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] Reference to an electronically submitted sequence listing The contents of the electronically submitted sequence listing (Name: 3817_053PC03_Seqlisting_ST25; Size: 7,104 bytes and Created: April 22, 2020) are incorporated herein by reference in their entirety.

[0002] 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. [Background technology]

[0003] Antibody-drug conjugates (ADCs), a rapidly growing class of targeted therapeutic agents, represent a promising new approach to improving drug selectivity and cytotoxic activity. These therapeutic agents are composed of an antibody (or antibody fragment) that can be conjugated to a payload drug to form an immunoconjugate. The antibody directs the ADC to bind to target cells. The ADC then internalizes and releases its payload, which provides cellular treatment. Because the ADC is directed to its target cells, the side effects of the conjugated drug may be less than those encountered when the same agent is administered systemically.

[0004] The adaptor protein STING (stimulator of interferon genes) 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 generation of specific killer T cells, which can shrink tumors and provide long-term immunity, preventing tumor recurrence. An activated STING pathway also contributes to the antiviral response by producing antiviral and pro-inflammatory cytokines that combat 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 function as STING agonists and are being investigated in clinical trials. However, their anionic properties result in low membrane permeability, which may limit their ability to engage STING within cells and often result in undesirable distribution of these compounds within the bloodstream.

[0005] There remains a need for new STING agonists as well as improved methods for delivering them to target cells. Summary of the Invention

[0006] In a first aspect, the present disclosure provides a compound of formula (I):

[0007] [ka]

[0008] or a pharmaceutically acceptable salt thereof,

[0009] During the ceremony,

[0010] a is an integer from 1 to 20;

[0011] b is an integer from 1 to 20;

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

[0013] n is 0 or 1;

[0014] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

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

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

[0018] L is a cleavable linker;

[0019] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0020] In a 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 a 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’ are hydrogen atoms.

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

[0023] [ka]

[0024] and

[0025] During the ceremony:

[0026] [ka]

[0027] is the point of attachment to the nitrogen atom;

[0028] [ka]

[0029] is the attachment point to Ab;

[0030] t is an integer from 1 to 10;

[0031] W is absent or a self-immolative group;

[0032] Z is absent or a peptide of 2 to 5 amino acids;

[0033] U and U' are independently absent or a spacer;

[0034] Q is a heterobifunctional group;

[0035] However, there is a condition that at least one of W and Z is present.

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

[0037] [ka]

[0038] During the ceremony:

[0039] [ka]

[0040] is the point of attachment to the carbonyl group;

[0041] [ka]

[0042] is the point of attachment to Z.

[0043] In a fifth embodiment of the first aspect, W is selected from:

[0044] [ka]

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

[0046] [ka]

[0047] is.

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

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

[0050] [ka]

[0051] is selected from

[0052] During the ceremony,

[0053] [ka]

[0054] is the point of attachment to Z;

[0055] [ka]

[0056] is the point of attachment to Q;

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

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

[0059] X is O or -CH2-;

[0060] Each r is independently 0 or 1.

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

[0062] [ka]

[0063] is.

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

[0065] [ka]

[0066] is selected from

[0067] During the ceremony,

[0068] [ka]

[0069] is the point of attachment to U, or if U is not present, to Z;

[0070] [ka]

[0071] is the point of attachment to U', or to Ab if U' is absent.

[0072] In a fifteenth 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] where t is 1; W is absent or is a self-immolative group; and Z is absent or is a two amino acid peptide.

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

[0076] In a seventeenth embodiment of the first aspect, the present 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 an eighteenth embodiment of the first aspect, the amino-substituted compound that modulates STING activity comprises a guanine derivative or an adenine derivative.

[0077] In a nineteenth embodiment, the amino-substituted compound that modulates STING activity has formula (II):

[0078] [ka]

[0079] or a pharmaceutically acceptable salt thereof,

[0080] During the ceremony,

[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 In this case, R a is C1-C4 alkyl the law of nature;

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

[0086] R 20 is hydrogen or fluoro;

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

[0088] R 50 is hydrogen or fluoro;

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

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

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

[0092] However, any of the rings A 10 or ring B 10 is attached to the carbonyl group of formula (I) via an -NH- group.

[0093] In a twentieth embodiment, the amino-substituted compound that modulates STING activity is

[0094] [ka]

[0095] or a pharmaceutically acceptable salt thereof,

[0096] During the ceremony,

[0097] [ka]

[0098] is the point of attachment to the carbonyl group of formula (I).

[0099] In a twenty-first embodiment of the first aspect, the amino-substituted compound that modulates STING activity has the formula (III):

[0100] [ka]

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

[0102] X 10 is SH or OH;

[0103] X 20 is SH or OH;

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

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

[0106] B 100 is the formula (B 1 -A) or formula (B 1 -B) is a group represented by:

[0107] [ka]

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

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

[0110] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 are each independently N or CR 1a In this case, R 1a is hydrogen or a substituent;

[0111] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C;

[0112] R 105 is hydrogen or a substituent;

[0113] B 200 is the formula (B 2 -A) or formula (B 2 -B) is a group represented by

[0114] [ka]

[0115] R 23 , R 24 , R 25 , R 26 , and R 27 are each independently a hydrogen atom or a substituent,

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

[0117] Y 21 , Y 22 , Y 23 , Y 24 , Y 25 , and Y 26 are each independently N or CR 2a In this 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, where R 105 and R 205 are each independently bonded to the 2- or 3-position of the 5-membered ring to which they are attached;

[0120] however,

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

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

[0123] [ka]

[0124] or a pharmaceutically acceptable salt thereof, During the ceremony,

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

[0126] [ka]

[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 In this case, 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 the formula (B 2 -A) or formula (B 2 -B) is a group represented by

[0133] [ka]

[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 are each independently N or CR 2a In this case, R 2a is hydrogen or a substituent;

[0137] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

[0138] R 205 is a hydrogen atom or a substituent, where R 105 and R 205 are each independently bonded to the 2- or 3-position of the 5-membered ring to which they are attached;

[0139] however,

[0140] B 100 or B 200 On the other hand,

[0141] [ka]

[0142] and

[0143] During the ceremony,

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

[0145] R 19 is a halogen atom;

[0146] And the other is bound to the carbonyl group of formula (I) via an -NH- group.

[0147] In a twenty-third 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 point of attachment to the carbonyl group of formula (I).

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

[0153] [ka]

[0154] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0155] a is an integer from 1 to 20;

[0156] b is an integer from 1 to 20;

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

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

[0159] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

[0161] R 4 is selected from hydrogen and any naturally occurring amino acid side chain;

[0162] R 5 is selected from C1-C4 alkyl, and O-C1-C4 alkyl;

[0163] L is a cleavable linker;

[0164] Ab is an antibody, antibody fragment, or antigen-binding fragment.

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

[0166] [ka]

[0167] and

[0168] During the ceremony:

[0169] [ka]

[0170] is the point of attachment to the carbonyl group;

[0171] [ka]

[0172] is the attachment point to Ab;

[0173] W is a self-immolative group;

[0174] Z is absent or a peptide of 2 to 5 amino acids;

[0175] U and U' are independently absent or a spacer;

[0176] Q is a heterobifunctional group.

[0177] In a third aspect, the present disclosure provides a compound of formula (V):

[0178] [ka]

[0179] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0180] D-NH-

[0181] [ka]

[0182] and;

[0183] X is O or CH2;

[0184] f is an integer from 1 to 10;

[0185] g is an integer from 1 to 20;

[0186] U and U' are independently absent or a spacer;

[0187] Q is a heterobifunctional group;

[0188] Ab is an antibody, and Ab is an antibody, antibody fragment, or antigen-binding fragment.

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

[0190] In a fourth aspect, the present disclosure provides a compound of formula (VI):

[0191] [ka]

[0192] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0193] a is an integer from 1 to 20,

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

[0195] n is 0 or 1;

[0196] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

[0198] R 2 is C1-C4 alkyl and -(CH2CH2O) s -CH3; wherein s is an integer from 1 to 10;

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

[0200] L is a cleavable linker;

[0201] LP is lipid.

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

[0203] In a sixth embodiment of the fourth aspect, the present 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 an eighth embodiment of the fourth aspect, the lipid complex further comprises one or more phospholipids. In a ninth embodiment, the one or more phospholipids are selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid, and egg yolk phospholipid. In a tenth embodiment, the phospholipid is phosphatidylcholine, and the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0205] In an eleventh embodiment of the fourth aspect the lipid-conjugate further comprises a fatty alcohol and / or cholesterol.

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

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

[0208] In a sixth aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound or conjugate described herein.

[0209] In a seventh aspect, the present disclosure provides a method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound or conjugate described herein. [Brief explanation of the drawings]

[0210] [Figure 1] FIG. 1 shows the preparation of Ab-STING agonist conjugates via stochastic cysteine ​​conjugation. [Figure 2] FIG. 1 shows the preparation of Ab-STING agonist conjugates via transglutaminase conjugation. [Figure 3] FIG. 1 shows the preparation of Ab-STING agonist conjugates via transglutaminase conjugation. [Figure 4] 1 shows the preparation of Ab-STING agonist conjugates via sortase conjugation (SEQ ID NO: 3 and SEQ ID NO: 4). [Figure 5] FIG. 1 shows payload release of ADC-1 in a tritosome assay. [Figure 6] FIG. 1 shows payload release of ADC-3 in a tritosome assay. [Figure 7] FIG. 1 shows payload release of ADC-9 in a tritosome assay. [Figure 8] FIG. 1 shows plasma PK of ADC-1 (0.1 mg / kg payload dose) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 9]FIG. 1 shows plasma PK of ADC-8 (0.055 mg / kg payload dose, released CDN BLQ) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 10] FIG. 1 shows plasma PK of ADC-9 (0.05 mg / kg payload dose, released CDN BLQ) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 11] FIG. 1 shows the efficacy of ADC-1 (payload dose indicated) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 12] FIG. 1 shows the efficacy of ADC-5 (payload dose indicated) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 13] FIG. 1 shows the efficacy of ADC-8 (payload dose indicated) in GCC-expressing CT26 tumor-bearing Balb / C mice. [Figure 14] FIG. 1 shows the efficacy of ADC-9 (payload dose indicated) in GCC-expressing CT26 tumor-bearing Balb / C mice. DETAILED DESCRIPTION OF THE INVENTION

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications and patents mentioned herein are incorporated by reference in their entirety.

[0212] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise.

[0213] As used herein, the term "or" is a logical or (i.e., and / or) and does not indicate an exclusive or, unless expressly indicated otherwise, such as by the terms "either," "otherwise," "alternatively," and words of similar effect.

[0214] As used herein, the term "about" refers to ±10%.

[0215] antibody-drug conjugates In some embodiments, the present disclosure provides a compound of formula (I):

[0216] [ka]

[0217] or a pharmaceutically acceptable salt thereof,

[0218] During the ceremony,

[0219] a is an integer from 1 to 20;

[0220] b is an integer from 1 to 20;

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

[0222] n is 0 or 1;

[0223] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

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

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

[0227] L is a cleavable linker;

[0228] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0229] In some embodiments, the present disclosure provides a compound represented by formula (IV):

[0230] [ka]

[0231] or a pharmaceutically acceptable salt thereof,

[0232] During the ceremony,

[0233] a is an integer from 1 to 20;

[0234] b is an integer from 1 to 20;

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

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

[0237] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

[0239] R 4 is selected from hydrogen and any naturally occurring amino acid side chain;

[0240] R 5 is selected from C1-C4 alkyl, and O-C1-C4 alkyl;

[0241] L is a cleavable linker;

[0242] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0243] In some embodiments, the present disclosure provides compounds of formula (V):

[0244] [ka]

[0245] or a pharmaceutically acceptable salt thereof,

[0246] During the ceremony,

[0247] D-NH

[0248] [ka]

[0249] and;

[0250] X is O or CH2;

[0251] f is an integer from 1 to 10;

[0252] g is an integer from 1 to 20;

[0253] U and U' are independently absent or a spacer;

[0254] Q is a heterobifunctional group;

[0255] Ab is an antibody, antibody fragment, or antigen-binding fragment.

[0256] In the compounds of formula (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 generates an immune response in a subject in need thereof. Examples of immunomodulators include IDO inhibitors, MEK inhibitors, STING inhibitors, and the like. In some embodiments, the D-NH2 is a STING modulator. In some embodiments, the D-NH2 comprises a guanine derivative or an adenine derivative. In some embodiments, the STING modulator has the formula:

[0257] [ka]

[0258] is a cyclic dinucleotide or cyclic dinucleotide-like compound (respectively, CDN) of wherein A' and A" are each independently a nucleoside or a synthetic analog thereof; Q, Q', Q 2 , and Q 2’ Each of Q is independently oxygen or sulfur. ’ and Q 2’ is sulfur. Examples of CDNs, including cyclic dinucleotides and cyclic dinucleotide-like compounds, within the scope of the present 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, US20180281 32, US20180002369, US20180186828, US20190016750, US20180162899, US20180369268, US2018273578, US20180092937, WO2017 / 027646, and WO2018 / 100558; as well as compounds such as, but not limited to, c-di-GMP, 2'3'-cGAMP, and SB 11285 (Spring Bank Pharmaceuticals).

[0259] In certain embodiments, the STING modulator has formula (II):

[0260] [ka]

[0261] 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 is -O-, -S-, -NH-, or -NR a - and then R a is C1-C4 alkyl;

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

[0268] R 20 is hydrogen or fluoro;

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

[0270] R 50 is hydrogen or fluoro;

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

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

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

[0274] As described herein, ring A 10 and Ring B 10 can contain one or more substituents and therefore can be optionally substituted. Suitable substituents on the unsaturated carbon atom of a heteroaryl group include -halo, -NO, -CN, -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 and generally selected therefrom, wherein R + are independently hydrogen or an optionally substituted aliphatic aryl, heteroaryl, or heterocyclyl group, or R + two independent occurrences of together with the intervening atom(s) form an optionally substituted 5- to 7-membered aryl ring; In some embodiments, R + are independently hydrogen, C 1-6 Aliphatic, or C 3-6 Each R° is independently an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic, or heterocyclyl group.

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

[0276] In some embodiments, the STING modulator has formula (IIA):

[0277] [ka]

[0278] or a pharmaceutically acceptable salt thereof,

[0279] In the formula, R 10 and R 40は each independently being hydrogen, fluoro, -OH, or -OCH2CF3; and ring A 10 and B 10 is as defined for compounds of formula (II), with the proviso that ring A 10 or ring B 10 is attached to the carbonyl group of the parent molecular moiety of compound (I), (IV), or (V) through an -NH- group.

[0280] In some embodiments, ring A 10 is an optionally substituted 6-membered monocyclic heteroaryl ring containing 1, 2 or 3 nitrogen atoms.

[0281] In some embodiments, ring B 10 is as follows:

[0282] [ka]

[0283] During the ceremony:

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

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

[0286] R 230 is hydrogen or -NH2;

[0287] R 200 , R 220 , and R 240 are each 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] is the point of attachment to the carbonyl group of the parent molecular moiety.

[0294] In certain embodiments, the STING modulator has formula (X):

[0295] [ka]

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

[0297] Formula (A-1):

[0298] [ka]

[0299] is a partial structure represented by formula (ZA), or a partial structure represented by formula (ZB), i.e.,

[0300] [ka]

[0301] and

[0302] R 105 and R 205 are each independently a hydroxy group or a halogen atom;

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

[0304] [ka]

[0305] is a group represented by

[0306] R 13 , R 14 , R 15 , R 16 , and R 17 are each independently a hydrogen atom or a substituent;

[0307] Y 11 , Y 12 , Y 13 , Y 14 , Y 15 , and Y 16 are each independently N or CR 1a and

[0308] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16 are each independently N or C;

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

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

[0311] [ka]

[0312] is a group represented by

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

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

[0315] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

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

[0317] X 1 and X 2 are each independently an oxygen atom or a sulfur atom;

[0318] Q 1a , Q 2a , Q3a, and Q 4a are each independently an oxygen atom or a sulfur atom;

[0319] B 100 or B 200 One of them is attached 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 alkyl; and the other is attached to the 5-membered ring of the parent structure via an -NH- group;

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

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

[0327] As described herein, specific R groups are 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 hydroxy groups, optionally substituted sulfanyl (SH) groups, and optionally substituted silyl groups.

