g, 89% was obtained. LCMS(AA): m / z = 1452.2(M+H).

[0911] Step 3: [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purine-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purine-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid Diphenyl phosphite (0.225 mL, 1.17 mmol) was added to a solution of N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purine-9-yl-tetrahydrofuran-2-yl]methyl]sulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (792 g, 0.55 mmol) in pyridine (2 mL) at 0°C. Upon completion of the additions, the reaction mixture was stirred at room temperature for 1 hour. Next, triethylamine (0.62 mL, 4.37 mmol) and water (0.51 mL, 28.3 mmol) were added, and the reaction mixture was stirred at room temperature for a further 60 minutes, after which it was concentrated to dryness. The residue was dissolved in acetic acid (1.25 mL, 21.8 mmol) and water (0.33 mL, 18.6 mmol) and stirred at room temperature for 1 hour. The reaction of the reaction mixture was stopped by adding triethylsilane (4.3 mL, 26.2 mmol), and the mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was concentrated to dryness, and the residue was purified by reverse-phase column chromatography (10-100% ACN / ammonium bicarbonate aqueous solution (5 mM)) to obtain [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purine-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purine-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid (396 mg, 59%) as an ammonium salt. LCMS(AA): m / z = 1213.4(M+H).

[0912] Step 4: [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl, intermediate 33

[0913] 0.055 mL of phosphorochloride diphenyl (0.26 mmol) was added to pyridine (1.6 mL), and the solution was cooled to -35°C. [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purine-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purine-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid (80 mg, 0.065 mmol) was added dropwise over 15 minutes to a cooled reaction mixture containing DCM (2.5 mL) and pyridine (3.2 mL). The resulting solution was stirred at -35°C for 30 minutes. The solids 3H-1,2-benzodithiol-3-one (25 mg, 0.15 mmol) and water (0.056 mL) were added to the cooled reaction mixture, and the mixture was stirred at room temperature for 1 hour. The reaction solution was then cooled to 0°C in an ice bath, and the reaction was stopped by adding an aqueous solution of sodium thiosulfate (0.2 M aqueous solution, 0.14 mL, 0.70 mmol). The mixture was stirred at room temperature for 5 minutes and concentrated under reduced pressure.The resulting residue was purified by reverse-phase column chromatography (0-50% ACN / triethylammonium acetate aqueous solution (10 mM)) to obtain the N,N-diethylethaneamine salt [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]- 3-methylbutanoyl}amino)propanoyl]aminobenzyl or [2-({9-[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purine-9-yl)-2-sulfanyldecahydro-5,8-methanofl[2,3-m][1,3,6,9,10,11,2]tetrao Xathiazafosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]aminobenzyl (intermediate 33, 27 mg, 31%) were obtained. LCMS(AA): m / z = 1227.9 (M + H). 1 H NM R(400MHz,CD3OD)δ9.14(s,1H),8.75(s,1H),8.63(s,1H),8.30(s,1H),7.53-7.36(m,5H),7.34-7.17(m,3H),6.47(d,J=19.3Hz,1H ),6.16(d,J=5.1Hz,1H),5.87-5.82(m,1H),5.76-5.55(m,1H),5.50-5.40(m,1H),5.00(s,2H),4.79-4.75(m,2H),4.68-4.61(m,1H) ,4.58-4.47(m,2H),4.44-4.39(m,1H),4.09-4.02(m,1H),3.92-3.89(m,1H),3.67-3.62(m,1H),3.57-3.53(m,1H),3.01-2.94(m,1 H),2.87(s,3H),2.74-2.68(m,1H),2.11-2.03(m,1H),1.45-1.43(m,3H),1.44(s,9H),0.98(d,J=6.7Hz,3H),0.93(d,J=6.7Hz,3H). 31 P NMR(162MHz,CD3OD)δ57.35(s,1P).

[0914] Example 38A [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5(6NS)-yl)-4-oxobutanoyl]amino}-29-isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatritriacontan-33-yl]amino}benzyl(C34)

[0915] [ka]

[0916] [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl] -9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl (intermediate 35, 17.4 mg, 0.0154 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.49 mg, 0.0182 mmol), and (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin- Triethylamine (0.004 mL, 0.0266 mmol) in DCM (0.37 mL) was added to a suspension of 5(6H-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (intermediate 27A) (10.4 mg, 0.0140 mmol) with 5(6H-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (intermediate 27A) (10.4 mg, 0.0140 mmol) and 5(6H-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (intermediate 27A) (10.4 mg, 0.0140 mmol). The mixture was stirred at room temperature for 20 minutes, then DMF (0.15 mL, 2.23 mmol) was added. The mixture was left to stand at room temperature for 2 hours. After stirring, the solvent was concentrated. The crude residue was purified by reverse-phase flash column chromatography (0-50% ACN / triethylammonium acetate aqueous solution (10 mM)) to obtain the N,N-diethylethaneamine salt [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxyl C-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofloxacin[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azosin-5( 6H-yl)-4-oxobutanoyl]amino}-29-isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatritriacontan-33-yl]aminobenzyl(C-34) (2.0 mg, 6.9%) was obtained. LCMS(AA): m / z = 938.1 (M+H). HRMS(m / z): [M+H]+, C 80 H 101 F2N 15 O 26 Calculated value for P2S2: 1852.6000; Measured value: 1852.6006.

[0917] Example 38B {(25S)-27-[(2,5-dioxopyrrolidine-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyl (Intermediate 36)

[0918] [ka]

[0919] Step 1: (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate The title compound was prepared by starting with (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidine-1-yl)oxy]-4-oxobutanoate tert-butyl (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol), respectively, instead of intermediate 15 and (tert-butoxycarbonyl)glycinate 2,5-dioxopyrrolidine-1-yl, and following the procedure described in Step 1 of Example 26. Purification by silica gel chromatography (0-10% MeOH / DCM) was performed on (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11, 14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (3.8g, 100%) was obtained.

[0920] Step 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added, and the mixture was stirred under hydrogen (15 psi) for 3 hours. The reaction mixture was filtered, washed with methanol, and the filtrate was evaporated to dryness to obtain (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-tert-butyl euicate (2.56 g, 85%).