[0328] As used herein, examples of "hydrocarbon groups" (including the "hydrocarbon group" of "optionally substituted hydrocarbon groups") include C 1-6 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 groups and C 7-16 Examples include aralkyl groups.

[0329] As used herein, examples of "optionally substituted hydrocarbon groups" include hydrocarbon groups optionally bearing substituent(s) selected from the following Substituent Group A, including:

[0330] (1) a halogen atom,

[0331] (2) a nitro group,

[0332] (3) a cyano group,

[0333] (4) an oxo group,

[0334] (5) a hydroxy group,

[0335] (6) Optionally halogenated C 1-6 alkoxy groups,

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

[0337] (8) C 7-16 aralkyloxy groups (e.g., benzyloxy),

[0338] (9) 5- to 14-membered aromatic heterocyclyloxy groups (e.g., pyridyloxy),

[0339] (10) 3- to 14-membered non-aromatic heterocyclyloxy groups (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) 5- to 14-membered aromatic heterocyclylcarbonyloxy groups (e.g., nicotinoyloxy),

[0346] (17) 3- to 14-membered non-aromatic heterocyclylcarbonyloxy groups (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy),

[0347] (18) Optionally halogenated C 1-6 alkylsulfonyloxy groups (e.g., methylsulfonyloxy, trifluoromethylsulfonyloxy);

[0348] (19)C 1-6 C optionally substituted with alkyl groups 6-14 arylsulfonyloxy groups (e.g., phenylsulfonyloxy, toluenesulfonyloxy);

[0349] (20) Optionally halogenated C 1-6 alkylthio groups,

[0350] (21) a 5- to 14-membered aromatic heterocyclic group,

[0351] (22) a 3- to 14-membered non-aromatic heterocyclic group,

[0352] (23) a formyl group,

[0353] (24) a carboxy group,

[0354] (25) Optionally halogenated C 1-6 alkyl-carbonyl groups,

[0355] (26)C 6-14 aryl-carbonyl groups,

[0356] (27) A 5- to 14-membered aromatic heterocyclylcarbonyl group,

[0357] (28) a 3- to 14-membered non-aromatic heterocyclylcarbonyl group,

[0358] (29)C 1-6 alkoxy-carbonyl groups,

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

[0360] (31)C 7-16 aralkyloxy-carbonyl groups (e.g., benzyloxycarbonyl, phenethyloxycarbonyl),

[0361] (32) a carbamoyl group,

[0362] (33) a thiocarbamoyl group,

[0363] (34) Mono- or di-C 1-6 alkyl-carbamoyl groups,

[0364] (35)C 6-14 aryl-carbamoyl groups (e.g., phenylcarbamoyl);

[0365] (36) 5- to 14-membered aromatic heterocyclylcarbamoyl groups (e.g., pyridylcarbamoyl, thienylcarbamoyl),

[0366] (37) 3- to 14-membered non-aromatic heterocyclylcarbamoyl groups (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),

[0367] (38) Optionally halogenated C 1-6 alkylsulfonyl groups,

[0368] (39)C 6-14arylsulfonyl groups,

[0369] (40) 5- to 14-membered aromatic heterocyclylsulfonyl groups (e.g., pyridylsulfonyl, thienylsulfonyl),

[0370] (41) Optionally halogenated C 1-6 alkylsulfinyl groups,

[0371] (42)C 6-14 arylsulfinyl groups (e.g., phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl);

[0372] (43) 5- to 14-membered aromatic heterocyclylsulfinyl groups (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 groups (e.g., phenylamino);

[0376] (47) 5- to 14-membered aromatic heterocyclylamino groups (e.g., pyridylamino),

[0377] (48)C 7-16 aralkylamino groups (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 groups (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 aralkyloxy-carbonylamino groups (e.g., benzyloxycarbonylamino),

[0384] (55)C 1-6 alkylsulfonylamino groups (e.g., methylsulfonylamino, ethylsulfonylamino);

[0385] (56)C 1-6 C optionally substituted with alkyl groups 6-14 arylsulfonylamino groups (e.g., phenylsulfonylamino, toluenesulfonylamino);

[0386] (57) Optionally halogenated C 1-6 alkyl groups,

[0387] (58)C 2-6 alkenyl groups,

[0388] (59)C 2-6 alkynyl groups,

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

[0390] (61) C 3-10 cycloalkenyl groups, and

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

[0392] The number of the above-mentioned substituents in the "optionally substituted hydrocarbon group" is, for example, 1 to 5, usually 1 to 3. When the number of substituents is 2 or more, the respective substituents may be the same or different.

[0393] In this specification, examples of the "heterocyclic group" (including the "heterocyclic group" of the "optionally substituted heterocyclic group") include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group, and (iii) a 7- to 10-membered bridged heterocyclic group, each of which contains 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom as ring-constituting atoms other than carbon atoms.

[0394] In this specification, examples of the "aromatic heterocyclic group" (including "aromatic heterocyclic group having a 5- to 14-membered ring") include 5- to 14-membered (usually 5- to 10-membered) aromatic heterocyclic groups containing, as ring-constituting atoms other than carbon atoms, 1 to 4 heteroatoms selected from nitrogen atoms, sulfur atoms, and oxygen atoms.

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

[0396] Benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazo and 8- to 14-membered fused polycyclic (usually bicyclic or tricyclic) aromatic heterocyclic groups such as pyrrolopyridine, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.

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

[0398] Preferable examples of the "non-aromatic heterocyclic group" include aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisothiazolyl ... 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as isoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl, and diazocanyl; and

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

[0400] In the present specification, suitable examples of the "7- to 10-membered bridged heterocyclic group" include quinuclidinyl and 7-azabicyclo[2.2.1]heptanyl.

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

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

[0403] The number of substituents in the "optionally substituted heterocyclic group" is, for example, 1 to 3. When the number of substituents is 2 or more, the respective substituents may be the same or different.

[0404] As used herein, examples of "acyl groups" include formyl groups, carboxy groups, carbamoyl groups, thiocarbamoyl groups, sulfino groups, sulfo groups, sulfamoyl groups, and phosphono groups, each of which is optionally 1-6 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 has one or two substituents selected from an aralkyl group, a 5- to 14-membered aromatic heterocyclic group, and a 3- to 14-membered non-aromatic heterocyclic group, each of which is a halogen atom, an optionally halogenated C 1-6 It optionally has 1 to 3 substituents selected from an alkoxy group, a hydroxy group, a nitro group, a cyano group, an amino group and a carbamoyl group.

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

[0406] Here, the term "hydrocarbon-sulfonyl group" refers to a sulfonyl group having a hydrocarbon group bonded thereto, the term "heterocyclylsulfonyl group" refers to a sulfonyl group having a heterocyclic group bonded thereto, the term "hydrocarbon-sulfinyl group" refers to a sulfinyl group having a hydrocarbon group bonded thereto, and the term "heterocyclylsulfinyl group" refers to a sulfinyl group having a heterocyclic group bonded thereto.

[0407] Suitable examples of the "acyl group" include a formyl group, a carboxy group, and C 1-6 Alkyl-carbonyl group, C 2-6 Alkenyl-carbonyl groups (e.g., crotonoyl), C 3-10 Cycloalkyl-carbonyl groups (e.g., cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), C 3-10 Cycloalkenyl-carbonyl groups (e.g., 2-cyclohexenecarbonyl), C 6-14 Aryl-carbonyl group, C 7-16 Aralkyl-carbonyl group, 5- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, C 6-14 Aryloxy-carbonyl groups (e.g., phenyloxycarbonyl, naphthyloxycarbonyl), C 7-16 Aralkyloxy-carbonyl group (e.g., benzyloxycarbonyl, phenethyloxycarbonyl), carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 2-6 Alkenyl-carbamoyl groups (e.g., diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl groups (e.g., cyclopropylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl groups (e.g., phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl group, 5- to 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 groups (e.g., diallylthiocarbamoyl), mono- or di-C 3-10Cycloalkyl-thiocarbamoyl groups (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14 Aryl-thiocarbamoyl groups (e.g., phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl groups (e.g., benzylthiocarbamoyl, phenethylthiocarbamoyl), 5- to 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 Alkylsulfonyl group, C 6-14 Arylsulfonyl group, phosphono group and mono- or di-C 1~6 Examples include alkylphosphono groups (for example, dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

[0408] In the present specification, examples of the "optionally substituted amino group" include C 1 ... 1-6 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- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, 5- to 14-membered aromatic heterocyclic group, carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 7-16 Aralkyl-carbamoyl group, C 1-6 Alkylsulfonyl group and C 6-14 and an amino group optionally having one or two substituents selected from arylsulfonyl groups.

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

[0410] In the present specification, examples of the "optionally substituted carbamoyl group" include C 1 ... 1-6 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- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, 5- to 14-membered aromatic heterocyclic group, carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group and mono- or di-C 7-16 Examples include a carbamoyl group optionally having one or two substituents selected from an aralkyl-carbamoyl group.

[0411] Suitable 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 groups (e.g., diallylcarbamoyl), mono- or di-C 3-10 Cycloalkyl-carbamoyl groups (e.g., cyclopropylcarbamoyl, cyclohexylcarbamoyl), mono- or di-C 6-14 Aryl-carbamoyl groups (e.g., phenylcarbamoyl), mono- or di-C 7-16 Aralkyl-carbamoyl group, mono- or di-C 1-6 Alkyl-carbonyl-carbamoyl groups (e.g., acetylcarbamoyl, propionyl Nylcarbamoyl), mono- or di-C 6-14 Examples thereof include aryl-carbonyl-carbamoyl groups (for example, benzoylcarbamoyl) and 5- to 14-membered aromatic heterocyclylcarbamoyl groups (for example, pyridylcarbamoyl).

[0412] In the present specification, examples of the "optionally substituted thiocarbamoyl group" include C thiocarbamoyl groups each of which optionally has 1 to 3 substituents selected from Substituent Group A. 1-6 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- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, 5- to 14-membered aromatic heterocyclic group, carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group and mono- or di-C 7-16 and thiocarbamoyl groups optionally having one or two substituents selected from aralkyl-carbamoyl groups.

[0413] Suitable 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 groups (e.g., diallylthiocarbamoyl), mono- or di-C 3-10 Cycloalkyl-thiocarbamoyl groups (e.g., cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), mono- or di-C 6-14Aryl-thiocarbamoyl groups (e.g., phenylthiocarbamoyl), mono- or di-C 7-16 Aralkyl-thiocarbamoyl groups (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 thereof include aryl-carbonyl-thiocarbamoyl groups (for example, benzoylthiocarbamoyl) and 5- to 14-membered aromatic heterocyclylthiocarbamoyl groups (for example, pyridylthiocarbamoyl).

[0414] In the present specification, examples of the "optionally substituted sulfamoyl group" include C 1 ... 1-6 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- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, 5- to 14-membered aromatic heterocyclic group, carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group and mono- or di-C 7-16 and a sulfamoyl group optionally 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 groups (e.g., diallylsulfamoyl), mono- or di-C 3-10 Cycloalkyl-sulfamoyl groups (e.g., cyclopropylsulfamoyl, cyclohexylsulfamoyl), mono- or di-C 6-14 Aryl-sulfamoyl groups (e.g., phenylsulfamoyl), mono- or di-C 7-16 Aralkyl-sulfamoyl group (e.g., benzylsulfamoyl, phenethylsulfamoyl), mono- or di-C 1-6 Alkyl-carbonyl-sulfamoyl group (e.g., acetylsulfamoyl, propionylsulfamoyl), mono- or di-C 6-14 Aryl-carbonyl-sulfamoyl groups (e.g. , benzoylsulfamoyl) and 5- to 14-membered aromatic heterocyclylsulfamoyl groups (for example, pyridylsulfamoyl).

[0416] As used herein, examples of the "optionally substituted hydroxy group" include C 1 ... 1-6 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- to 14-membered aromatic heterocyclylcarbonyl group, 3- to 14-membered non-aromatic heterocyclylcarbonyl group, C 1-6 Alkoxy-carbonyl group, 5- to 14-membered aromatic heterocyclic group, carbamoyl group, mono- or di-C 1-6 Alkyl-carbamoyl group, mono- or di-C 7-16 Aralkyl-carbamoyl group, C 1-6 Alkylsulfonyl group and C 6-14 and hydroxy groups optionally having a substituent selected from arylsulfonyl groups.

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

[0418] In the present specification, examples of the "optionally substituted sulfanyl group" include C 1111, each of which optionally has 1 to 3 substituents selected from Substituent Group A. 1-6 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 a sulfanyl group and a halogenated sulfanyl group optionally having a substituent selected from an aryl-carbonyl group and a 5- to 14-membered aromatic heterocyclic group.

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

[0420] In the present specification, examples of the "optionally substituted silyl group" include C 1111, each of which optionally has 1 to 3 substituents selected from Substituent Group A. 1-6 Alkyl group, C 2-6 Alkenyl group, C 3-10 Cycloalkyl groups, C 6-14 Aryl groups and C 7~16 Examples of the optionally substituted silyl group include a silyl group having 1 to 3 substituents selected from aralkyl groups. Examples of the optionally substituted silyl group include tri-C 1-6 Examples include alkylsilyl groups (for example, trimethylsilyl, tert-butyl(dimethyl)silyl).

[0421] In some embodiments, the STING modulator has formula (III):

[0422] [ka]

[0423] or a pharmaceutically acceptable salt thereof,

[0424] During the ceremony,

[0425] R 105 and R 205 are each independently a hydroxyl group or a halogen atom;

[0426] B 100 is the formula (B 1 -A) or formula (B 1 -B) is a group represented by

[0427] [ka]

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

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

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

[0431] Z 11 , Z 12 , Z 13 , Z 14 , Z 15 , and Z 16are each independently N or C;

[0432] R 105 and R 205 are each independently a hydroxyl group or a halogen atom;

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

[0434] B 200 is the formula (B 2 -A) or formula (B 2 -B) is a group represented by:

[0435] [ka]

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

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

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

[0439] Z 21 , Z 22 , Z 23 , Z 24 , Z 25 , and Z 26 are each independently N or C;

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

[0441] X 1 and X 2 are each independently an oxygen atom or a sulfur atom;

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

[0443] however,

[0444] B 100 or B 200 One of the

[0445] [ka]

[0446] and

[0447] During the ceremony,

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

[0449] R 19 is a halogen atom, and B 100 or B 200 The other must be attached 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, wherein R 205 is F and B 100 teeth

[0451] [ka]

[0452] is.

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

[0454] [ka]

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

[0456] [ka]

[0457] is the point of attachment to the carbonyl group of the parent molecular moiety.

[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 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, light-induced cleavage, enzymatic cleavage, etc. under conditions that allow the drug and / or antibody to remain active. 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, VAGP tetrapeptide, etc. In certain embodiments, the linker can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0460] In certain embodiments, "L" is of the formula:

[0461] [ka]

[0462] and

[0463] During the ceremony:

[0464] [ka]

[0465] is the point of attachment to the nitrogen atom;

[0466] [ka]

[0467] is the point of attachment to the Ab.

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

[0469] [ka]

[0470] and

[0471] During the ceremony:

[0472] [ka]

[0473] is the point of attachment to the nitrogen atom;

[0474] [ka]

[0475] is the point of attachment to the antibody.

[0476] The "W" group is absent or a self-immolative group. As used herein, the term "self-immolative" refers to a group that undergoes an electronic cascade that results in the release of the group to which it is attached. In some embodiments, the self-immolative group comprises one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cycloelimination, 1,5-cycloelimination, 1,3-cycloelimination, intramolecular 5-exo-trigger cyclization, and / or 6-exo-trigger cyclization. In certain embodiments, the self-immolative group can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0477] The "Z" group is absent or is a peptide of 2-5 amino acids. In certain embodiments, the peptide is the site of cleavage of the linker, thereby facilitating 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-homolysine (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 be present in the reverse order (ie, cit-val).

[0478] The peptides of the present disclosure may include naturally occurring and / or non-naturally occurring amino acid residues. The term "naturally occurring amino acid" refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. "Non-naturally occurring amino acids" (i.e., amino acids not occurring in nature) include, by non-limiting example, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalene, ornithine ("Orn"), and the like. 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 also include D-forms of natural and unnatural amino acids. "D-" refers to an amino acid having a "D" (dextrorotatory) configuration as opposed to the configuration in naturally occurring ("L-") amino acids. Natural and unnatural amino acids can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0480] The "U" and "U'" groups are independently absent or a spacer. As used herein, the term "spacer" refers to a chemical moiety that functions as a connecting group. In the present disclosure, the spacer can connect an antibody, antibody fragment, or antigen fragment to the heterobifunctional group and / or connect the heterobifunctional group to the peptide "Z", or to the "W" group if "Z" is absent. 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 the present disclosure, "U," when present, can 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 one "-C(O)-WZ-" group. In certain embodiments, the spacer can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0483] The "Q" group is a heterobifunctional group. In the present disclosure, the term "heterobifunctional group" refers to a chemical moiety that connects a linker of which it is a part to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO2017 / 191579. Heterobifunctional groups are characterized by having different reactive groups at both ends of the chemical moiety. The heterobifunctional group may be directly attached to the "Ab" or alternatively connected via a linker "U'". Attachment to the "Ab" can be achieved via chemical or enzymatic coupling, or a combination of both. Chemical attachment involves the controlled reaction of accessible amino acid residues on the surface of the antibody with a reactive handle at "Q" or "U'". Examples of chemical attachment include, but are not limited to, lysine amide coupling, cysteine ​​coupling, and coupling via unnatural amino acids incorporated by genetic engineering, whereby unnatural amino acid residues with the desired reactive handle are introduced into the "Ab". In enzymatic attachment, an enzyme mediates the coupling of the linker to accessible amino acid residues on the antibody, antibody fragment, or antigen-binding fragment. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can be used to introduce a unique reactive handle into an "Ab" that is utilized in subsequent chemical conjugation. In certain embodiments, heterobifunctional groups are described in PCT Publication WO 2018 / 20081, which is incorporated by reference in its entirety. 2, can be any of those disclosed in WO2018 / 100558.

[0484] In some embodiments, "Q" is

[0485] [ka]

[0486] is selected from

[0487] During the ceremony,

[0488] [ka]

[0489] is the point of attachment to U, or if U is not present, to Z;

[0490] [ka]

[0491] is the point of attachment to U', or to Ab if U' is absent.

[0492] In some embodiments, the present disclosure provides a compound of formula (XX):

[0493] [ka]

[0494] or a pharmaceutically acceptable salt 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 * is a reactive functional group capable of binding to an antibody, antibody fragment, or antigen-binding fragment. *Exemplary 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 cyclooctynes, oxanoborazines, norbornenes, azides, diaryltetrazines, monoaryltetrazines, aldehydes, ketones, hydroxylamines, vinyl sulfones, and aziridines. In certain embodiments, the reactive functional groups can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are incorporated by reference in their entireties.

[0495] An "Ab" group refers to an antibody, antibody fragment, or antigen-binding fragment. An antibody is a protein generated by the immune system that can recognize and bind to a specific antigen. A target antigen generally has multiple binding sites, also called epitopes, 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" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, or chimeric, or may be derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001), Immuno Biology, 5th Ed., Garland Publishing, New York)

[0496] The term "antibody," as used herein, also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, a cancer cell or cells that produce autoimmune antibodies associated with autoimmune disease. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulin can be derived from any species. However, in one aspect, the immunoglobulin is of human, murine, or rabbit origin.

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

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

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

[0500] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975), Nature, 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0501] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity, as well as fragments of such antibodies (U.S. Pat. No. 4,816,567 and Morrison, J. Med. Chem. Soc. 1999, 10, 144-145). (Eds., et al., (1984), Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

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

[0503] Chimeric and humanized antibodies reduce the likelihood of a HAMA reaction by minimizing the non-human portion of the administered antibody, and can have the added benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

[0504] An intact antibody may possess one or more "effector functions," which refer to biological activities attributable to the Fc region of an antibody (a native-sequence Fc region or an 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, down-regulation of cell surface receptors (e.g., B-cell receptor; BCR), and the like.

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

[0506] Useful non-immunoreactive protein, polypeptide, 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 apoproteins derived from low-density lipoproteins.

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

[0508] Useful monoclonal antibodies are homogeneous populations of antibodies directed against a particular antigenic determinant (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) directed against an antigen of interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous 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. Liss). 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 producing mAbs used in this disclosure may be cultivated 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 a number of 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] The antibody can also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed using affinity chromatography steps, is rather cumbersome and results in low product yields. Similar procedures are described in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0511] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion may be with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2, and CH3 regions. The first heavy chain constant region (CH1) may contain the site necessary for light chain binding, present in at least one of the fusions. Nucleic acids encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction result in optimal yields. However, if expression of at least two polypeptide chains in equal ratios results in high yields, or if their ratios are not particularly critical, it is possible to insert the coding sequences for 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 chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a facile separation method (WO 94 / 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, the immunoglobulin light chains as defined herein can be separated from undesired immunoglobulin chain combinations. Bispecific antibodies can be prepared for conjugation as ADCs in the treatment or prevention of diseases such as those described herein.

[0513] Hybrid or bifunctional antibodies can be derived biologically, i.e., by cell fusion techniques, or chemically, in particular by cross-linking or disulfide bridge-forming reagents, and may comprise whole antibodies or fragments thereof (EP 105360; WO 83 / 03679; EP 217577).

[0514] The antibody can be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a cancer cell antigen, a viral antigen, or a microbial antigen, or to another antibody bound to a tumor cell or matrix. In this context, "functionally active" means that the fragment, derivative, or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen recognized by the antibody from which the fragment, derivative, or analog is derived. Specifically, in exemplary embodiments, the idiotypic antigenicity of an immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences C-terminal to the CDR sequences that specifically recognize the antigen. To determine which CDR sequences bind to an antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., BIAcore 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 antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments, which contain the variable region, light chain constant region, and CH1 domain of the heavy chain and can be produced by pepsin digestion of an antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of F(ab')2 fragments. Other useful antibodies are antibody heavy and light chain dimers, or any minimal fragments thereof, such as Fv or single-chain antibodies (SCAs) (e.g., as described in U.S. Pat. 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 an antibody.

[0516] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions, which can be produced using standard recombinant DNA techniques, are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397.) Humanized antibodies are antibody molecules derived from non-human species having one or more complementarity-determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089.) Such chimeric and humanized monoclonal antibodies are described in, e.g., WO 87 / 02671; EP 184,187; EP 171496; EP 173494; WO 86 / 01533; U.S. Pat. No. 4,816,567; EP 12023; 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;U.S. 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 by recombinant DNA techniques known in the art using the methods described in et al., 1988, J. Immunol. 141:4053-4060.

[0517] Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes but 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 a portion of a polypeptide of the present disclosure. Monoclonal antibodies against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Other human antibodies are commercially available from, for example, Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.).

[0518] Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al. (1994), Biotechnology, 12:899-903). Human antibodies can also be produced 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 also be a fusion protein of an antibody, or a functionally active fragment thereof, for example, wherein the antibody is fused at either the N- or C-terminus via a covalent bond (e.g., a peptide bond) to the amino acid sequence of another protein (or portion thereof, e.g., at least a 10, 20, or 50 amino acid portion of the protein) that is not an antibody. The antibody or fragment thereof may also be covalently linked to the other protein at the N-terminus of the constant domain.

[0520] Antibodies include analogs and derivatives that are modified, i.e., by the covalent attachment of any type of molecule, so long as such covalent attachment enables the antibody to retain its antigen-binding immunospecificity. For example, and not by way of limitation, antibody derivatives and analogs include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular antibody units or other proteins, etc. Thus, further modifications are included. Numerous chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

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

[0522] The antibody of the ADC may be a monoclonal antibody, e.g., a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody may also be an antibody fragment, e.g., a Fab fragment.

[0523] Known antibodies for cancer treatment or prevention can be combined as ADCs. Antibodies immunospecific for cancer cell antigens can be commercially available or can be produced by any method known to those skilled in the art, such as recombinant expression technology. The nucleotide sequence encoding an antibody immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a similar database, literature publications, or by routine cloning and sequencing.Examples of antibodies available for the treatment of cancer include a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; RITUXAN® (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA), a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY), an anti-EGFR IgG 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-CD33 IgG antibody for the treatment of acute myeloid leukemia (AML). Labs, Inc., CA); LymphoCide, a humanized anti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma (Immunomedics, Inc., NJ); Smart ID10, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma (Protein Design Labs, Inc., CA); Oncolym, a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma (Techniclone, Inc., CA); Allomune, a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma (BioTransplant, CA); Avastin, a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer (Genentech, Inc., CA); Epratuzamab, an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma (Immunomedics, Inc., NJ and Amgen, CA); and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0524] Other antibodies useful in treating cancer include those directed against the following antigens: CA125 (ovarian), 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). Examples of antibodies that may be used include, but are not limited to, antibodies against: human leukemia (T-cell leukemia), breast cancer (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphoma), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu oncogene product (carcinoma). 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 internalizing antibodies that bind to tumor-associated antigens can be used and have been reviewed (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. Antibodies immunospecific for cellular antigens involved in the production of autoimmune antibodies can be obtained by any method known to those skilled in the art, such as chemical synthesis or recombinant expression technology. SLE is characterized by overexpression of the interferon-α (IFN-α) 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, erythroblastosis, hemolytic anemia, anti-DNA autoantibodies, renal disease, and mortality. These results suggest that type 1 IFN mediates lupus in mice and that reducing their activity in human counterparts may be beneficial. Anti-IFN Abs conjugated to bis-1,8 naphthalimide drug moieties may be effective therapeutic agents for SLE and other autoimmune diseases.

[0527] In another embodiment, antibodies useful in ADCs are immunospecific for the treatment of autoimmune diseases, including, but not limited to, antinuclear antibodies, anti-dsDNA; anti-ssDNA, anticardiolipin antibodies IgM, IgG; antiphospholipid antibodies IgM, IgG; anti-SM antibodies; antimitochondrial antibodies; thyroid antibodies; microsomal antibodies; thyroglobulin antibodies; anti-SCL-70; anti-Jo; anti-U1RNP; anti-La / SSB; anti-SSA; anti-SSB; anti-pericyte antibodies; anti-histone; anti-RNP; C-ANCA; P-ANCA; anti-centromere; anti-fibrillarin, and anti-GBM antibodies.

[0528] The antibodies of the ADCs can bind to both receptors or receptor complexes expressed on activated lymphocytes. The receptors or receptor complexes can 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 suitable 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 are 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 are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitable lectins are C-type, S-type, and I-type lectins.

[0529] As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoproteins (e.g., Gb, Gc, Gd, and Ge), and hepatitis B surface antigen) that can elicit an immune response. As used herein, the term "microbial antigen" includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., bacterial, fungal, pathogenic protozoan, or yeast polypeptides, including LPS and capsular polysaccharide 5 / 8) that can elicit an immune response.

[0530] Antibodies immunospecific for viral or microbial antigens can be obtained commercially, 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 of skill in the art, such as, for example, chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for viral or microbial antigens can be obtained, for example, from the GenBank database or a database similar thereto, literature publications, or by routine cloning and sequencing.

[0531] In some embodiments, antibodies useful in the current ADCs are those that 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; C-C1000 (MedImmune, Inc., MD), a humanized anti-RSV monoclonal antibody useful for treating patients with RSV infection; Examples of antibodies 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 in 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). 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).

[0533] Other antibodies useful in ADCs for the treatment of viral diseases include those from the Poxviridae, Herpesviridae, herpes simplex virus 1, herpes simplex virus 2, Examples of antibodies include, but are not limited to, antibodies against antigens of pathogenic viruses, including, but not limited to, 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 virus, 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 a native sequence polypeptide. Typically, an amino acid sequence variant will have at least about 70% sequence identity with at least one receptor-binding domain of a native antibody or at least one ligand-binding domain of a native receptor, and usually will have at least about 80%, and more usually at least about 90%, sequence identity with such receptor or ligand-binding domain. Amino acid sequence variants have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, one-letter codes, and three-letter codes.

[0535] "Sequence identity" is defined as the percentage of residues of amino acid sequence variants that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. 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, along with user documentation, on December 10, 1991.

[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-sequence human FcR. Furthermore, FcRs may bind IgG antibodies (gamma receptors) and include receptors for FcγRI, FcγRII, and Fcγ. The RIII subclass, including allelic variants and alternatively spliced ​​forms of these receptors, includes FcγRII receptors, FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review M in Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed 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 yet to be identified, are encompassed by the term "FcR" herein. The term also encompasses FcRn, the neonatal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., 2001). al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0537] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, For example, a CDC assay such as that 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 a 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 end. 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. Particular amino acid residues are believed 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 portions of the variable domains differ widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light-chain variable domains each contain four FRs that adopt a primarily beta-sheet configuration connected by three hypervariable regions, which form loops that connect, and in some cases form, part of the beta-sheet structure. The hypervariable regions in each chain are closely adjacent by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC).

[0540] When used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., supra), and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 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). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0541] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0542] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region comprises one heavy-chain and one light-chain variable domain in tight, non-covalent association. It consists of a dimer of six hypervariable regions. It is in this configuration that the three hypervariable regions of each variable domain interact to define an 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 an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0543] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(Ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

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

[0545] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide further contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, 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 WO 93 / 16185, U.S. Patent Nos. 5,571,894, and 5,587,458.

[0546] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a variable heavy domain (VH) connected to a variable light domain (VL) on the same polypeptide chain (VH-VL). The domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites, by using a linker that is too short to allow pairing between the two domains on the same chain. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448.

[0547] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain 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 a number of therapeutically useful drugs. The humanization process generally begins by transferring all six mouse complementarity-determining regions (CDRs) into a human antibody framework (Jones et al., (1986) Nature 321:522-525). Antibodies with these CDRs grafted generally do not retain their original affinity for antigen binding; in fact, affinity is often severely impaired. In addition to the CDRs, selected non-human antibody framework residues must also be incorporated to maintain proper CDR configuration (Chothia et al., (1989) Nature 342:877). Transfer of key murine framework residues to support the structural alignment of the grafted CDRs into the human acceptor 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, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see U.S. Patent No. 6,407,213; 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 comprising an amino acid sequence derived from a sequence in which one or more amino acid residues have been replaced with one or more cysteine ​​residues. The parent antibody may comprise a native or wild-type sequence. The parent antibody may have modifications (such as additions, deletions, and / or substitutions) to the existing amino acid sequence relative to other native, wild-type, or modified forms of the antibody. The parent antibody is directed against a target antigen of interest. Antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens, see U.S. Pat. No. 5,091,178) are also contemplated.