[0921] Step 3: (25S)-25-({[(1R,8S,9s)-Bicyclo[6.1.0]nonano-4-in-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oate (587 mg, 1.15 mmol) was dissolved in DCM (11.3 mL) and TFA (11.3 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction product was concentrated, azeotropically dissolved in toluene, and then dried under high vacuum to obtain a crude intermediate as oil. 1-({[(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (279 mg, 0.959 mmol), DCM (9.8 mL), and triethylamine (1.34 mL, 9.59 mmol) were added to the crude oil intermediate at room temperature. The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with HCl (50 mL) and potassium carbonate solution (10 mL, 4.0 M aqueous solution). The separated organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by reverse-phase column chromatography (0-100% ACN / ammonium bicarbonate aqueous solution (5 mM)). The desired fraction was concentrated under vacuum to obtain (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid (19 mg, 3.1% yield). LCMS(AA):m / z=629.2(MH);1HNMR(400MHz,METHANOL-d4)δ=4.46(dd,J=5.7,7.4Hz,1H),4.25-4.12(m,2H),3.68-3.51(m,28 H),3.41-3.34(m,5H),2.80-2.60(m,2H),2.33-2.12(m,4H),1.69-1.54(m,2H),1.40(quin,J=8.7Hz,1H),1.02-0.89(m,2H)

[0922] Step 4: {(25S)-27-[(2,5-dioxopyrrolidine-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyl (intermediate 36) The title compound was prepared by starting with (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-euic acid instead of 2,2-dimethyl-4-oxo-3,8,11,14,17,19-pentaoxa-5-azanonadecane-19-euic acid and following the procedure described in Example 29. The reaction mixture was stirred at room temperature for 30 minutes to obtain {(25S)-27-[(2,5-dioxopyrrolidine-1-yl)oxy]-24,27-dioxo-2,5,8, 11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyl (intermediate 36, 22 mg, 100%) was obtained. LCMS(AA): m / z = 728.4 (M + H). Example 38C The compounds listed in Table 22A (intermediate 37 and intermediate 42) were prepared as described in Example 10 using the starting materials shown in Steps 1 and 2 (intermediate 37 and intermediate 42) and (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenoxy}tetrahydro-2H-pyran-2-carboxylate methyl instead of 2,5,8,11,14,17,20-heptaoxadocosan-22-amine. (Intermediate 42)

[0923] [Table 22A]

[0924] Example 38D [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl] 4-Methylcarbamate {[(2S,5S)-57-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6-diazaheptaspentacontan-1-oil]aminobenzyl(C-37)

[0925] [ka]

[0926] Step 1: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-di 4-{[(53S,56S)-1-azido-53-isopropyl-56-methyl-51,54,57-trioxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecaoxa-52,55-diazaheptapentacontan-57-yl]aminobenzyl Intermediate 8 (18.68 mg, 0.01674 mmol), 1-[(1-azido-51-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecaoxahenpentacontan-51-yl)oxy]pyrrolidine-2,5-dione (32.3 mg, 0.03348 mmol), and DIEA (0.044 mL, 0.2511 mmol) were added to a solution of THF (0.6 mL) and DMF (0.2 mL). The reaction mixture was stirred at room temperature for 3 hours, and then stopped with MeOH (5 mL). After evaporating most of the solvent, the residue was purified by reverse-phase column chromatography (0-50% ACN / ammonium bicarbonate aqueous solution (5 mM)). The desired fraction was concentrated and freeze-dried to obtain the title compound (17.8 mg, 55%) as a white powder. LC-MS (AA): m / z = 1915 (MH).

[0927] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15 aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S,5S)-57-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6-diazaheptaspentacontan-1-oil]amino}benzyl(C-37) [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methyl 4-{[(53S,56S)-1-azido-53-isopropyl-56-methyl-51,54,57-trioxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecaoxa-52,55-diazaheptapentacontan-57-yl]aminobenzyl carbamate (17.7 mg, 0.00923 mmol), THF (1.0 mL), and triphenylphosphine (24.2 mg, 0.0923 mmol) were added. This mixture was stirred overnight at room temperature. Sodium hydroxide (0.0554 mL, 1.0 mol / L aqueous solution) was added to the reaction mixture. The mixture was stirred at room temperature for 1 hour, and then concentrated. The residue was purified by reverse-phase column chromatography (0-50% ACN / ammonium bicarbonate aqueous solution (5 mM)). The desired fraction was recovered, freeze-dried, and obtained as a fluffy white powder [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo- 6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S,5S)-57-amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6-diazaheptaspentacontan-1-oil]amino}benzyl (C-37) (5.7 mg, 32%) was obtained. LCMS (AA): m / z = 1889 (MH). HRMS (m / z): [M + H]+ , C 80 H 124 N 12 O 32 Calculated value for P2S2: 1891.7434; Measured value: 1891.7433.

[0928] Example 38E N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 39)

[0929] [ka]

[0930] Step 1: (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-euic acid To a mixture of (2S)-2-amino-6-(2,5-dioxopyrrole-1-yl)hexanoate (1.05 g, 4.00 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)oxy]pyrrolidine-2,5-dione (2.08 g, 4.04 mmol) in pyridine (21.0 mL), DIEA (1.43 mL, 8.20 mmol) was added, the mixture was stirred at room temperature for 30 minutes, then cooled to 0°C and stirred overnight. After removing the volatile solvent, the residue was azeotropically analyzed with toluene (3 × 20 mL) and acetonitrile (10 mL) to obtain (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-euic acid (4.00 g, 100% yield) as a colorless oil. LCMS(AA): m / z = 619.3 (MH).

[0931] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 39) A mixture of N-hydroxysuccinimide (469 mg, 4.08 mmol) and N,N'-dicyclohexylcarbodiimide (824 mg, 4.00 mmol) in 76.7 mL, 1200 mmol of DCM, and crude (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-euic acid (4.00 g, 4.00 mmol) derived from step 1 was heated at room temperature for 16 minutes. The mixture was stirred for a specified time. The precipitated solid from this reaction was filtered, and the filtrate was concentrated to obtain crude oil. Purification by silica gel chromatography (0-20% IPA / DCM) yielded N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 39) (1.70 g, 52%) as a colorless oil. 1 H_NMR (400MHz, METHANOL-d4)δppm1.42-1.55(m,2H)1.64(m,2H)1.81-1.93(m,1H)1.94-2.01(m,1H)2.52(m,2H) )2.81-2.86(m,4H)3.36(s,3H)3.51-3.57(m,4H)3.60-3.66(m,26H)3.68-3.76(m,2H)4.03-4.1 7(m,2H)4.74-4.80(m,1H)6.74-6.86(s,2H).