[0549] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight, or greater than 99% by weight, as determined by the Lowry method, (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas-phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

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

[0551] The term "treat" or "treatment" refers to both therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent or slow (alleviate) an undesired physiological change or disorder, such as the onset or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of disease, stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, palliation or remission of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

[0552] "Phage display" is a technique in which mutant polypeptides are displayed as fusion proteins with coat proteins on the surface of phage, e.g., filamentous phage, particles. One of the usefulness of phage display lies in the fact that large libraries of random protein variants can be rapidly and efficiently screened for sequences that bind to target molecules with high affinity. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Polyvalent phage display methods are typically used to display small random peptides and small proteins via fusion to either PIII or PVIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992) and references cited therein. In monovalent phage display, a protein or peptide library is fused to a phage coat protein or a portion thereof and expressed at low levels in the presence of the wild-type protein. Avidity effects are reduced for polyvalent phage, as selection is based on intrinsic ligand affinity, and phagemid vectors are used, simplifying DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991). Phage display includes techniques for generating antibody-like molecules (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p627-628).

[0553] A "phagemid" is a plasmid vector containing a bacterial origin of replication, e.g., Co1E1, and a copy of the intergenic region of a bacteriophage. Phagemids can be used with any known bacteriophage, including filamentous and lambda bacteriophages. Plasmids will also generally contain a selectable marker for antibiotic resistance. DNA segments cloned into these vectors can be propagated as plasmids. Providing all the genes necessary for phage particle production in cells harboring these vectors changes the replication mode of the plasmid to rolling circle replication, producing single-stranded copies of the plasmid DNA and packaged phage particles. Phagemids can form infectious or non-infectious phage particles. The term includes phagemids containing a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion, such that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein can 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 reviews of suitable salts, see, e.g., 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 the present disclosure, an "Ab" group (i.e., an antibody, antibody fragment, and / or antigen fragment) can be conjugated to more than one drug-containing moiety. In some embodiments, an "Ab" can be conjugated to 1-20 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1-10 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1-5 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1-2 drug-containing moieties. In some embodiments, an "Ab" can be conjugated to 1 The compound can be attached to two drug-containing moieties.

[0555] The compounds described herein can be in the form of pharmaceutical or pharmaceutically acceptable salts. In some embodiments, such salts are derived from inorganic or organic acids or bases. For reviews 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] Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate salts.

[0557] Examples of suitable 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, dicyclohexylamine salts, salts with organic bases such as N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, and the like.

[0558] For example, Berge lists the following FDA-approved commercially available salts: the anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), and edisylate (1,2-ethanedisulfonate). , estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycolyl arsanilate (glycolamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxybenzoate) The following cations are also included: benzoate (8-chlorotheophylline), ...

[0559] Berge goes on to list the following commercially available (outside the United States) salts that are not FDA approved: the anions adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, and butyl bromide. , camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), napadisilate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate, and undecanoate; the organic cations benethamine (N-benzylphenethylamine), clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations 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 into 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 muscle, or directly into an organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle syringes, needleless syringes, and infusion techniques.

[0562] Parenteral compositions are usually aqueous solutions which may contain excipients such as salts, carbohydrates, and buffers. However, the compositions may also be formulated as sterile nonaqueous solutions or in dry form for use in combination with a suitable vehicle such as sterile, pyrogen-free water.

[0563] The preparation of parenteral compositions under sterile conditions, for example, by lyophilization, may be readily accomplished 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-, sustained-, pulsed-, controlled-, targeted-, and programmed-release. Thus, compositions can be formulated as solids, semi-solids, or thixotropic liquids for administration as implanted depots that provide modified release of the active agent.

[0565] The parenteral preparation may 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 as a suitable oral dosage form such as, for example, a tablet, capsule, powder, pellet, suspension, solution, emulsion, etc. Other suitable carriers can be present such as, for example, disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, bulking agents, anti-adherents, etc.

[0567] The oral dosage formulations may also contain other suitable pharmaceutical excipients such as, for example, sweeteners, vehicles / humectants, colorants, flavoring agents, preservatives, viscosity enhancers / thickeners, and the like.

[0568] Doses of the pharmaceutical compositions of the present disclosure can be adjusted to suit the individual patient.

[0569] lipid complex In certain embodiments, the present disclosure provides a compound of formula (VI):

[0570] [ka]

[0571] or a pharmaceutically acceptable salt thereof, During the ceremony,

[0572] a is an integer from 1 to 20,

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

[0574] n is 0 or 1;

[0575] D-NH- is part of an amino-substituted compound, the amino-substituted compound having the formula D-NH2;

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

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

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

[0579] L is a cleavable linker;

[0580] LP is lipid.

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

[0582] In certain embodiments, the present disclosure provides compounds of formula (VI), wherein L is of formula

[0583] [ka]

[0584] and wherein W, Z, t, U, Q, and U' are as described herein.

[0585] In some embodiments, the compound of formula (VI) is associated with a lipid conjugate. The term "lipid conjugate" is an art-recognized term for 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 bound by any particular theory, the compound of formula (VI) can be associated with the lipid complex through electrostatic interaction, hydrophilic interaction, hydrophobic interaction, or any combination thereof.

[0586] In some embodiments, the lipid complexes described herein are in the form of lipid particles. In one embodiment, the complexes are in the form of lipid nanoparticles. In one embodiment, the complexes are in the form of liposomes. In some embodiments, the liposomes of the present disclosure comprise a lipid monolayer or a lipid multilayer. In some embodiments, the liposomes comprise 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, the LP can be cholesterol or a phospholipid. In some embodiments, the LP is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, soybean phospholipid, and egg yolk phospholipid. 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 present disclosure provides a lipid complex comprising a compound of formula (VI): In some embodiments, the lipid complex can be a liposome.

[0589] In some embodiments, the lipid complexes comprise about 0.5 to about 40 mole percent of a compound of Formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complexes comprise about 1 to about 35 mole percent of a compound of Formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complexes comprise about 1 to about 30 mole percent of a compound of Formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complexes comprise about 1 to about 20 mole percent of a compound of Formula (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complexes comprise about 1 to about 15 mole percent of a 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, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipids, and egg yolk phospholipids.

[0591] In some embodiments, the lipid complexes comprise from about 30 to about 90 mole percent of one or more phospholipids. In some embodiments, the lipid complexes comprise from about 40 to about 85 mole percent of one or more phospholipids. In some embodiments, the lipid complexes comprise from about 50 to about 80 mole percent of one or more phospholipids.

[0592] In some embodiments, the lipid complexes further comprise a fatty alcohol or cholesterol. In some embodiments, the lipid complexes comprise about 5 to about 35 mole percent cholesterol. In some embodiments, the lipid complexes comprise about 5 to about 30 mole percent cholesterol. In some embodiments, the lipid complexes comprise about 5 to about 25 mole percent 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 PEGylated phospholipid 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, a pharmaceutically acceptable salt thereof, or a mixture thereof. In some embodiments, the PEGylated phospholipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0594] In some embodiments, lipid complexes comprise about 0 to about 6 molar percent of one or more PEGylated phospholipids. In some embodiments, lipid complexes comprise about 0.5 to about 5 molar percent of one or more PEGylated phospholipids. In some embodiments, lipid complexes comprise about 1 to about 5 molar percent of one or more PEGylated phospholipids. In some embodiments, lipid complexes comprise about 3 molar percent of one or more PEGylated phospholipids. In some embodiments, lipid complexes comprise about 4 molar percent of one or more PEGylated phospholipids. In some embodiments, lipid complexes comprise about 5 molar percent of one or more PEGylated phospholipids. Methods of Use of the Compounds and Compositions

[0595] Certain compounds described herein are STING agonists and are therefore useful for stimulating an immune response in a subject. The compositions can be used to treat viruses.

[0596] The compounds of the present disclosure exhibit STING modulatory / agonist activity. Certain compounds of the present disclosure may be excellent 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 toxicity), and / or stability (e.g., chemical stability, enzymatic stability), and may be useful as drugs.

[0597] The compounds of the present disclosure can be used to increase STING activity in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans).

[0598] The compounds of the present disclosure are useful in treating diseases that can be affected by STING (sometimes abbreviated herein as "STING-associated diseases"), such as cancer, for example, colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), 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 tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestinal cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low malignant potential tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver carcinoma, extrahepatic bile duct carcinoma), 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 carcinoma, uterine sarcoma), gestational choriocarcinoma, brain tumor (e.g., medulloblastoma, glioma, pineal astrocytoma, hairy cell The compounds can be used as drugs for preventing or treating cancers of unknown primary origin (e.g., basal cell carcinoma, malignant melanoma), sarcomas (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumors, bladder cancer, blood cancers (e.g., multiple myeloma, leukemia (e.g., acute myeloid leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), and other cancers; cancer growth inhibitors; cancer metastasis inhibitors; apoptosis promoters; and drugs for treating 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 compound of the present disclosure or the combination of the present disclosure can be used simultaneously with non-drug therapies. More precisely, the compound of the present disclosure or the combination of the present disclosure can be combined with non-drug therapies, such as (1) surgery, (2) hypertension chemotherapy using angiotensin II or the like, (3) gene therapy, (4) hyperthermia, (5) cryotherapy, (6) laser ablation, and (7) radiation therapy.

[0601] For example, by using the compounds of the present disclosure before or after the above-mentioned surgery or before or after a combination of two or three of these treatments, effects such as preventing the development of resistance, extending disease-free survival, suppressing cancer metastasis or recurrence, and prolonging life may be obtained.

[0602] In addition, treatment with the compound of the present disclosure or the combination drug of the present disclosure can be combined with supportive care: (i) administration of antibiotics (e.g., β-lactams such as pansporin, macrolides such as clarithromycin) for various concurrent infections, (ii) administration of high-calorie infusions, amino acid preparations, or multivitamins to improve malnutrition, (iii) administration of morphine to relieve pain, (iv) administration of pharmaceutical agents to improve side effects such as nausea, vomiting, loss of appetite, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, liver damage, kidney damage, DIC, fever, etc., and (v) administration of pharmaceutical agents to suppress multidrug resistance in cancer, etc. [Example]

[0603] General Synthetic Methods and Intermediates The compounds of the present disclosure can be prepared by one of ordinary skill in the art in light of this disclosure and knowledge in the art, and / or by reference to the schemes and synthetic examples set forth below. Exemplary synthetic routes are described in the schemes and examples below. It should be understood that variables, e.g., "R" groups, appearing in the schemes and examples below should be read independently of their appearance elsewhere in this application. One of ordinary skill in the art will readily understand how the schemes and examples set forth below illustrate the preparation of the compounds described herein.

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

[0605] [ka]

[0606] Scheme 1 shows a general route for activating carboxylic acids i to the corresponding acid chlorides or (isobutylcarbonate) propionic anhydrides such as ii, 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 base. When E is Cl, formation of ii is achieved by treating i with oxalyl chloride or Ghosez's reagent (α-chloroenamine, a reactive intermediate in the synthesis: 1-chloro-N,N,2-trimethylpropenylamine; see Haveaux B., et al. Org. Synth. 1980, 59, 26.).

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

[0608] [ka]

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

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

[0611] [ka]

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

[0613] Scheme 4: 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 such as ii to the amino group of a drug moiety such as iii-b, following the general method described in Scheme 3.

[0616] Scheme 5: General route for generating spacer peptide acid chlorides

[0617] [ka]

[0618] Scheme 5 shows a general method for preparing acid chlorides viii. Deprotection of Boc-amine i with an acid such as TFA or HCl, followed by treatment with activated carbonate v, affords 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 general route for attaching a spacer to the amino group of a drug moiety

[0620] [ka]

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

[0622] Scheme 7: Alternative general route for attaching a spacer to the amino group of a drug moiety

[0623] [ka]

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

[0625] Scheme 8: Alternative general route for attaching a spacer to the amino group of a 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 following the general method described in Scheme 6.

[0628] Scheme 9: General route for preparing linker-payloads

[0629] [ka]

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

[0631] Scheme 10: General route for preparing linker-payloads

[0632] [ka]

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

[0634] Scheme 11: General route for preparing linker-payloads

[0635] [ka]

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

[0637] Scheme 12: General route for preparing linker-payloads

[0638] [ka]

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

[0640] Scheme 13: General route for preparing linker-payloads

[0641] [ka]

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

[0643] Scheme 14: General route for preparing linker-payloads

[0644] [ka]

[0645] Scheme 14 shows a general method for preparing glucuronide-containing linker payloads xvi. Deprotection of the amine of iv with an acid such as TFA or HCl, followed by reaction with an activated carbonate xiii, affords xiv. Global acetic acid hydrolysis and removal of the FMOC group of xiv with a base such as lithium hydroxide affords xv. Treatment of xv with activated binding handle x affords glucuronide-containing linker payloads xvi.

[0646] Scheme 15: General route for preparing linker-payloads

[0647] [ka]

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

[0649] Scheme 16: General route for preparing linker-payloads

[0650] [ka]

[0651] Scheme 16 shows a general method for preparing terminal amine-containing linker payloads xviii, where Ye is a linear alkyl chain or a polyether-containing alkyl chain. Deprotection of the amine of vi with an acid such as TFA or HCl is followed by reaction with an activated linking handle xvii. Removal of the terminal protecting group with an acid such as TFA affords terminal amine-containing linker payloads xviii.

[0652] Scheme 17: General route for preparing linker-payloads

[0653] [ka]

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

[0655] Scheme 18: General route for preparing linker-payloads

[0656] [ka]

[0657] Scheme 18 shows a general method for preparing linker payloads such as xxii incorporating a dipeptide spacer, where R 7 is hydrogen, alkyl, or aromatic moiety. Compound iii is globally protected by treatment with trimethylsilyl chloride and pyridine, followed by treatment with Boc-proline succinimide ester. Removal of the silyl group with a fluoride source such as NEt3-3HF affords xix. Deprotection of the amine of xix with an acid such as TFA or HCl, followed by reaction with a succinimide ester-activated amino acid xx, affords xxi. Deprotection of the amine of xxi with an acid such as TFA or HCl, followed by introduction of the maleimide handle, affords the linker payload x. xii is obtained.

[0658] Scheme 19: General route for preparing linker-payloads

[0659] [ka]

[0660] Scheme 19 shows a general method for preparing linker payloads such as xxii-a following the general method described in Scheme 18.

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

[0662] Ac: Acetate

[0663] ACN: acetonitrile

[0664] ATM:Atmosphere

[0665] aq: aqueous solution

[0666] BCN: Bicyclo[6.1.0]nonyne

[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: Dibenzocyclooctyne

[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%: Extinction coefficient of 0.1% solution

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

[0689] EDTA: Ethylenediaminetetraacetic acid

[0690] Et: Ethyl

[0691] EtOH: ethanol

[0692] EtOAc: ethyl acetate

[0693] FA: LCMS method using formic acid

[0694] Fmoc: fluorenylmethyloxycarbonyl

[0695] h: time

[0696] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 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 per 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 H NMR spectra were obtained on a 400 MHz Bruker spectrometer unless otherwise stated. Obtained at. 31 PN MR spectra were obtained on a 400MHz Bruker spectrometer. Recorded in mHz, unless otherwise stated 1 Obtained with H decoupling. 19 F NMR Spectra were recorded at 376 mHz on a 400 MHz Bruker spectrometer unless otherwise stated.

[0734] HRMS criteria: HRMS spectra were acquired using an Agilent 1260 Infinity II Bio-inert Multisampler, a bio-inert column compartment, a bio-inert quaternary pump, a DAD multi-wavelength detector, and an OsakaSoda CapcelLPAK. Acquisition was performed using an Agilent 6545 LC-QTOF mass spectrometer equipped with a C1 UG120 (5 μm, 2.0 mm i.d. × 35 mm length) reversed-phase column. The solvent system for LC consisted of 0.1% formic acid in water for mobile phase A and 0.1% formic acid in acetonitrile for mobile phase B, or an equivalent solvent system. LC elution typically began with a mobile phase A / B ratio of 95% / 5%. After an isocratic run, typically 0.5 min later, the mobile phase The gradient was changed to 100% B over a 1.5 minute linear gradient at a flow rate of 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 were: gas temperature 35°C, drying 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 4500 V, nozzle voltage 2000 V, fragmentor voltage 75 V.

[0736] Data were analyzed using Agilent Mass Hunter Qualitative Analysis software version 8.07.00SP2. Mass error [ppm] was 1 × 10 6 Expressed as the difference between the accurate mass divided by the observed mass multiplied by .

[0737] LCMS conditions: LCMS 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 optimize compound characterization. The mobile phase was based on an ACN / water or MeOH / water gradient containing either 0.1% formic acid (method indicated as FA) or 10 mM ammonium acetate (method 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) at a flow rate of 1 mL / min over a 16.5-minute run.

[0738] In some cases, LCMS spectra were recorded using a reverse-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 optimize compound characterization. The mobile phase was based on an ACN / water or MeOH / water gradient containing either 0.1% formic acid (method indicated as FA) or 10 mM ammonium acetate (method indicated as 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) at a flow rate of 0.5 mL / min over a 5-minute run.

[0739] Preparative HPLC: Preparative HPLC separation was performed using an 18 x 150 mm Sunfire C-18 column eluted with a water-ACN gradient using a Gilson instrument operated with a 322 pump, with fraction collection set between 200 nm and 400 nm, driven by a UV / visible detector. Mass-gated fraction collection was performed on an Agilent 1100 LC / MSD instrument.

[0740] Those skilled in the art will recognize that variations in gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemical species being analyzed.

[0741] Preparative SFC: Preparative SFC was performed on 10, 20, or 30 mm x 250 mm Chira LPak columns (usually eluted with the appropriate proportions of supercritical carbon dioxide and alcohol containing 0.3% diethylamine, 0.3% TEA, 0.3% formic acid, or no acid or base additives). SFC was typically performed using 10 or 20 mm x 250 mm Phenomenex Lux Cellulose-4 or 2-ethylpyridine columns (typically IA, IB, IC, ID, IE, and IF). Isocratic conditions with flow rates ranging from 10 to 100 mL / min and a column temperature of 40 °C were typical. Preparative SFC was performed on a Jasco SFC purification system with UV / visible-driven fractionation set at 200 nm to 400 nm and backpressure regulation set at 10 MPa.

[0742] Those skilled in the art will recognize that variations in gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemical species being analyzed.

[0743] Analytical SEC Terms: SEC spectra were recorded at 280 nm on 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 / N 8541; 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 run was isocratic at a flow rate of 1 mL / min for 20 min.

[0744] Analytical HIC conditions: HIC spectra were recorded at 280 nm on a Hewlett Packard HP1100 or Agilent 1100 series LC system equipped with a diode array detector using an HIC column (typically a 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, a 20-minute run was used, although a 12-minute linear gradient from 95% / 5% A / B to 100% B was used between the first and last legs of the isocratic run.

[0745] LC-QTOF conditions: LCMS spectra were recorded on an Agilent 1260 Bioinert series LC system connected to an Agilent 6545QTOF mass spectrometer using a reversed-phase column (typically Agilent, PLRP-S, 5 μm, 1000 Å, 2.1 mm × 50 mm) heated to 80 °C. Various gradients and run times were selected to best characterize the compounds. The mobile phase was based on an ACN / water gradient containing 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 either intact or reduced (20 μL of 1–5 mg / mL ADC solution treated with 4 μL of 0.5 M DTT solution for 30 min at 37°C). Raw data were deconvoluted within the appropriate mass range using Agilent BioConfirm software to obtain the molecular weight(s) of the protein, and DARs were 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 and SIL-30ACMP autosampler system and an AB SCIEX Triple Quad 4500ESI mass spectrometer.

[0749] Typically, a 5 μL sample aliquot was injected into the LC / MS / MS after passing through a Waters Xselect C18 CSH 3.5 μL, 2.1 mm ID x 30 mm column. Mobile phase A contained 0.1% formic acid in water, and mobile phase B was 5% 0.1% formic acid in water 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 1.5 minute flow rate. First, the instrument was run with 100% aqueous mobile phase solvent for 0.5 minutes, followed by 100% organic solvent for the next 1.5 minutes.

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

[0751] ADC concentration: ADC concentrations were calculated from UV absorbance at 280 nm measured by NanoDrop (2000c; Fisher Scientific) coefficients after subtracting the UV absorbance from the corresponding linker-payload construct.

[0752] Example 1 tert-Butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (Intermediate 1)

[0753] [ka]

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

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

[0756] Example 2 tert-Butyl N-[[2-(2-chloro-2-oxo-ethyl)phenyl]methyl]-N-methyl-carbamate (Intermediate 2)

[0757] [ka]

[0758] Step 1: 2-[2-[[tert-butoxycarbonyl(`methyl)amino]methyl]phenyl]acetic acid To a solution of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid (1.50 g, 5.7 mmol) in THF (20 mL) was added iodomethane (2.8 mL, 45.2 mmol) at room temperature. The reaction mixture was cooled to 0°C and added with sodium hydride (mineral A solution of 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (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 mixture was quenched with water and the organic solvent was removed by concentration. The aqueous phase was acidified to pH 3 using 1 N HCl, and the crude product was extracted with EtO (2x). The combined organic phases were washed with brine, dried (NaSO), filtered, and evaporated to give 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: tert-butyl N-[[2-(2-chloro-2-oxo-ethyl)phenyl]methyl]-N-methyl-carbamate, intermediate 2 The title compound was prepared according to the procedure described in Example 1, Step 2, starting with 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (650 mg, 2.33 mmol) to give crude intermediate 2 (693 mg, 100%).

[0760] Example 3 4-[tert-Butoxycarbonyl(methyl)amino]butanoic acid isobutoxycarbonyl (Intermediate 3)

[0761] [ka]

[0762] Step 1: 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid The title compound was prepared according to the procedure described in Example 2, Step 1 using 4-(tert-butoxycarbonylamino)butanoic acid (2.92 g, 14.4 mmol) instead of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid to give 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]butanoic acid isobutoxycarbonyl (Intermediate 3) To a solution of 4-((tert-butyl-butoxycarbonyl)(methyl)amino)butanoic acid (360 mg, 1.57 mmol) and triethylamine (0.24 mL, 1.73 mmol) dissolved in DCM (7 mL) and cooled to 0 °C was added isobutyl chloroformate (0.22 mL, 1.73 mmol) dropwise. Upon complete addition, the reaction mixture was stirred at room temperature for 2 h. The mixture was then concentrated to dryness to give crude isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (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 in place 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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- tert-Butyl)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 2-Amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (compound I-5c, see PCT / IB2018 / 058846, 160 mg, 0.19 mmol) as the N,N-diethylethanamine salt was dissolved in anhydrous pyridine, concentrated to dryness (3 × 2 mL), and then placed under vacuum for 15 min. 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 at room temperature under an argon atmosphere 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 stirred for 30 minutes. The reaction mixture was concentrated to dryness, and the residue was dissolved in MeOH (15 mL). Triethylamine trihydrofluoride (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, and the crude residue was adsorbed onto Celite and purified by reverse-phase flash column chromatography (0–50% ACN / 10 mM triethylammonium acetate in water) to give tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (120 mg, 58%) as the N,N-diethylethanamine 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-propan-1 as the N,N-diethylethanamine salt tert-Butyl [(2-(2-methyl-2-yl)carbamoyl)benzyl]methylcarbamate (50 mg, 0.045 mmol) 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 for 30 min at 0 °C. The reaction mixture was then concentrated to dryness and placed under vacuum for 2 h to give Intermediate 6 (41 mg, 100%) as the 2,2,2-trifluoroacetic acid salt. LCMS (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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-7-yl 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]methylcarbamate, Compound 1 (C-1)

[0771] [ka]

[0772] Step 1: 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, 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) at room temperature was added a solution of Intermediate 6 (45 mg, 0.05 mmol) in DMF (1.5 mL). 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 on Celite was purified by reverse-phase flash column chromatography (0–40% ACN / aqueous triethylammonium acetate (10 mM)) to give the N,N-diethylethanamine salt of [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-diyloxy] ... 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl 4-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 7, 10 mg, 16%). 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 (162MH z,CD3OD)δ55.01(s,1P),53.03(s,1P).

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

[0774] The title compound was prepared according to the procedure described in Example 5, Step 2 using Intermediate 7 (10 mg, 0.008 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate and stirred at room temperature for 10 minutes to afford the 2,2,2- 4-{[(2NS)-2-{[(2NS)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (Intermediate 8, 9 mg, 100%) was obtained as the trifluoroacetate salt. 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl, compound C-1

[0775] To a solution of intermediate 8 (8 mg, 0.007 mmol) and N-succinimidyl 6-maleimidohexanoate (3.1 mg, 0.010 mmol) in THF (0.20 mL) and DMF (0.10 mL) was added N,N-diisopropylethylamine (2.5 μL, 0.014 mmol) dropwise. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0–100% ACN / aqueous ammonium acetate (10 mM)) to give the ammonium salt [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1, 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]methylcarbamate (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 Calculated value for P2S2: 1309.3607, measured value: 1309.3639.

[0776] Example 7 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonochloride (Intermediate 9)

[0777] [ka]

[0778] Step 1: 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonochloride, Intermediate 9 To a solution of tert-butyl N-(2-hydroxyethyl)-N-methylcarbamate (296 mg, 1.69 mmol) in THF (4.2 mL) and N,N-diisopropylethylamine (1 mL, 5.9 mmol) cooled to 0° C., 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 solid was washed with THF, and the filtrate was concentrated to dryness to give crude 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonochloride (Intermediate 9, 402 mg, 100%).

[0779] Example 8 N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate [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]benzoic acid 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-methyl-carbamate [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]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) in THF (1.5 mL) cooled to 0 °C was added oxalyl chloride (2.0 M solution in DCM, 0.25 mL, 0.50 mmol), followed by 3 drops of DMF. The reaction mixture was stirred at 0 °C for 45 min. The mixture was then concentrated to dryness to give crude intermediate 10 (304 mg, 100%).

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

[0784] [ka]

[0785] Step 1: methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate The title compound was prepared according to the procedure described in Example 6, Step 1, starting with methyl 4-(methylamino)butanoate HCl (1.44 g, 8.16 mmol) instead of intermediate 6. Following purification by silica gel chromatography (0-70% EtOAc / 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 To a solution of methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 6.27 mmol) in THF (40.7 mL) and water (20.3 mL) at 0° C. was added lithium hydroxide monohydrate (657 mg, 15.7 mmol). The homogeneous mixture was then warmed to room temperature and stirred for 2 h. 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 additional 1 N HCl. The solution was warmed to room temperature and diluted with EtOAc and water. The aqueous phase was further extracted with EtOAc. The combined organic phases were washed with brine and dried over magnesium sulfate. After filtration and removal of the solvent, the residue was purified by silica gel chromatography (0-10% MeOH / EtOAc). Removal of the solvent afforded 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%) 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-butoxycarbonyl The title compound was prepared according to the procedure described in Example 8, Step 2, 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-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid to give crude intermediate 34 (400 mg, 100%) upon concentration of the reaction mixture.

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

[0789] [ka]

[0790] [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl Intermediate 7 The title compound was prepared according to the procedure described in Example 5, Step 1, using Intermediate 10 instead of tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (Intermediate 1). The crude product was purified by reverse-phase flash column chromatography (0–50% ACN / ammonium bicarbonate (5 mM) in water) to afford Intermediate 7 as the ammonium salt. This material was then dissolved in MeOH (2 mL) and triethylamine (5 mL) was added. The mixture was shaken for 1 minute, the solvent removed, and lyophilized overnight to afford Intermediate 7 as the N,N-diethylethanamine salt of [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9, 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl]methylcarbamate (147 mg, 48%). LCMS (AA): m / z = 1216.3 (M+H). 1 H NMR Example 6, step Same as 1.

[0791] Example 10 [2-(Chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl (Intermediate 11)

[0792] [ka]

[0793] Step 1: 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid To a solution of 2-carboxybenzaldehyde (1.10 g, 7.3 mmol) in MeOH (15 mL) was added 2,5,8,11-tetraoxatridecan-13-amine (1.82 g, 8.8 mmol) and the reaction mixture was stirred at room temperature for 1 hour. Sodium (139 mg, 3.7 mmol) was added slowly in portions. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched 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 give 2-(2,5,8,11-tetraoxa-14-azapentadecan-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-azapentadecan-15-yl)benzoic acid The title compound was prepared according to the procedure described in Example 6, Step 1, using DCM as the solvent, starting with 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (764 mg, 2.24 mmol) instead of intermediate 6, and stirring for 16 hours. Purification by silica gel chromatography (0-25% MeOH / DCM) followed by 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (855 mg, 47%) was obtained. LCMS (AA): m / z=759.4 (MH).

[0795] Step 3: [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl, Intermediate 11 The title compound was prepared according to the procedure described in Example 8, Step 2, starting with 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)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) instead of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid to give crude intermediate 11 (605 mg, 100%) upon 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 material indicated in the table instead of tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate (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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide (Intermediate 17)

[0800] [ka]

[0801] Step 1: tert-butyl [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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate The title compounds were prepared as 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one and 7-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6- Prepared according to the procedure described in Example 5, step 1 using amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (compound number 14, see WO2018100558A2, 200 mg, 0.265 mmol) and intermediate 3 (503 mg, 1.59 mmol). Purification by reverse-phase flash column chromatography (0–50% ACN / 5 mM ammonium bicarbonate in water) afforded tert-butyl [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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate (148 mg, 59%) as the ammonium salt. 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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide, intermediate 17 The title compound is prepared as the ammonium salt of [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate instead of tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R, Prepared using tert-butyl 15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate (42 mg, 0.045 mmol) according to the procedure described in Example 5, step 2, 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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide (Intermediate 17, 41 mg, 100%) was obtained as the 2,2,2-trifluoroacetate salt. 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 material shown in Table 4 in place of isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (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 material shown in Table 6 in place 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 material indicated 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 in place of Intermediate 7.

[0812] [Table 9]

[0813] [Table 10]

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

[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 in place of intermediate 7 and N-succinimidyl 6-maleimidohexanoate, respectively.

[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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo- 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]-2,2-dimethyl-4-oxobutyl]methylcarbamate, 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl]-2,2-dimethyl-4-oxobutyl]methylcarbamate, Compound 20 A solution of (4-nitrophenyl)carbonate [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) in DMF (0.60 mL) and N,N-diisopropylethylamine (26 μL, 0.15 mmol) was added to the 2,2,2-trifluoroacetate salt of N-{9-[(2R,5 A solution of (R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 onto Celite was purified by reverse-phase flash column chromatography (0–100% ACN / aqueous ammonium acetate (10 mM)) to obtain the ammonium salt [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][ 1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl (C-20, 4 mg, 17%). LCMS (AA): m / z = 1287.4 (M−H).

[0836] Example 20 The compounds listed in Table 16 were prepared by substituting starting materials A and B shown in Table 15 for intermediate 14 and [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl)carbonate, respectively. 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-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecan-17-amide (Intermediate 20)

[0841] [ka]

[0842] Step 1: (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic 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) was added (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid hydrochloride (3.4 g, 12.9 mmol) dissolved in DMF (40 mL), followed by 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 give (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic acid (1.26 g, 24%). LCMS (AA): m / z = 489.3 (M+H).