[0932] Example 38F N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 40)

[0933] [ka]

[0934] Step 1: (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-euic acid The title compound was prepared according to the procedure described in Example 38E, Step 1, starting with N6-(tert-butoxycarbonyl)-L-lysine (0.49 g, 1.99 mmol) instead of (2S)-2-amino-6-(2,5-dioxopyrrole-1-yl)hexanoate. Purification by reverse-phase column chromatography (0-60% ACN / formic acid aqueous solution (0.1%)) yielded (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-euic acid (1.14 g, 90%) as a colorless oil. LCMS (AA): m / z = 639.4 (MH).

[0935] Step 2: (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-euic acid (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosane-29-euic acid (148 mg, 0.2310 mmol) was added to a solution of DCM (2.0 mL) with TFA (2.0 mL) at room temperature. The mixture was stirred at room temperature for 15 minutes and evaporated, then co-evaporated with toluene (×3) to obtain the crude TFA salt. After drying this crude product under high vacuum for a further 2 hours, maleimide acetate NHS ester (69.89 mg, 0.2772 mmol) and DIEA (0.121 mL, 0.6929 mmol) in DCM (2650 mg, 2.00 mL, 31.2 mmol) were added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was evaporated. Purification by reverse-phase column chromatography (10-30% ACN / ammonium acetate aqueous solution (10 mM)) yielded (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-euic acid (120 mg, 38%) as an oil. LC-MS (AA): m / z = 676.3 (MH).

[0936] Step 3: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 40) (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-euic acid (311 mg, 0.459 mmol) and DCM (2.94 mL) were added to a flask. N,N'-dicyclohexylcarbodiimide (94.7 mg, 0.459 mmol) and N-hydroxysuccinimide (53.9 mg, 0.459 mmol) were added. The suspension was stirred under an argon atmosphere at room temperature for 1 hour. The reaction mixture was then cooled to 0°C, filtered through Celite, and rinsed with MeCN. The filtrate was evaporated. The residue was redissolved in DCM, washed with water (x2), dried over MgSO4, and concentrated to obtain N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidine-1-yl)oxy]-1-oxohexane-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 40) (300 mg, 84.4%) as a viscous oil. LCMS(AA): m / z = 773.2 (MH).

[0937] Example 38G The compound (C-43) listed in Table 22C is (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidine-4-yloxy)-2,10-disulfani Instead of methyl tetrahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yl]-6-oxo-6,9-dihydro-1H-purine-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate, the preparations were made as described in steps 2 and 3 of Example 23 using the starting materials shown in the table.

[0938] [Table 22B]

[0939] [Table 22C]

[0940] Example 38H Compound intermediate 44, listed in Table 22D, was prepared as described in Example 38E, using the starting material (2R)-2-amino-6-(2,5-dioxopyrrole-1-yl)hexanoate shown in Table 22D instead of the (2S)-2-amino-6-(2,5-dioxopyrrole-1-yl)hexanoate shown in Step 1.

[0941] [Table 22D]

[0942] Example 39 Procedure for preparing the Ab-STING agonist complex by stochastic cysteine ​​conjugation (shown in Figure 1) A solution of anti-GCC antibody (clone 5F9, see PCT publication number WO2011 / 050242, 60 mg / mL) was prepared in 50 mM histidine, 100 mM arginine, and pH 6.0 buffer. 0.5 M Tris / 25 mM EDTA pH 8.0 buffer was added to the solution until the pH was 7.5–8.0. TCEP (10 mM solution in H2O, 3–5 equivalents) was added to the solution. The reaction mixture was purged with argon and incubated at room temperature or 37°C with gentle shaking. Next, the desired linker payload construct (10 in DMA) was prepared. A mM solution (5-9 equivalents) was slowly added to the above mixture. The reaction mixture was purged with argon and incubated at 37°C for a further 1.5-2 hours with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to obtain ADC. The ADC concentration, aggregation rate, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0943] Other antibody complexes can be prepared using the procedure described above.

[0944] Example 40 The antibody-drug conjugates listed in Table 24 were prepared as described in Example 39 using the starting linker-payload constructs indicated as starting materials.

[0945] [Table 24]

[0946] Example 41 Procedure for preparing the Ab-STING agonist complex via transglutaminase conjugation (shown in Figure 2) Deglycosylation: A solution of anti-GCC antibody (clone 5F9, see PCT publication number WO2011 / 050242, 60 mg / mL) in 50 mM histidine, 100 mM arginine, and pH 6.0 buffer was diluted with an equal volume of pH 7.2 PBS. N-glycosidase F (New England BiolAbs, P0704S, 500,000 units / mL, 300 units per 1 mg of antibody) was added to the solution, and the reaction mixture was heated to 37°C overnight while gently mixing. The resulting deglycosylated 5F9 was used to exchange buffer with PBS pH 7.2.

[0947] Transglutaminase conjugation: 29-azido-3,6,9,12,15,18,21,24,27-nononaoxanonacosan-1-amine (azido-PEG9-amine, BroadPharm, BP-23556, 40 equivalents) was added to the deglycosylated 5F9 solution in PBS (10-20 mg / mL) prepared above, followed by the addition of transglutaminase (ACTIVA®, Ajinomoto, 5-10 mg per 1 mg of antibody). The reaction mixture was heated to 37°C with gentle mixing overnight. The product was purified according to the preparative SEC method described herein to obtain 5F9-NH-PEG-azide.

[0948] Strain-promoted azide-alkyne cycloaddition: A solution of the 5F9-NH-PEG-azide complex prepared above (2–15 mg / mL in PBS) was added to a 4–10 mM DMSO solution of the linker-payload construct containing the strained alkyne (3–5 equivalents of DMSO at <10% of the total solvent volume). The resulting mixture was gently stirred overnight at room temperature. The product was purified according to the preparative SEC method described herein to obtain ADC. The ADC concentration, aggregation rate, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0949] Other antibody complexes can be prepared using the procedure described above.

[0950] Example 42 The antibody-drug conjugates listed in Table 25 were prepared as described in Example 41 using the starting linker-payload constructs indicated as starting materials in the table.

[0951] [Table 25]

[0952] Example 43 Preparation of the Ab-STING agonist complex via transglutaminase conjugation (shown in Figure 3). To a deglycosylated 5F9 solution in PBS (10-20 mg / mL, prepared according to the deglycosylation procedure described in Example 41), 1 M Tris, 5 M NaCl, and pH 8.0 buffer (10-20% of the total volume) adjusted to pH 8.0 were added. To this solution, 10 mM DMSO solution of a linker payload construct (20 equivalents) containing a primary amine was added. The solution was added, followed by transglutaminase (ACTIVA®, Ajinomoto, 100-150 mg per 1 mg of antibody). The reaction mixture was heated to 37°C overnight with gentle mixing. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01) by first washing with 20 mM phosphate pH 7.0 and then eluting the ADC with 0.1 M citrate pH 4.0.

[0953] Other antibody complexes can be prepared using the procedure described above.

[0954] Example 44 The antibody-drug conjugates listed in Table 26 were prepared as described in Example 43 using the starting linker-payload constructs indicated as starting materials in the table.

[0955] [Table 26]

[0956] Example 45 2-amino-N-(2-((2-(2-azidoacetamido)ethyl)amino)-2-o Xoethyl acetamide

[0957] [ka]

[0958] Step 1: (2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecane-13-yl)benzyl carbamate Triethylamine (0.340 mL, 2.44 mmol) was added at 0°C to a solution of Boc-Gly-Gly-OH (550 mg, 2.32 mmol), EDC·HCl (437 mg, 2.28 mmol), and HOAt (317 mg, 2.33 mmol) in DMF (8.0 mL). The resulting yellow heterogeneous mixture was stirred for 15 minutes. A solution of (2-aminoethyl)carbamate benzyl (500 mg, 2.44 mmol) in DMF (6.0 mL) was slowly added over 10 minutes, and the mixture was warmed to room temperature and stirred for 3 days. The reaction mixture was partitioned into water (100 mL) and pharmaceutically acceptable (150 mL). The separated aqueous layer was extracted with pharmaceutically acceptable (50 mL). The combined organic layers were washed with 50 mL of water, then brine, dried over Na2SO4, and concentrated. The crude product was purified by silica gel chromatography (0-6% MeOH / CH2Cl2) to obtain N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate tert-butyl (617 mg, 1.51 mmol, 65% yield) as a white solid. LCMS(FA): m / z = 431.2 (M + Na). 1 1H NMR (400MHz,DMSO-d6)δ7.97-7.97(m,1H),7.86-7.84(m,1H),7.39-7.29(m,5H),7.27-7.24(m,1H),7.01-6 .98(m,1H),5.02(s,2H),3.67(d,J=5.62Hz,2H),3.58(d,J=5.87Hz,2H),3.03-3.18(m,4H),1.39(s,9H).

[0959] Step 2: (2-((2-((2-aminoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl) tert-butyl carbamate To a solution of N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate tert-butyl (615 mg, 1.51 mmol) in ethanol (20.0 mL), 10% Pd / C (62.0 mg, 0.0583 mmol) was added. The mixture was purged with H2 (1 atm) and stirred at room temperature for 3 hours. Celite was added to the reaction mixture, and the slurry was filtered through a short bed of Celite. The filtrate was concentrated, and the residue was dried by co-evaporation with toluene followed by high vacuum to obtain a whitish solid N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl] [L]amino]-2-oxo-ethyl]carbamate tert-butyl (407 mg, 1.48 mmol, 98.5% yield) was obtained. LC-MS (FA): m / z = 275.2 (M + H). 1 HNMR(400MHz,DMSO-d6)δ8.05-7.89(m,1H),7.80-7.63(m,1H),7.08-6.96(m,1H),3.67(d,J=5.75Hz,2 H),3.56(d,J=5.87Hz,2H),3.06(q,J=6.24Hz,2H),2.59-2.53(m,2H),1.87-1.61(m,2H),1.39(s,9H).

[0960] Step 3: N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl] tert-butyl carbamate A mixture of EDC·HCl (57.6 mg, 0.300 mmol), HOAt (43.6 mg, 0.320 mmol), and 2-azidoacetic acid (27.0 μL, 0.315 mmol) in dichloromethane (2.70 mL) was stirred for 10 minutes. The mixture was cooled in an ice bath, and then N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate tert-butyl (75.0 mg, 0.273 mmol) was added, followed by N,N-diisopropylethylamine (55.8 μL, 0.320 mmol) over several minutes. The mixture was stirred at 0°C for 5 minutes, and then stirred overnight at room temperature. Water (2 mL) and ethyl acetate (5 mL) were added, and the mixture was vigorously stirred for 20 minutes. The mixture was diluted with ELISA (25 mL) and then washed with 50% brine / water (20 mL) and brine. The separated organic layer was dried over Na₂SO₄ and concentrated. Purification by silica gel chromatography (0-8% MeOH / CH₂Cl₂) yielded N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate tert-butyl (88.0 mg, 0.246 mmol, 90.1% yield) as a glassy solid. LCMS (FA): m / z = 356.2 (MH). 1 1H NMR (400MHz, DMSO-d6) δ8.1 6-8.08(m,1H),8.04-7.92(m,1H),7.92-7.81(m,1H),7.05-6.94(m,1H),3.82(s,2 H),3.67(d,J=5.62Hz,2H),3.58(d,J=5.87Hz,2H),3.10-3.19(m,4H),1.39(s,9H).

[0961] Step 4: 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide (intermediate 34) To a solution of N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate tert-butyl (86.0 mg, 0.241 mmol) in dichloromethane (4.0 mL), trifluoroacetic acid (1.00 mL, 8.77 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The mixture was diluted with 5 mL of toluene and concentrated. The residue was taken into 5 mL of water and freeze-dried to obtain a colorless syrup, 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide 2,2,2-trifluoroacetate (102 mg, 0.275 mmol, quantitative). LCMS(FA): m / z = 258.1 (M + H). 1 H NMR(400MHz,DMSO-d6)δ8.55-8.6 2(m,1H),8.12-8.20(m,1H),8.02-8.09(m,1H),7.90-8.02(m,3H),3.74-3.82(s,2H),3.80(m,2H),3.55-3.68(m,2H),3.11-3.19(m,4H).