[0843] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecan-17-amide, intermediate 20 To a solution of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic acid (65 mg, 0.133 mmol) and N,N'-dicyclohexylcarbodiimide (30 mg, 0.145 mmol) in 1,4-dioxane (0.68 mL) was added N-hydroxysuccinimide (15.3 mg, 0.133 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. Celite was added, and the mixture was filtered, rinsed with 1,4-dioxane, and concentrated to dryness. The crude residue absorbed on Celite was purified by silica gel chromatography (0-10% MeOH / DCM) to give 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 material indicated in the table in place 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-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotet

[0033] -6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, compound 24 (C-24)

[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-(pyrimidin-4-yl)amino] 2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate methyl ester The title compound was prepared according to the procedure described in Example 6, Step 1, starting with Intermediate 6 (104 mg, 0.094 mmol) and methyl (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 (150 mg, 0.16 mmol) using N,N-diisopropylethylamine as the base (for synthesis see Jeffrey, S.C. et al. Bioconjugate Chem. 2006, 17, 831-840), 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-(pyrimidin-4-yloxy)-2,10-disulfa Nildecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate (71 mg, 48%). 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8- To a solution of methyl methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate (66 mg, 0.042 mmol) was added LiOH (0.5 M aqueous solution, 1.25 mL, 0.625 mmol) at room temperature, and the reaction mixture was stirred for 2.5 h. 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 afford intermediate 22 (26 mg, 50%) as the ammonium salt. This material was then 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 lyophilized overnight to give (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-(pyrimidin-4-yloxy)-2,10-disulfanyldecanoic acid as the N,N-diethylethanamine salt. Hydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (intermediate 22, 30 mg, 100%) was 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-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14 -(Pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 according to the procedure described in Example 6, Step 3 using Intermediate 22 (24 mg, 0.02 mmol) instead of Intermediate 8 to give (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10 14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 Each of the compounds listed in Table 18 (Compound 25) was prepared as described in Example 23, Step 3, using the starting material indicated in the table in place 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate, Compound 26 (C-26)

[0855] [ka]

[0856] Step 1: 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]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) was added (4-nitrophenyl)carbonate [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (220 mg, 0.34 mmol) 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 the addition of formic acid. The mixture was concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0–100% ACN / formic acid (0.1%) in water) to give 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]- 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl 6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate, Compound 26 To a solution of 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-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) was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46 mg, 0.12 mmol) at room temperature, and the reaction mixture was stirred for 10 minutes. To this solution was added dropwise a solution of (2R)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide (Intermediate 15, 50 mg, 0.047 mmol) as the 2,2,2-trifluoroacetate salt in DMF (0.60 mL). The reaction mixture was stirred at room temperature for 30 min. The mixture was then cooled to 0 °C, water (1 mL) was added, and the solution was acidified to pH 4 by the addition of formic acid. The reaction mixture was concentrated to dryness, and the crude residue was purified by reverse-phase flash column chromatography (0–100% ACN / aqueous ammonium acetate (10 mM)) to give [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9]-methyl ... ,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl(C-2 6, 32 mg, 50%) was 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-pyrrol-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-Dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide, Compound 27 (C-27)

[0859] [ka]

[0860] Step 1: tert-butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate To a mixture of Intermediate 15 (60 mg, 0.05 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate (19 mg, 0.07 mmol) in DMF (1.6 mL) was added triethylamine (50 μL, 0.355 mmol), and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water, Celite was added, and the reaction mixture was concentrated to dryness. The crude residue absorbed on Celite was purified by reverse-phase column chromatography (0-100% ACN / 5 mM aqueous ammonium bicarbonate). Triethylamine (14 μL, 0.1 mmol) was added to the aqueous solution of the product. Lyophilization overnight gave tert-butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (49 mg, 87%) as the N,N-diethylethanamine salt. 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}pyrrolidine-2-carboxamide {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxide as the N,N-diethylethanamine salt To a solution of tert-butyl [diphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (37 mg, 0.033 mmol) in MeOH (1 mL) was added hydrochloric acid (4 M solution in dioxane, 1 mL, 4 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated to dryness and placed under vacuum for 2 hours to give (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-(9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydro Oxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-ethanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-7-yloxy] as the hydrochloride salt Example 26 starting with (2S)-2-[[(2NS)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoic acid (2,5-dioxopyrrolidin-1-yl) (2S)-2-[[(2NS)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methyl-butanoyl]amino]propanoic acid (2,5-dioxopyrrolidin-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 / aqueous formic acid (0.1%)) afforded (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10 ]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 tert-butyl ester The title compound was prepared according to the procedure described in Example 6, Step 3, starting with Intermediate 8 (23 mg, 0.016 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate (11 mg, 0.039 mmol) instead of N-succinimidyl 6-maleimidohexanoate. The reaction mixture was stirred at room temperature for 30 minutes. Purification by reverse-phase flash column chromatography (0-100% ACN / aqueous triethylammonium acetate (10 mM)) afforded (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidinyl)-2-methyl-1H-pyrimidinyl)-2 ...-2H-pyrimid 4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3 tert-Butyl (1-methyl-1-oxobutan-2-yl)amino}-2-oxoethyl)carbamate (13 mg, 57%) was obtained. LCMS (AA): m / z = 1273.1 (M+H).

[0866] Step 2: 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, compound 28 [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-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 according to the procedure described in Example 5, Step 2, starting with tert-butyl 2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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 (6.6 mg, 0.005 mmol). The reaction mixture was stirred at room temperature for 5 minutes to give 4-{[(2S)-2-({(2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (C-28, 5.6 mg, 91%) as the 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 material shown in Table 19 in place of 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate.

[0867] [Table 19]

[0868] [Table 20]

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

[0870] [ka]

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

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

[0873] [ka]

[0874] Step 1: 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid The title compound was prepared according to the procedure described in Example 10, Step 1, starting with 2-carboxybenzaldehyde (6.0 g, 40 mmol) and 2,4-dimethoxybenzylamine (12 mL, 80 mmol) to give 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 To a solution of 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (380 mg, 1.26 mmol) and triethylamine (0.5 mL, 3.55 mmol) in DMF (6 mL) was added 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)-L-alaninate (640 mg, 2.2 mmol). Water (2 mL) was added to help solubilize the reaction mixture, which 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%) in ACN / formic acid (0.1%) to give 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: tert-Butyl N-[(1S)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]-1-methyl-2-oxo-ethyl]carbamate (Intermediate 24)

[0876] The title compound was prepared according to the procedure described in Example 1, Step 2 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 to give crude intermediate 24 (280 mg, 100%).

[0877] Example 31 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide, compound 31 (C-31)

[0878] [ka]

[0879] Step 1: tert-butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](amino)-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate The title compound was prepared according to the procedure described in Example 26, Step 1, using intermediate 16 (45 mg, 0.53 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)-L-valine acid (100 mg, 0.32 mmol) instead of intermediate 15 and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate, respectively. Purification by reverse-phase flash column chromatography (0-100% ACN / ammonium acetate (10 mM) in water) afforded [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanilide] as the ammonium salt. tert-Butyl rudecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate (24 mg, 44%). 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide tert-Butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate ammonium salt of [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purine- The title compound was prepared according to the procedure described in Example 5, Step 2, starting with tert-butyl 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide (24 mg, 97%). LCMS (AA): m / z = 953.1 (M+H).

[0881] Step 3: 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide, compound 31 The title compound was obtained according to the procedure described in Example 6, Step 3, 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide (24 mg, 0.022 mmol) as the 2,2,2-trifluoroacetic acid salt instead of intermediate 8. and prepared as the ammonium salt 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy 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-Nitrophenylcarbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl (Intermediate 25)

[0883] [ka]

[0884] Step 1: 6-(11,12-didehydrodibenzo[b,f]azocin-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 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) in DMF (2 mL) and THF (5.5 mL) was added N,N-diisopropylethylamine (0.39 mL, 2.24 mmol). The reaction mixture was cooled to 0 °C, and 1-{[6-(11,12-didehydrodibenzo[b,f]azocin-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 EtOAc (3x). The combined organic phase was washed with saturated aqueous sodium bicarbonate and brine, dried (NaSO), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (50-100% EtOAc / DCM) to give 6-(11,12-didehydrodibenzo[b,f]azocin-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-Nitrophenylcarbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl, Intermediate 25 A solution of 6-(11,12-didehydrodibenzo[b,f]azocin-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) in THF (4.2 mL) and N,N-diisopropylethylamine (0.37 mL, 2.1 mmol) was cooled to 0 °C. To a solution of 4-nitrophenyl chloroformate (362 mg, 1.69 mmol) was added in one portion. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was diluted with water and extracted with DCM (3×). The combined organic phases were washed with saturated aqueous sodium bicarbonate and brine, dried (NaSO), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0–100% EtOAc / DCM) to give 4-nitrophenylcarbonate 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl (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 material indicated in the table instead of 1-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione.

[0887] [Table 21]

[0888] Example 34 (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-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]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Intermediate 27A)

[0889] [ka]

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

[0891] Step 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,2 0-Heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester (25S)-tert-butyl 25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (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 give tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (2.56 g, 85%).

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

[0893] Step 4: (25S)-25-{[4-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methyl The title compound was prepared according to the procedure described in Step 2 of Example 5, starting with tert-butyl (25S)-25-{[4-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (278 mg, 0.35 mmol) instead of tert-butyl ethylcarbamate. Purification by silica gel chromatography (0-20% MeOH / DCM) gave (25S)-25-{[4-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Intermediate 27A, 180 mg, 70%). LCMS (AA): m / z = 742.4 (M+H).

[0894] Step 5: (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide, intermediate 27 The title compound was prepared according to the procedure described in Example 29, starting with (25S)-25-{[4-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (60 mg, 0.08 mmol) instead of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-oic acid. The reaction mixture was stirred at room temperature for 30 minutes to give (2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-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 in place of N-succinimidyl 6-maleimidohexanoate.

[0896] [Table 22]

[0897] [Table 23]

[0898] Example 36 tert-Butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}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-azatriacontan-30-oic acid The title compound was prepared according to the procedure described in Example 21, Step 1, starting with (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (238 mg, 1.17 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)oxy]pyrrolidine-2,5-dione (300 mg, 0.58 mmol) instead of (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid hydrochloride and 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione, using DMF as solvent and stirring at room temperature for 2 hours. Purification by reverse-phase flash column chromatography (0–100% ACN / 10 mM triethylammonium acetate in water) gave (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-oic acid (344 mg, 99%). LCMS (AA): m / z = 599.4 (M+H).

[0901] Step 2: tert-Butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}carbamate, Intermediate 30 The title compound was prepared according to the procedure described in Example 21, step 2, starting with (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-30-oic acid (100 mg, 0.166 mmol) instead of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic acid, using DCM as the solvent and stirring at room temperature for 1 hour. The reaction mixture was filtered and concentrated to give crude tert-butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}carbamate (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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamic acid [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonyl) (3-methyl-butanoylamino)-3-methyl-butanoylamino)propanoylamino)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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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) (for synthesis see Prakash, TPJ Med. Chem. 2005, 48, 1199-1210) was dissolved in anhydrous pyridine, concentrated to dryness (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 dryness. MeOH (100 mL) and ammonium hydroxide (28-30% aqueous solution, 50 mL) were added, and the resulting mixture was stirred for 60 min. The reaction mixture was concentrated to dryness, and the crude residue was adsorbed onto Celite and purified by silica gel chromatography (0-100% EtOAc / hexanes) to give N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-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, 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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]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 trihydrofluoride (1.19 mL, 7.33 mmol), and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was slowly quenched by the addition of saturated aqueous sodium bicarbonate and then diluted with EtOAc. The organic phase was separated, washed with brine, dried (NaSO), filtered, and evaporated to dryness. The crude residue was purified by silica gel chromatography (0–20% MeOH / DCM) to give N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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% NEt) to give N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl 4-{[(2S)-2-({(2S)-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-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (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-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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] [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-N-methyl-carbamate (Intermediate 32, 1.1 g, 0.97 mmol) and N-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro- A mixture of 4-nitrophenyl 5-purin-9-yl-tetrahydrofuran-2-yl]methyl]sulfamate (667 mg, 1.17 mmol) (for synthesis, see PCT Application No. PCT / IB2019 / 052364), 4-dimethylaminopyridine (122 mg, 0.97 mmol), and 4 Å molecular sieves (1.74 g) in anhydrous DCM (13.6 mL) was stirred under argon at room temperature for 10 minutes. Triethylamine (0.60 mL, 4.3 mmol) was then 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 subjected to purification by reverse-phase column chromatography (10-100% ACN / aqueous ammonium bicarbonate (5 mM)) to give 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 [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-N-methyl-carbamate (0.94 g, 62%). 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-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [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-[[(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]carbohydrate was placed in a polypropylene test tube. To a solution of [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl]-N-methyl-carbamate (939 g, 0.60 mmol) in THF (40 mL) was added triethylamine trihydrofluoride (0.52 mL, 3.17 mmol) and triethylamine (0.98 mL, 6.95 mmol). The reaction solution was stirred overnight at room temperature, then diluted with dichloromethane and washed successively with saturated aqueous 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 / aqueous ammonium bicarbonate (5 mM)) to give N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl ( Yield: 795 g, 89%). 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-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid Diphenyl phosphite (0.225 mL, 1.17 mmol) was added to a 0° C. 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-purin-9-yl-tetrahydrofuran-2-yl]methyl]sulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (792 g, 0.55 mmol) in pyridine (2 mL). Upon completion of the addition, the reaction mixture was stirred at room temperature for 1 hour. Triethylamine (0.62 mL, 4.37 mmol) and water (0.51 mL, 28.3 mmol) were then added, and the reaction mixture was stirred at room temperature for an additional 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 mixture was quenched by the addition of triethylsilane (4.3 mL, 26.2 mmol) and stirred at room temperature for 1 hour. The reaction mixture was then concentrated to dryness, and the residue was purified by reverse-phase column chromatography (10-100% ACN / aqueous ammonium bicarbonate (5 mM)) to give [(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-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid (396 mg, 59%) as the 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-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2NS)-2-({(2NS)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl or 4-{[(2S)-2-({(2S)-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-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate 33

[0913] Diphenyl phosphorochloride (0.055 mL, 0.26 mmol) was added to pyridine (1.6 mL) and the solution was cooled to -35°C. A solution of [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid (80 mg, 0.065 mmol) as the ammonium salt in DCM (2.5 mL) and pyridine (3.2 mL) was added dropwise over 15 minutes to the cooled reaction mixture. The resulting solution was stirred at −35° C. for 30 minutes. The solid 3H-1,2-benzodithiol-3-one (25 mg, 0.15 mmol) and water (0.056 mL) were added to the cooled reaction mixture, which was then stirred at room temperature for 1 hour. The reaction mixture was then cooled to 0°C in an ice bath and quenched by the addition of aqueous 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 / 10 mM aqueous triethylammonium acetate) to give [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxide-14-(9H-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate 4-{[(2S)-2-({(2S)-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-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetrahydro- 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl]methylcarbamate (intermediate 33, 27 mg, 31%) was obtained. LCMS (AA): m / z=1227.9 (M+H). 1 HNM 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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3 ,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-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 (C-34)

[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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl] 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl (2S)-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate (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]azocine- To a suspension of {5(6H-yl)-4-oxobutanoyl}amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Intermediate 27A) (10.4 mg, 0.0140 mmol) was added triethylamine (0.004 mL, 0.0266 mmol) in DCM (0.37 mL). The mixture was stirred at room temperature for 20 minutes, and then DMF (0.15 mL, 2.23 mmol) was added. This mixture was stirred 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 / aqueous triethylammonium acetate (10 mM)) to give the N,N-diethylethanamine salt 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]pyrimidin-7-yl)-16-hydroxybenzoate]. 4-(2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocine-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]amino}benzyl (C-34) (2.0 mg, 6.9%). 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-dioxopyrrolidin-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]non-4-yn-9-ylmethyl ester (Intermediate 36)

[0918] [ka]

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

[0920] Step 2: (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid tert-butyl ester (25S)-tert-butyl 25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (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 give tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (2.56 g, 85%).