[0962] Example 46 Manufacturing and purification of 5F9-LPETGG-His6 The 5F9-LPETGG-His6 protein is a transient Expi29F™ cell, Expi293™ expression growth medium, and Expifectamine293™ translocation kit from ThermoFisher, cat.no.A14635. Prepared using the 3F Mammalian Protein Expression System Kit. 2.8 × 10⁶ in a 5L vented shaking flask. 6Expi293FTM cells at a density of cells / mL were transfused with 0.666 mg of light chain DNA and 0.334 mg of heavy chain DNA mixed with 5.4 mL of Expifectamine293TM translocation reagent in 1.8 liters of water. The cells were incubated overnight in an 8% CO2 incubator at 37°C with shaking at 100 rpm. The following day, 10 mL of enhancer 1 and 100 mL of enhancer 2 from the kit were added to the flask. The flask was incubated under the above conditions and harvested on day 5 after translocation. The cells were centrifuged at 9000 rpm at 4°C for 10 minutes, filtered and sterilized through a 0.22 μm filter unit, and stored at 4°C until purified on a freshly packed 30 mL MabSelect SuRe LX Protein A column (GE Healthcare, catalog #17-5474-03). After loading the supernatant onto the column using a peristaltic pump, the resin was washed with 60 mL [2 column volumes (CV)] of low-salt buffer (25 mM trisodium citrate, pH 7.6, 125 mM NaCl), then with 60 mL (2 CV) of high-salt buffer (25 mM trisodium citrate, pH 7.6, 2 M NaCl), followed by another wash with 60 mL (2 CV) of low-salt buffer. Proteins were eluted by pH gradient elution using 25 mM citrate, 125 mM NaCl, pH 8 to pH 2.2, and peak fractions were collected. Each fraction was neutralized with 1 M trisodium citrate, pH 8.2 to approximately pH 6 and 70 mM. The fraction of the major peaks (2A5-2C5) was pooled. The pooled fractions were concentrated and buffered with 25 mM sodium citrate, pH 5.5, 125 mM NaCl buffer using a Sartorius VIVAFLOW 200 cartridge with a molecular weight cutoff of 50 kDa. The samples were then filtered through a 0.22 μm syringe filter unit, dispensed, and stored at -80°C. The final yield was 623 mg of purified 5F9-LPETGG-His6 antibody with a purity of >95% and an endotoxin content of 0.64 EU / mL.

[0963] Heavy chain sequence:

[0964]

number

[0965] Light chain sequence:

[0966]

number

[0967] Example 47 The procedure for preparing the Ab-STING agonist complex via saltase conjugation is shown in Figure 4. Saltase conjugation: A 5F9 antibody containing LPETGG-His6 at the C-terminus of the heavy chain was generated according to the procedure described in Example 46. The solution of 5F9 containing the LPETG-His6 tag at the C-terminus of the heavy chain (5F9-LPETGG-His6) in 25 mM citrate, 125 mM NaCl, pH 5.5 buffer was diluted with 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer to a protein concentration of 48 μM. To this solution, CaCl2 (4 mM aqueous solution, 80-100 equivalents) and 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide (intermediate 34, 800 μM solution in 50 mM HEPES containing 0.1% DMSO, 150 mM NaCl, pH 7.45 buffer, 20 equivalents) were added, followed by the addition of saltase A Q60-K206-P94S-D160N-D165A-K196 His6 (Antos, JM, Ingram, J., Fang, T., Pishesha, N., Truttmann, MC, Ploegh, HL (2017). Site-specific protein labeling via sortase-mediated transpeptidation. Current Protocols in Protein A 2.0 μM solution (0.03 equivalents) in 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer was prepared as described in Science, 89, 15.3.1-15.3.19. The reaction mixture was stirred at room temperature for 4 hours. The product was purified to obtain 5F9-LPETG-azide by filtration by Capturem nickel column (from Takara, washed with PBS), followed by dialysis, or according to the preparative SEC method described herein.

[0968] Strain-promoted azide-alkyne cycloaddition: A solution of the 5F9-LPETGG-NH(CH2)2NHCOCH2-azide complex prepared above (2-15 mg / mL in PBS) was added to a 4-10 mM DMSO solution of the strain-alkyne-containing linker-payload construct (3-5 equivalents of DMSO at <10% of the total solvent volume). The resulting mixture was gently stirred overnight at room temperature. The product was purified to obtain ADC according to the preparative SEC method described herein. The ADC concentration, aggregation rate, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

[0969] Other antibody complexes can be prepared using the procedure described above.

[0970] Example 48 The antibody-drug conjugates listed in Table 27 were prepared as described in Example 47 using the starting linker-payload constructs indicated as starting materials in the table.

[0971] [Table 27]

[0972] Example 49 Biological protocols and STING cell data Conditions for the tritosome assay The test compound (121 μM linker-payload construct solution in DMSO or ADC solution in PBS, 4.65 μL) was added to a mixture of 125 μL rat liver tritosomes (purchased from XenoTech) and 433 μL of buffered aqueous solution (54.7 mg / mL phosphate, 1.7 mg / mL EDTA, pH 6.0), and the solution was incubated at 37°C. 40 μL of the sample was taken at 10 and 30 minutes, and at 1, 3, 5, and 24 hours, treated with 160 μL of 0.1% formic acid in methanol solution in a 96-well plate, and then stored at -80°C. After collection at the final time point, each sample was thawed and treated with 200 μL of 0.1% formic acid in methanol solution containing 150 nM carbutamide (internal standard). The samples were centrifuged at 4000 g for 10 minutes and analyzed according to the LC / MS / MS method described herein. Using the Excel-Fit program 1 / 2 The calculated t 1 / 2 This is reported in Table 28. As shown in Table 28, in this tritosome assay, no release of the free payload from ADC-3 was observed within 24 hours, whereas ADC-9 with the same payload was observed within 7 hours. 1 / 2 The successful release of the payload indicates that the ADC-9 linker resulted in a significantly improved rate of payload release under surrogate cell release conditions compared to ADC-3.

[0973] [Table 28]

[0974] Figures 5, 6, and 7 show graphs of payload release over time for ADC-1, ADC-3, and ADC-9, as shown in Table 28.

[0975] Conditions for plasma stability assay The test compound was added to 1 mL of plasma at a concentration of 10 μg / mL, and then five equal aliquots were dispensed into 2 mL Eppendorf microcentrifuge tubes (labeled 0, 24, 48, 72, and 96 hours). The sample centrifuge tube at time 0 was immediately stored at -80°C, and the remaining centrifuge tubes were incubated at 37°C with moderate shaking. The aliquots were removed from the incubator at the corresponding time points and stored at -80°C. After all samples were recovered, they were... The samples were thawed at room temperature and placed on moist ice. 50 μL of each sample was divided into three sets into a 96-well microtiter plate. The reaction of the samples was stopped with 200 μL of ice-cold methanol containing a 50 nM internal standard. The samples were vortexed for 2 minutes and then centrifuged at 3000 rpm for 10 minutes. 185 μL of the supernatant was transferred to a clean injection plate and dried under N2 gas at 40°C. The dried sample extracts were reconstituted with 100 μL of LCMS-grade water and then vortexed for 1 minute in preparation for LC-MS / MS analysis.

[0976] Each sample was separated at 40°C by reverse-phase HPLC using a Synergi 2.5μ and a Polar-RP 100A C18 column (2.0 mm × 30 mm) (Phenomenex®) with a gradient consisting of a 0.1% formic acid aqueous solution (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The analytes were analyzed using SCIEX API 4500. Detection was performed using a cation spray in multi-reaction monitoring (MRM) mode with a QTRAP instrument. The payload loss rates in human, primate, and mouse plasma at various time points are reported in Table 29.

[0977] [Table 29]

[0978] Conditions for the THP1 Dual Lucia Reporter Gene Assay THP1-Dual(TM) KI-hSTING-R232 cells (InvivoGen The #thpd-r232 variant was derived from human THP-1 monocyte cell lines by stable biallelic knockout of the endogenous human HAQ STING gene and knock-in of the R232 mutant of human STING. These cells also stably express the inducible secretory Lucia luciferase reporter gene under the control of the ISG54 (interferon-stimulating gene) minimal promoter in conjunction with five IFN-stimulated response elements (ISREs). Reporter gene expression allows for the study of the IFN regulatory (IRF) pathway by evaluating the activity of Lucia luciferase. In addition to human STING and luciferase... These cells were engineered to stably express human guanylyl cyclase C (GCC), enabling studies of target-mediated IRF pathway activation. Non-GCC-expressing vector cells were used as a control.

[0979] On the day of the experiment, cells were placed in a white 384-well plate (Corning 356661) at a well density of 15,000 cells / 25 μl using growth medium (RPMI1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg / mL Normocin®, 100 U / mL-100 μg / mL Pen-Strep, 10 μg / mL blastosidine, 100 μg / mL zeosin, and 1 μg / mL promycin). 5 μL of GCC-targeted ADC sample was added to the cell plate, and the plate was incubated at 37°C for 20 hours. At the end of incubation, 10 μL / well of QUANTI-Luc® (InvivoGen #rep-qlc1) was added, and luminescence was immediately measured using a LeadSeeker.

[0980] For the assay method described above, the luminescence signal induction percentage of each test ADC at various concentrations was calculated compared with untreated and control samples. The compound concentration vs. signal induction percentage curve was fitted, and the EC 50 A value was generated. A person skilled in the art would know the EC 50 You will understand that the values ​​generated are affected by experimental variations. 50The Emax values ​​are reported in Table 30. As shown in Table 30 below, ADC-16 was approximately 21 times more potent than ADC-15 in vector THP1 cells, which is likely due to the more rapid release of the intracellular payload from ADC-16 compared to ADC-15.

[0981] [Table 30]

[0982] Example 50 Pharmacokinetic evaluation in mice For the in vivo evaluation of ADCs in tumor-bearing Balb / C mice expressing GCC and CT26 colon cancer, female Balb / C mice aged 6-8 weeks (Jackson (Purchased from the laboratory) was used. CT26.WT parental cells were obtained from ATCC, catalog number CRL-2638. GCC-expressing CT26 cells were constructed by transduction of CT26.WT with the lentiviral vector pLenti6.3 containing the human GCC gene at Takeda, and CT26 pLenti6.3 GCC clone A5 was obtained. CT26 pLenti6.3 GCC clone A5 cells were grown under sterile conditions in a 37°C incubator with 5% CO2 for 12-14 days. Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum. Cells were passaged every 3-4 days, and cells were detached using 0.05% trypsin / EDTA. On the day of transplantation, cells were removed and resuspended in serum-free RPM-1640 medium at a concentration of 0.5 × 10⁶ cells / 100 μL. A 27-gauge needle (0.5 × 10⁶ per mouse) was used. 6 CT26 pLenti6.3 GCC clone A5 cells were transplanted by subcutaneous injection into the lower flank of mice using a cell / 100 μI injection dose. After cell transplantation, the tumor was measured with a caliper, and once the tumor was palpable, the body weight of the mice was measured twice a week. The tumor was measured in two dimensions. The caliper measurement was (L × W 2The calculation was performed using ) / 2. Mice were fed a normal diet and housed in an SPF animal facility in accordance with the guidelines for the management and use of laboratory animals and the rules of the facility's animal management and use committee. The animals were kept at a temperature of 18–26°C, a relative humidity of 50±20%, and a 12-hour intermittent light-dark cycle, and were given free access to food and water.

[0983] The pharmacokinetics of ADCs are as follows: 3 Upon reaching this point, the study examined Balb / C mice (n=3 / treatment group) bearing CT26-GCC tumors following injection of ADC. Serum samples were collected at various time points and frozen for analysis.

[0984] Mouse plasma levels of total antibody and complexed payload were measured by a two-component immunocapture-based LC / MS assay on a Shimadzu UHPLC system connected to a Sciex 6500QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated with anti-human IgG coated magnetic beads at room temperature for 45 minutes, after which nonspecifically bound proteins were removed by washing the magnetic beads with PBST (PBS buffer at pH 7.4 containing 0.05% Tween 20) and subsequently with PBS buffer. Then, both the naked antibody (DAR=0) and ADC (DAR≧1) were eluted from the magnetic beads in 0.1% trifluoroacetic acid. After neutralizing the eluent and adding it to a stable isotope-labeled internal standard, one aliquot of the sample was pipettered and digested with papain at 37°C for 1 hour, and then used for LC / MS analysis of the complexed payload. The remaining sample was subjected to trypsin / lys-C digestion at 70°C for 1 hour, and then used for LC / MS analysis of the total antibody.

[0985] The circulating free payload was also measured by LC / MS after plasma protein precipitation. In short, mouse plasma was mixed with 8 volumes of methanol-containing stable isotope-labeled internal standards, and the supernatant was evaporated to dryness at 40°C under a gentle nitrogen stream. Finally, the residue was reconstituted with LC / MS-grade water before LC / MS analysis. The PK profiles of ADC-1, ADC-8, and ADC-9 are summarized in Table 31. Graphs of plasma PK are shown in Figures 12, 13, and 14. As shown in Table 24, the DAR values ​​for ADC-1, ADC-8, and ADC-9 are 4.5, 3.9, and 4.3, respectively, which leads to higher molar concentrations of complexed CDN obtained at time 0 than the total Ab shown in Figures 8-10, respectively. However, as shown in Figure 8, the molar concentration of complexed CDN derived from ADC-1 decreases faster than the molar concentration of total Ab, and the two concentrations become the same on day 7 (leading to approximately 1 DAR on that day). This indicates that the DAR of ADC-1 is due to the low stability of the linker over time. This indicates a decrease in both. In contrast, as shown in Figures 9 and 10, the molar concentrations of the complexed CDN derived from both ADC-8 and ADC-9 remained higher than the molar concentrations of their respective total Ab throughout the experiment. In addition, the longer half-lives of the complexed payload / CDN derived from ADC-8 and ADC-9 compared to ADC-1 are likely a result of improved linker stability in ADC-8 and ADC-9, as shown in Table 31. In summary, this experiment demonstrates that the linkers of ADC-8 and ADC-9 have improved stability in mouse plasma compared to those of ADC-1.