[0921] Step 3: (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid To a solution of (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (587 mg, 1.15 mmol) in DCM (11.3 mL) was added TFA (11.3 mL). The mixture was stirred at room temperature for 1 hour. The reaction was concentrated, azeotroped with toluene, and dried under high vacuum to give the crude intermediate as an oil. 1-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-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 EtOAc (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 / 5 mM aqueous ammonium bicarbonate). The desired fractions were concentrated in vacuo to give (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic 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: (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {(25S)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (Intermediate 36) The title compound was prepared according to the procedure described in Example 29, starting with (25S)-25-({[(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid instead of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-oic acid. The reaction mixture was stirred at room temperature for 30 minutes to afford {(25S)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8, (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (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 methyl (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 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-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl] 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-diazaheptapentacontan-1-oyl]amino}benzyl methylcarbamate (C-37)

[0925] [ka]

[0926] Step 1: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-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-diazaheptacontan-57-yl]amino}benzyl]methylcarbamate To a solution of 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) was added in THF (0.6 mL) and DMF (0.2 mL). The reaction mixture was stirred at room temperature for 3 h and then quenched with MeOH (5 mL). After evaporation of most of the solvent, the residue was purified by reverse-phase column chromatography (0–50% ACN / 5 mM aqueous ammonium bicarbonate). The desired fractions were concentrated and lyophilized to give the title compound (17.8 mg, 55%) as a white powder. LCMS (AA): m / z=1915 (M−H).

[0927] Step 2: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15 aS,16R)-16-Hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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-diazaheptapentacontan-1-oyl]amino}benzyl (C-37) In a 50 mL round-bottom flask was placed [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-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]amino}benzyl carbamate (17.7 mg, 0.00923 mmol), THF (1.0 mL), and triphenylphosphine (24.2 mg, 0.0923 mmol) were added. The mixture was stirred at room temperature overnight. 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 / aqueous ammonium bicarbonate (5 mM)). The desired fractions were collected, lyophilized, and obtained as a fluffy white powder: [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo- 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-diazaheptapentacontan-1-oyl]amino}benzyl]methylcarbamate (C-37) (5.7 mg, 32%). 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-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (Intermediate 39)

[0929] [ka]

[0930] Step 1: (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-oic acid To a mixture of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride (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) was added DIEA (1.43 mL, 8.20 mmol), and the mixture was stirred at room temperature for 30 minutes, then cooled to 0° C. and stirred overnight. The volatile solvents were removed and the residue was azeotroped with toluene (3 × 20 mL) and acetonitrile (10 mL) to give (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-oic acid (4.00 g, 100% yield) as a colorless oil. LCMS (AA): m / z = 619.3 (M−H).

[0931] Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (Intermediate 39) A mixture of N-hydroxysuccinimide (469 mg, 4.08 mmol), N,N'-dicyclohexylcarbodiimide (824 mg, 4.00 mmol), and crude (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-oic acid (4.00 g, 4.00 mmol) from step 1 in DCM (76.7 mL, 1200 mmol) was stirred at room temperature for 16 h. The mixture was stirred for 1 hour. The solids that precipitated from the reaction were filtered, and the filtrate was concentrated to give a crude oil. Purification by silica gel chromatography (0-20% IPA / DCM) gave N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (Intermediate 39) (1.70 g, 52%) as a colorless oil. 1 H NMR (400 MHz, 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-pyrrol-1-yl)acetyl]amino}-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosane-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-azanonacosan-29-oic 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 grams, 1.99 mmol) instead of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride. Purification by reverse-phase column chromatography (0-60% ACN / aqueous formic acid (0.1%)) afforded (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-oic acid (1.14 g, 90%) as a colorless oil. LCMS (AA): m / z = 639.4 (M−H).

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

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

[0937] Example 38G 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-(pyrimidin-4-yloxy)-2,10-disulfanilic acid Prepared as described in Steps 2 and 3 of Example 23 using the starting material shown in the table instead of methyl rubecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate.

[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-dioxopyrrol-1-yl)hexanoic acid hydrochloride shown in Table 22D instead of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride in step 1.

[0941] [Table 22D]

[0942] Example 39 Preparation procedure of Ab-STING agonist conjugates by stochastic cysteine ​​conjugation (shown in Figure 1) To a solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO2011 / 050242, 60 mg / mL) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer was added 0.5 M Tris / 25 mM EDTA pH 8.0 buffer until the pH of the solution reached 7.5-8.0. TCEP (10 mM solution in HO, 3-5 equivalents) was added to the above 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 mM in DMA) was added. A solution of 100 mM HCl (5-9 equiv.) was slowly added to the above mixture. The reaction was purged with argon and incubated at 37 °C for an additional 1.5-2 h with gentle shaking. The reaction mixture was purified according to the preparative SEC method described herein to obtain the 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 conjugates can be prepared using the above procedure.

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

[0945] [Table 24]

[0946] Example 41 Preparation procedure for Ab-STING agonist complex via transglutaminase conjugation (shown in Figure 2) Deglycosylation: A solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO2011 / 050242, 60 mg / mL) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer was diluted with an equal volume of PBS, pH 7.2. N-glycosidase F (New England BiolAbs, P0704S, 500,000 units / mL, 300 units per mg of antibody) was added to the solution, and the reaction mixture was heated to 37°C overnight with gentle mixing. The resulting deglycosylated 5F9 was buffer exchanged with PBS, pH 7.2.

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

[0948] Strain-promoted azide-alkyne cycloaddition: To a solution of the 5F9-NH-PEG-azide conjugate prepared above (2–15 mg / mL in PBS), a 4–10 mM DMSO solution of the linker-payload construct containing the strained alkyne (3–5 equivalents in <10% DMSO of the total solvent volume) was added. The resulting mixture was gently stirred overnight at room temperature. The product was purified according to the preparative SEC method described herein to obtain the 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 conjugates can be prepared using the above procedure.

[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 Ab-STING agonist complexes via transglutaminase conjugation (shown in Figure 3) To a solution of deglycosylated 5F9 in PBS (10-20 mg / mL, prepared according to the deglycosylation procedure described in Example 41), 1 M Tris, 5 M NaCl, pH 8.0 buffer (10-20% of the total volume) was added, adjusted to pH 8.0. To this solution was added a 10 mM DMSO solution of a linker-payload construct (20 equiv.) containing a primary amine. The solution was added, followed by transglutaminase (ACTIVA™, Ajinomoto, 100-150 mg per 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 citric acid pH 4.0.

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

[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: Benzyl (2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-yl)carbamate 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) was added triethylamine (0.340 mL, 2.44 mmol) at 0 °C. The resulting yellow heterogeneous mixture was stirred for 15 min. A solution of benzyl (2-aminoethyl)carbamate (500 mg, 2.44 mmol) in DMF (6.0 mL) was added slowly over 10 min, and the mixture was warmed to room temperature and stirred for 3 days. The reaction mixture was partitioned between water (100 mL) and EtOAc (150 mL). The separated aqueous layer was extracted with EtOAc (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 / CHCl) to give tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (617 mg, 1.51 mmol, 65% yield) as a white solid. LCMS (FA): m / z = 431.2 (M+Na). 1 H 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: tert-butyl (2-((2-((2-aminoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate To a solution of tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (615 mg, 1.51 mmol) in ethanol (20.0 mL) was added 10% Pd / C (62.0 mg, 0.0583 mmol). 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 co-evaporated with toluene followed by drying under high vacuum to give N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate as an off-white solid. tert-Butyl [2-(2-methyl-2-oxo-ethyl)]amino-2-oxo-ethylcarbamate (407 mg, 1.48 mmol, 98.5% yield). LCMS (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: tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]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 min. The mixture was cooled in an ice bath, and then tert-butyl N-[2-[[2-(2-aminoethylamino)-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (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 min and then at room temperature overnight. Water (2 mL) and EtOAc (5 mL) were added, and the mixture was stirred vigorously for 20 min. The mixture was diluted with EtOAc (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 / CHCl2) afforded tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (88.0 mg, 0.246 mmol, 90.1% yield) as a glassy solid. LCMS (FA): m / z = 356.2 (M−H). 1 H NMR (400 MHz, DMSO-d) δ 8.1 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 tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (86.0 mg, 0.241 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (1.00 mL, 8.77 mmol). 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 up in 5 mL of water and lyophilized to give 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxo-ethyl]acetamide 2,2,2-trifluoroacetate (102 mg, 0.275 mmol, quantitative yield) as a colorless syrup. 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 Production and purification of 5F9-LPETGG-His6 The 5F9-LPETGG-His6 protein was transfected with transient Expi293F™ cells, Expi293™ expression growth medium, and the Expifectamine 293™ transfection kit from ThermoFisher, cat. no. A14635. Produced using the 3F Mammalian Protein Expression System Kit. 2.8 x 10 cells in a 5 L vented shake flask. 61.8 liters of Expi293F™ cells at a density of 1000 cells / mL were transfected with 0.666 mg of light chain DNA and 0.334 mg of heavy chain DNA mixed with 5.4 mL of Expifectamine 293™ transfection reagent. The cells were incubated overnight at 37°C in an 8% CO2 incubator 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 5 days post-transfection. The cells were spun down at 9000 rpm at 4°C for 10 minutes, filter-sterilized through a 0.22 μm filter unit, and stored at 4°C until purification 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 60 mL (2 CV) of high-salt buffer (25 mM trisodium citrate pH 7.6, 2 M NaCl), followed by another 60 mL (2 CV) of low-salt buffer. Protein was eluted by pH gradient elution with 25 mM citric acid, 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. Fractions of the main peak (2A5-2C5) were pooled. The pooled fractions were concentrated and buffer-exchanged into 25 mM sodium citrate pH 5.5, 125 mM NaCl buffer using a Sartorius VIVAFLOW200 cartridge with a 50 kDa molecular weight cutoff. The sample was then filtered through a 0.22 μm syringe filter unit, aliquoted, and stored at -80°C. The final yield was 623 mg of purified 5F9-LPETGG-His6 antibody, with a purity of >95% and 0.64 EU / mL endotoxin.

[0963] Heavy chain sequence:

[0964]

number

[0965] Light chain sequence:

[0966]

number

[0967] Example 47 Preparation procedure for Ab-STING agonist complex via sortase conjugation (shown in Figure 4) Sortase conjugation: The 5F9 antibody containing LPETG-His6 at the C-terminus of the heavy chain was generated according to the procedure described in Example 46. A solution of 5F9 containing an 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 the solution was added CaCl2 (4 mM solution in water, 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, 150 mM NaCl, pH 7.45 buffer containing 0.1% DMSO, 20 equivalents), followed by sortase 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 (2.0 μM solution in 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer, 0.03 equivalents, expressed 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 by a Capturem nickel column (from Takara, washed with PBS) followed by dialysis or according to the preparative SEC method described herein to give 5F9-LPETG-azide.

[0968] Strain-promoted azide-alkyne cycloaddition: To a solution of the 5F9-LPETGG-NH(CH2)2NHCOCH2-azide conjugate prepared above (2–15 mg / mL in PBS), a 4–10 mM DMSO solution of the linker-payload construct containing the strained alkyne (3–5 equivalents in <10% DMSO of the total solvent volume) was added. The resulting mixture was gently stirred overnight at room temperature. The product was purified according to the preparative SEC method described herein to obtain the 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.

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

[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 Tritosome assay conditions Test compounds (4.65 μL of a 121 μM linker-payload construct solution in DMSO or an ADC solution in PBS) were added to a mixture of 125 μL of rat liver tritosomes (purchased from XenoTech) and 433 μL of aqueous buffer solution (54.7 mg / mL phosphate, 1.7 mg / mL EDTA, pH 6.0), and the solution was incubated at 37 °C. 40 μL samples were removed at 10 and 30 min, 1, 3, 5, and 24 h, treated with 160 μL of 0.1% formic acid in methanol in a 96-well plate, and then stored at -80 °C. After the final time point, each sample was thawed and treated with 200 μL of 0.1% formic acid in methanol containing 150 nM carbutamide (internal standard). Samples were centrifuged at 4000 g for 10 min and analyzed according to the LC / MS / MS method described herein. Using the Excel-Fit program 1 / 2 The calculated t 1 / 2 The release of free payload from ADC-3 was not observed within 24 hours in this tritosome assay, while ADC-9 with the same payload showed a 7 hour t 1 / 2 The successful release of the payload at 1000 kJ / min indicates that the linker in ADC-9 resulted in a significantly improved rate of payload release under surrogate cellular release conditions compared to ADC-3.

[0973] [Table 28]

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

[0975] Plasma stability assay conditions Test compounds were added to 1 mL of plasma at a concentration of 10 μg / mL, and then five equal aliquots were distributed into 2 mL Eppendorf microcentrifuge tubes (labeled 0, 24, 48, 72, and 96 hours). The time 0 sample tube was immediately stored at -80°C, while the remaining tubes were incubated at 37°C with moderate shaking. At the corresponding time points, aliquots were removed from the incubator and stored at -80°C. After all samples were collected, they were The samples were thawed at room temperature and placed on wet ice. 50 μL of each sample was distributed in triplicate into a 96-well microtiter plate. Samples were quenched with 200 μL of ice-cold methanol containing 50 nM internal standard. 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 N gas at 40°C. The dried sample extract was 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 by reversed-phase HPLC using a Synergi 2.5μ, Polar-RP 100A C18 column (2.0 mm × 30 mm) (Phenomenex®) at 40°C using a gradient consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Analytes were analyzed by SCIEX API 4500 Detection was by positive ion spray in multiple reaction monitoring (MRM) mode using a QTRAP instrument. The percentage of payload loss in human, primate, and mouse plasma at various time points is reported in Table 29.

[0977] [Table 29]

[0978] THP1 Dual Lucia reporter gene assay conditions THP1-Dual(TM) KI-hSTING-R232 cells (InvivoGen The THP-1 monocytic cell line (#thpd-r232) was derived from the human THP-1 monocytic cell line by stable biallelic knockout of the endogenous human HAQ STING gene and knock-in of the R232 mutant form of human STING. These cells also stably express an inducible secreted Lucia luciferase reporter gene under the control of the ISG54 (interferon stimulated gene) minimal promoter in conjunction with five IFN-stimulated response elements (ISREs). Expression of the reporter gene allows for the study of the IFN regulatory factor (IRF) pathway by assessing the activity of Lucia luciferase. In addition to human STING and luciferase, These cells were engineered to stably express human guanylyl cyclase C (GCC), allowing for the study of target-mediated activation of the IRF pathway. Non-GCC-expressing vector cells were used as a control.

[0979] On the day of the experiment, cells were plated into white 384-well plates (Corning 356661) at a density of 15,000 cells / 25 μl per well in growth medium (RPMI 1640, 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 blasticidin, 100 μg / mL Zeocin, and 1 μg / mL puromycin). Five μL of GCC-targeting ADC sample was dosed onto the plate of cells and then incubated at 37°C for 20 hours. At the end of the incubation, 10 μL / well of QUANTI-Luc™ (InvivoGen #rep-qlc1) was added, and luminescence was immediately measured using a LeadSeeker.