[0986] [Table 31]

[0987] Example 51 Evaluation of antitumor activity in mice GCC-expressing CT26 colon cancer mouse tumor-bearing Balb / C mice were created as described in Example 50.

[0988] The average tumor volume (MTV) is approximately 75 mm. 3 When the tumor reached a certain stage, the animals were randomized into several treatment groups (n=8 / group) and administered a single intravenous dose of various ADCs. Tumors were measured twice weekly. The effect of ADC treatment on tumor growth was evaluated by measuring tumor volume and comparing it to vehicle-treated tumors.

[0989] The endpoint of the efficacy test was achieved when the tumor size reached 10% of the animal's body weight in volume or 2 cm in either direction. Mice were also closely monitored for signs of clinical deterioration after injection. Mice were euthanized if, for any reason, they showed signs of a pathological condition including difficulty breathing, hunched posture, decreased activity, hind limb paralysis, tachypnea as a sign of pleural effusion, weight loss close to 20% or 15%, or other signs, or if they were unable to perform normal activities (feeding, mobility).

[0990] Graphs showing the observed antitumor activity for ADC-1, ADC-5, ADC-8, and ADC-9 are shown in Figures 7, 8, 9, and 10, respectively. lipid complex Abbreviation: LC-MS method using AA ammonium acetate AC Acetate ACN Acetonitrile ATM atmosphere AQ water-based BBN Boravicyclo (3.3.1) Nonan Bn Benzyl Boc tert-butoxycarbonyl (Bpin) 2-screw (Pinacolato) Diboron tBu tert-ptil Bz benzoyl Celsius CAD-based electrostatic aerosol detection cGAMP Cyclic Guanosine Monophosphate / Adenosine Monophosphate DBAD azodicarboxylate di-tert-butyl DBU 1,8-Diazabicyclo[5.4.0]Undec-7-Endichloroacetic acid DCA (Dichloroacetic Acid) DCE Dichloroethane DCM Dichloromethane DEAD Diethyl Azodicarboxylate DIAD (Diisopropyl Azodicarboxylate) DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF (N,N-dimethylformamide) DMSO (Dimethyl Sulfoxide) DMTr 4,4'-Dimethoxytrityl DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane DPPC 1,2-Dipalmitoyl-sn-Glycerol-3-Phosphocholine DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine Et ethyl EtOH Ethanol HCl ethyl acetate LC-MS method using FA formic acid h time Int intermediate HPLC (High-Pressure Liquid Chromatography) HRMS high resolution mass spectrometry I C 50 50% inhibitory concentration IPA Isopropyl Alcohol IPC Diisopinocamphal LCMS (Liquid Chromatography Mass Spectrometry) LC / MS / MS Liquid Chromatography / Tandem Mass Spectrometry LDA Lithium Diisopropylamide LHMDS Lithium bis(trimethylsilyl)amide m / z mass-to-charge ratio MHz (megahertz) Me methyl MeOH methanol min mL (milliliter) MMP Methylmorpholin MS Mass Spectrum nBu n-butan NMP 1-methyl-2-pyrrolidinone NMR nuclear magnetic resonance PE (Petroleum Ether) Ph Phenyl psi (pounds per square inch) Pyr Pyridine rt room temperature SFC Supercritical Fluid Chromatography T3P 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphospholinane-2,4,6-trioxide TDA-1 Tris[2-(2-methoxyethoxy)ethyl]amine TBAF Tetrabutylammonium Fluoride TBS tert-butyldimethylsilyl TBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TEA (Triethylamine) TFA (Trifluoroacetic Acid) TIDPSi 1,1,3,3-Tetraisopropyldisiloxane TIPS Triisopropylsilyl THF (Tetrahydrofuran) UPLC Ultra-High-Speed ​​Liquid Chromatography Xantphos 4,5-bis(diphenylphosphin)-9,9-dimethylxanthene

[0991] Analysis method NMR conditions: Unless otherwise specified, NMR spectra are measured using a Bruker Avance III 400 spectrometer at 400 MHz. 1 Recorded at H frequency. 31 P NMR and 19 F NMR spectra were obtained using the same spectrometer at 162 MHz and 376 MHz, respectively. Unless otherwise specified, all heteronuclear experiments were performed. 1 It was obtained using H decoupling.

[0992] LCMS requirements: LC-MS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 series LC system connected to a Micromass mass spectrometer using a reversed-phase C18 column. Various gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained either 0.1% formic acid (as indicated as FA) or 10 mM ammonium acetate (as indicated as AA). One example of a solvent gradient used was from 100% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid), with a flow rate of 1 mL / min over a 16.5 minute run.

[0993] In some cases, LC-MS spectra were recorded using a reversed-phase C18 column on an Agilent 1290 Infinity UPLC system connected to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system connected to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 series HPLC system connected to a Waters Micromass ZQ mass spectrometer. Various gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water or MeOH / water gradient and contained either 0.1% formic acid (as indicated by FA) or 10 mM ammonium acetate (as indicated by AA). One example of a solvent gradient used was a flow rate of 0.5 mL / min over a 5-minute run from 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid).

[0994] Preparative HPLC: Preparative HPLC was performed using a Gilson instrument operated by a 322 pump with fractionation set to 200 nm to 400 nm using a UV / Visible 155 detector, eluting with a solvent gradient of solvent A (10 mM ammonium acetate 99% / 1% acetonitrile) / solvent B (10 mM ammonium acetate 5% / 95% acetonitrile). Fractionation was performed using a Phenomenex 5 micron C5 21.2 × 250 mm microscope. Mass-gated fractionation was performed using Agi. This will be performed using a lent 1100LC / MSD instrument.

[0995] Those skilled in the art will recognize that the gradient, column length, and flow rate can be modified, and that, depending on the chemical species being analyzed, some conditions may be more favorable than others for characterizing the compound.

[0996] Preparative SFC: Preparative SFC is performed using 10, 20, or 30 mm × 250 mm ChiralPak columns (typically IA, IB, IC, ID, IE, and IF), 10 or 20 mm × 250 mm Phenomenex Lux Cellulose-4, or 2-ethylpyridine columns that elute with appropriate proportions of supercritical carbon dioxide and alcohol, containing 0.3% diethylamine, 0.3% TEA, or 0.3% formic acid, or without acid or base additives. Typical conditions include constant composition conditions with flow rates in the range of 10–100 mL / min and a column temperature of 40°C. Preparative SFC is performed using a Jasco SFC preparative purification system with UV / visible light fractionation set to 200 nm–400 nm and back pressure adjusted to 10 MPa.

[0997] Those skilled in the art will recognize that the gradient, column length, and flow rate can be modified, and that, depending on the chemical species being analyzed, some conditions may be more favorable than others for characterizing the compound.

[0998] HPLC conditions for Example B HPLC analysis was performed using a gradient of solvent A (80% water / 10% ammonium acetate / 10% acetonitrile) / solvent B (10% ammonium acetate / 90% acetonitrile), a flow rate of 1 mL / min, a column temperature of 40°C, and UV detection at 254 nm using a SHISEIDO 3 micron C18 4.6×75 mm column. The test was performed using DZULC-2010C.

[0999] LC / MS / MS conditions for Example B: LC / MS / MS analysis was performed using Shimadzu UltraFast liquid chromatography and an automated sample collection system connected to an AB SCIEX 4500 or AB SCIEX 5500 triple quadrupole mass spectrometer or an equivalent system.

[1000] For the analysis of compound number 14 and compound I-5c, a 10 mM ammonium acetate or 0.1% formic acid aqueous solution in 99% water and 1% acetonitrile was prepared as mobile phase A, and a 10 mM ammonium acetate or 0.1% formic acid in 5% water and 95% acetonitrile was prepared as mobile phase B. Liquid chromatography was performed at 1.5 mL / min on a gradient for 3 minutes. The HPLC column was a Waters Xselect C18 CSH 3.5 micron column with an inner diameter of 2.1 mm and a length of 30 mm, and the column temperature was room temperature.

[1001] For the analysis of compound CP-1, 10 mM ammonium acetate in 99% water and 1% acetonitrile was prepared as mobile phase A, and 10 mM ammonium acetate in 5% water and 95% acetonitrile was prepared as mobile phase B. Liquid chromatography was performed at 1.5 mL / min on a gradient for 3.5 minutes. The HPCL column used was Osaka Soda Ca The pcell PAK C1 UG120 column was 5 microns in diameter, with an inner diameter of 2.0 mm and a length of 35 mm. The column temperature was room temperature.

[1002] The sample concentration was calculated by analyzing the MS / MS peak area using Analyst software 1.7.

[1003] UPLC-CAD requirements: UPLC-CAD analysis was performed using a Thermo Scientific®, Dionex®, Corona®, Veo®, RS (Rapid Separation) charged aerosol detector and a Waters ACQUITY UPLC system equipped with an ACE Excel C4 2 micron 2.1 × 100 mm column or a similar column, eluted by gradient elution using solvent A (10 mM ammonium acetate aqueous solution, 2.6 mL ammonium hydroxide in 4 L) / solvent B (10 mM ammonium acetate in methanol, 2.6 mL ammonium hydroxide 95%) at a flow rate of 0.8 mL / min and a column temperature of 35°C. Variations of up to ±20% may occur from UPLC-CAD analysis.

[1004] Example A1 Step 1: [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofl[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-14-yl]-9H-purine-6-yl}amino)-4-oxobutyl]methylcarbamate tert-butyl TEA salt, intermediate 1

[1005] [ka]

[1006] Compound No. 14 (300 mg, 0.40 mmol) was dissolved in anhydrous pyridine and concentrated to dryness (5 mL x 3 times). The residue was dissolved in pyridine (6.41 mL) under an argon atmosphere and treated with chlorotrimethylsilane (0.30 mL, 2.38 mmol) cooled in an ice bath. The reaction mixture was heated to room temperature and stirred for 2 hours to produce compound No. 14 protected by TMS. Separately (about 30 minutes after stirring the above reaction mixture at room temperature), 4-methylmorpholine (0.28 mL, 2.50 mmol) was added to a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (520.0 mg, 2.27 mmol) cooled in a tetrahydrofuran (10.7 mL) ice bath, followed by the slow addition of isobutyl chloroformate (0.29 mL, 2.27 mmol) in THF. The turbid suspension was warmed to room temperature and stirred for 1 hour, then slowly added to the TMS protection intermediate via syringe. The reaction mixture was stirred overnight at room temperature. MeOH (10 mL) was added to the reaction mixture and the reaction mixture was concentrated. The residue was redissolved in MeOH (3.8 mL). Triethylamine trifluoride (0.26 mL, 1.59 mmol) was added to the reaction mixture cooled in an ice bath and warmed to room temperature. The mixture was stirred for 30 minutes. The reaction mixture was absorbed onto diatomaceous earth (Celite®) (1 g) and purified by reverse-phase flash column chromatography (0-50% ACN with 10 mM triethylammonium acetate aqueous solution) to obtain intermediate 1 (214.0 mg, 48.5%) as the TEA salt. LCMS(AA): m / z = 910.2(M+H).1H NMR(D2O)δ8.55(s,1H),8.20(s,1H),7.87(s,1 H),7.14(d,,J=4.0Hz,1H),6.42(d,J=16.0Hz,1H),6.29(d,J=8.0Hz,1H),5.5d(dd,J=4.0,52.0Hz ,1H),5.04-4.93(m,1H),4.91-4.86(m,1H),4.66(d,J=4.0Hz,1H),4.46(d,J=8.0Hz,1H),4.33-4.2 5(m,3H),4.14(dd,J=4.0,12.0Hz,1H),3.94(dd,J=4.0,8.0Hz,1H),3.25(br,m,2H),3.05(q,J=8. 0Hz,15H),2.73(s,3H),2.59-2.54(m,1H),1.90-1.82(m,1H),1.20(s,9H),1.13(t,J=8.0Hz,22H). 31 P-NMR(D2O)δ55.46(s,1P),52.06(s,1P). 19 F-NMR(D2O)δ-164.19(s,br,1F),-200.75to-200.99(m,1F).

[1007] Example A2 Octadecanoic acid [(2R)-3-[hydroxy-[2-[[5-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoyl]amino]ethoxy]phosphoryl]oxy-2-octadecanoyloxypropyl], intermediate-2

[1008] [ka]

[1009] (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide (112.1 mg, 0.38 mmol) was suspended in toluene in vials and evaporated (1 mL x 3). The residue was dissolved in DMF (1.5 mL) and treated with DIPEA (88.9 μ...

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