[0980] For the assay described above, the percent luminescence signal induction of each test ADC at various concentrations was calculated relative to untreated and control-treated samples. Compound concentration vs. percent signal induction curves were fitted to determine the EC 50 Those skilled in the art will appreciate that EC 50 It will be understood that the values ​​generated as observed EC 50and Emax are reported in Table 30. As shown in Table 30 below, ADC-16 was approximately 21-fold more potent than ADC-15 in vector THP1 cells, 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 in vivo evaluation of the ADC in Balb / C mice bearing CT26 colon carcinoma mouse tumors expressing GCC, 6- to 8-week-old female Balb / C mice (Jackson We used CT26.WT parental cells (purchased from the ATCC Laboratory). CT26.WT parental cells were obtained from ATCC, catalog number CRL-2638. GCC-expressing CT26 cells were constructed by transducing CT26.WT with the lentiviral vector pLenti6.3 containing the human GCC gene at Takeda, resulting in CT26 pLenti6.3 GCC clone A5. CT26 pLenti6.3 GCC clone A5 cells were grown in a 37°C incubator with 5% CO2 under sterile conditions for 12–14 days. Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum. Cells were passaged every 3–4 days and detached using 0.05% trypsin / EDTA. On the day of transplantation, cells were removed and resuspended in RPM-1640 serum-free medium at a concentration of 0.5 × 106 cells / 100 μL. Using a 27-gauge needle (0.5 × 106 cells per mouse), the cells were removed and resuspended in RPMI-1640 serum-free medium. 6 Mice were implanted with CT26 pLenti6.3 GCC clone A5 cells by subcutaneous injection into the lower flank using an injection volume of 100 μL of cells / 100 μL. After cell implantation, tumors were measured with a caliper and mice were weighed twice weekly once tumors were palpable. Tumors were measured in two dimensions. Caliper measurements were (L × W). 2) / 2. Mice were fed a normal diet and housed in a SPF animal facility in accordance with the Guide for the Care and Use of Laboratory Animals and the regulations of the Institutional Animal Care and Use Committee. Animals were maintained at a temperature of 18–26°C, a relative humidity of 50 ± 20%, and a 12-h intermittent light / dark cycle with free access to food and water.

[0983] The pharmacokinetics of ADCs was evaluated in patients with mean tumor volumes (MTVs) of 500–800 mm 3 When the IL-16 expression level reached 1.0, IL-16 expression was investigated following injection of the ADC into CT26-GCC tumor-bearing Balb / C mice (n=3 / treatment group). Serum samples were collected at various time points and stored frozen for analysis.

[0984] Mouse plasma levels of total antibody and conjugated payload were measured by a two-component immunocapture-based LC / MS assay on a Shimadzu UHPLC system interfaced with a Sciex 6500 QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated with anti-human IgG-coated magnetic beads for 45 min at room temperature, and nonspecifically bound proteins were removed by washing the magnetic beads with PBST (PBS buffer, pH 7.4, containing 0.05% Tween 20) and subsequently with PBS buffer. Both naked antibody (DAR = 0) and ADC (DAR ≥ 1) were then eluted from the magnetic beads with 0.1% trifluoroacetic acid. After neutralization of the eluate and addition of stable isotope-labeled internal standards, one aliquot of the sample was pipetted and digested with papain at 37 °C for 1 h before being used for LC / MS analysis of the conjugated payload. The remaining sample was subjected to trypsin / Lys-C digestion at 70 °C for 1 h before being used for LC / MS analysis of the total antibody.

[0985] Free payload in circulation was also measured by LC / MS after plasma protein precipitation. Briefly, mouse plasma was mixed with 8 volumes of methanol-containing stable isotope-labeled internal standard, and the supernatant was then evaporated to dryness at 40°C under a gentle stream of nitrogen. Finally, the residue was reconstituted with LC / MS-grade water prior to LC / MS analysis. The PK profiles of ADC-1, ADC-8, and ADC-9 are summarized in Table 31. Graphical representations 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 were 4.5, 3.9, and 4.3, respectively, which led to higher molar concentrations of conjugated CDN obtained at time 0 than the total Abs shown in Figures 8–10, respectively. However, as shown in Figure 8, the molar concentration of the conjugated CDN from ADC-1 decreases faster than that of the total Ab, and the two concentrations become equal on day 7 (leading to a DAR of approximately 1 on that day), suggesting that the DAR of ADC-1 decreases over time due to the less stable linker. The results show that the concentration of conjugated payload / CDN decreased with increasing ion concentration. In contrast, as shown in Figures 9 and 10, the molar concentrations of conjugated CDNs from both ADC-8 and ADC-9 remained higher than the molar concentrations of their respective total Abs throughout the course of the experiment. In addition, the longer half-lives of conjugated payload / CDNs from ADC-8 and ADC-9 compared to ADC-1 are likely a result of the enhanced stability of the linker in ADC-8 and ADC-9, as shown in Table 31. In summary, this experiment demonstrates that the linker in ADC-8 and ADC-9 has improved stability in mouse plasma compared to that of ADC-1.

[0986] [Table 31]

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

[0988] Mean tumor volume (MTV) was approximately 75 mm 3 When tumor volume reached 100 mg / kg, animals were randomized into treatment groups (n=8 / group) and administered various doses of ADC once intravenously. Tumors were measured twice weekly. The effect of ADC treatment on tumor growth was assessed by measuring tumor volume and comparing it with vehicle-treated tumors.

[0989] The efficacy endpoint was achieved when tumor size reached 10% of the animal's body weight or 2 cm in either direction. Following injection, mice were closely monitored for signs of clinical deterioration. If, for any reason, mice showed signs of morbidity, including respiratory distress, hunched posture, decreased activity, hind limb paralysis, tachypnea as an indication of pleural effusion, weight loss approaching 20% ​​or 15%, as well as other signs, or if their ability to perform normal activities (eating, mobility) was impaired, they were euthanized.

[0990] Graphical representations of the antitumor activity observed for ADC-1, ADC-5, ADC-8, and ADC-9 are shown in Figures 7, 8, 9, and 10, respectively. lipid complex Abbreviation: AA LCMS method using ammonium acetate AC Acetate ACN Acetonitrile ATM atmosphere aq water-based BBN Borabicyclo(3.3.1)nonane Bn Benzyl Boc tert-butoxycarbonyl (Bpin)2bis(pinacolato)diboron tBu tert-butyl Bz Benzoyl C Celsius CAD Charged Aerosol Detection cGAMP cyclic guanosine monophosphate-adenosine monophosphate DBAD Di-tert-butyl azodicarboxylate DBU 1,8-diazabicyclo[5.4.0]undec-7-ene dichloroacetic 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-glycero-3-phosphocholine DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine Et Ethyl EtOH ethanol EtOAc ethyl acetate FA LCMS method using 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 Diisopinocampheil 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 methylmorpholine MS mass spectrum nBu n-butane 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-trioxatriphosphorinane-2,4,6-trioxide TDA-1 Tris[2-(2-methoxyethoxy)ethyl]amine TBAF Tetrabutylammonium Fluoride TBS tert-butyldimethylsilyl TBTU Tetrafluoroborate O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium TEA Triethylamine TFA trifluoroacetic acid TIDPSi 1,1,3,3-Tetraisopropyldisiloxane TIPS Triisopropylsilyl THF tetrahydrofuran UPLC Ultra High Performance Liquid Chromatography Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

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

[0992] LCMS conditions: LCMS 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 containing either 0.1% formic acid (method denoted as FA) or 10 mM ammonium acetate (method denoted 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) at a flow rate of 1 mL / min over a 16.5 minute run.

[0993] In some cases, LCMS spectra were recorded using a reverse-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 containing either 0.1% formic acid (method indicated as FA) or 10 mM ammonium acetate (method indicated as 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) at a flow rate of 0.5 mL / min in a 5-minute run.

[0994] Preparative HPLC: Preparative HPLC was performed using a Phenomenex 5 micron C5 21.2 x 250 mm column eluting with a gradient of solvent A (10 mM ammonium acetate 99% / 1% acetonitrile) / solvent B (10 mM ammonium acetate 5% / 95% acetonitrile) using a Gilson instrument operated with a 322 pump with fraction collection set at 200 nm to 400 nm, provided by a UV / Vis 155 detector. Mass-gated fraction collection was performed using Agilson. The analysis is carried out on a Lent 1100 LC / MSD instrument.

[0995] Those skilled in the art will recognize that variations in gradient, column length, and flow rate are possible, and that depending on the chemical species being analyzed, some conditions may be more suitable for compound characterization than others.

[0996] Preparative SFC: Preparative SFC is performed using 10, 20, or 30 mm x 250 mm ChiralPak columns (typically IA, IB, IC, ID, IE, and IF), 10 or 20 mm x 250 mm Phenomenex Lux Cellulose-4, or 2-ethylpyridine columns eluted with the appropriate ratio of supercritical carbon dioxide and alcohol containing 0.3% diethylamine, 0.3% TEA, or 0.3% formic acid, or without acid or base additives. Isocratic conditions with flow rates ranging from 10 to 100 mL / min and a column temperature of 40 °C are typical. Preparative SFC is performed on a Jasco SFC purification system with UV / visible light-driven fractionation set at 200 nm to 400 nm and backpressure regulation set at 10 MPa.

[0997] Those skilled in the art will recognize that variations in gradient, column length, and flow rate are possible, and that depending on the chemical species being analyzed, some conditions may be more suitable for compound characterization than others.

[0998] HPLC conditions for Example B HPLC analysis was performed using a SHIMA 3 micron C18 4.6 × 75 mm column eluted with a gradient of solvent A (80% water / 10% 50 mM ammonium acetate / 10% acetonitrile) / solvent B (10% 50 mM ammonium acetate / 90% acetonitrile), a flow rate of 1 mL / min, a column temperature of 40 °C, and UV detection at 254 nm. This was carried out using DZULC-2010C.

[0999] LC / MS / MS conditions for Example B: LC / MS / MS analysis was performed using a Shimadzu UltraFast liquid chromatograph and autosampler interfaced to an AB SCIEX 4500 or AB SCIEX 5500 triple quadrupole mass spectrometer or equivalent system.

[1000] For the analysis of Compound No. 14 and Compound I-5c, mobile phase A was prepared using 10 mM ammonium acetate or 0.1% formic acid in 99% water and 1% acetonitrile, and mobile phase B was prepared using 10 mM ammonium acetate in 5% water and 95% acetonitrile or 0.1% formic acid in 5% water and 95% acetonitrile. Liquid chromatography was performed with a 3-minute gradient at 1.5 mL / min. The HPLC column was a Waters Xselect C18 CSH 3.5 micron column with an internal diameter of 2.1 mm and 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 with a gradient of 3.5 min. The HPLC column was Osaka Soda Ca. pcell PAK C1 UG120 5 micron, 2.0 mm i.d. × 35 mm length, and the column temperature was room temperature.

[1002] Analyst software 1.7 was used to analyze MS / MS peak areas and calculate the concentrations of the samples.

[1003] UPLC-CAD conditions: UPLC-CAD analysis was performed using a Waters ACQUITY UPLC system equipped with a Thermo Scientific™ Dionex™ Corona™ Veo™ RS (rapid separation) charged aerosol detector and an ACE Excel C4 2 micron 2.1 x 100 mm column or similar, eluted with a gradient of Solvent A (10 mM aqueous ammonium acetate, 2.6 mL ammonium hydroxide in 4 L) / Solvent B (10 mM ammonium acetate in methanol, 2.6 mL ammonium hydroxide 95% in 4 L), using 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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamic acid 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). 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-water bath. The reaction was allowed to warm to room temperature and stirred for 2 hours to produce TMS-protected Compound No. 14. Separately (approximately 30 minutes after stirring the above reaction at room temperature), 4-methylmorpholine (0.28 mL, 2.50 mmol) was added to an ice-water bath-cooled solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (520.0 mg, 2.27 mmol) in tetrahydrofuran (10.7 mL), followed by the slow addition of isobutyl chloroformate (0.29 mL, 2.27 mmol) in THF. The cloudy suspension was warmed to room temperature, stirred for 1 hour, and then slowly added to the above TMS-protected intermediate via syringe. The reaction mixture was stirred overnight at room temperature. MeOH (10 mL) was added to the above reaction mixture, and the reaction mixture was concentrated. The residue was redissolved in MeOH (3.8 mL). Triethylamine trihydrofluoride (0.26 mL, 1.59 mmol) was added to the reaction mixture cooled in an ice-water bath and allowed to warm to room temperature. The reaction mixture was absorbed onto diatomaceous earth (Celite®) (1 g) and purified by reverse-phase flash column chromatography (0-50% ACN in 10 mM aqueous triethylammonium acetate) to give Intermediate 1 (214.0 mg, 48.5%) as the TEA salt. LCMS (AA): m / z = 910.2 (M+H). 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-octadecanoyloxy-propyl], 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 a vial and evaporated to dryness (1 mL × 3). The residue was dissolved in DMF (1.5 mL) and treated with DIPEA (88.9 μL, 0.51 mmol). The DMF solution was then added to sodium [(2R)-2,3-di(octadecanoyloxy)propyl]phosphate 2-[[5-(2,5-dioxopyrrolidin-1-yl)oxy-5-oxo-pentanoyl]amino]ethyl (250.0 mg, 0.25 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 4 h. Most of the solvent (DCM) was evaporated, and the remaining solution (DMF) was poured into ice-cold aqueous HCl (0.1 N, 5 mL). The solid was collected by filtration, washed with water (3 mL × 3) and DCM (3 mL × 3), and dried under vacuum to give Intermediate-2 (247.0 mg, 85.2%). LCMS (AA): m / z = 1137.8 (M+H). 1 H NMR (MeOD / CDCl3) δ 7.55 (buried within the CDCl3 peak, 2H), 7.29 (d, J = 8.0 Hz, 1H), 5.25-5.22 (m, 1H), 4.53-4.49 (hidden within the MeOD residue water peak, 3H), 4.40 (dd, J = 4.0, 12.0 Hz, 1H), 4.19-4.00 (m, 6H), 3.49-3.39 (m, 2H), 2 .35-2.30(m,6H),2.22(t,J=8.0Hz,2H),2.17-2.09(m,1H),1.97-1.89(m,2H),1.65-1.58 (m,4H),1.45(d,J=8.0Hz,3H),1.35-1.28(br,m,56H),0.99-0.97(m,6H),0.89-0.86(6H). 31 P-NMR(MeOD / CDCl3)δ0.77(s,1P).

[1010] Example A3 Octadecanoic acid [(2R)-3-[hydroxy-[2-[[5-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-(4-nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-5-oxo-pentanoyl]amino]ethoxy]phosphoryl]oxy-2-octadecanoyloxy-propyl], intermediate-3

[1011] [ka]

[1012] To a vial was added Intermediate-2 (247.0 mg, 0.22 mmol), THF (4.0 mL), and DIPEA (83.3 μL, 0.48 mmol). The solution was cooled in an ice-water bath under a N atmosphere, and then bis(4-nitrophenyl)carbonate (97%, 74.9 mg, 0.24 mmol) was added in one portion. The pale yellow solution was stirred at 50° C. for 1 h. Additional bis(4-nitrophenyl)carbonate (97%, 145.3 mg, 0.48 mmol) was added and stirred at 50° C. for 1 h, then overnight at room temperature. The reaction mixture was poured into ice-cold aqueous HCl (0.05 N, 30 mL). The solid was collected by filtration and washed with water (10 mL × 3) and EtOAc (8 mL × 6) to give solid Intermediate-3 (282.8 mg, 79.5%). LCMS(FA): m / z=1302.8(M+H). 1 H NMR (MeOD / CDCl3) δ 8.28 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.42-7.38 (m, 4H), 5.25-5.22 (m, 3H), 4.53-4.49 (hidden within MeOD residual water peak, 1H), 4.38 (dd, J = 4.0, 12.0 Hz, 1H), 4.19-4.02 (m, 6H), 3.61 -3.387(m,2H),2.35-2.29(m,6H),2.22-2.20(m,2H),2.17-2.08(m,1H),1.98-1.90(m,2H),1.64 -1.56(m,4H),1.46(d,J=8.0Hz,3H),1.35-1.28(br,m,56H),0.99-0.97(m,6H),0.89-0.85(6H). 31 P-NMR(MeOD / CDCl3)δ0.74(s,1P).

[1013] Example A4 Octadecanoic acid (2S,5S,19R)-2-({4-[({[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]pyrimidin-7-yl)-16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphospha Cyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl](methyl)carbamoyl}oxy)methyl]phenyl}carbamoyl)-16-hydroxy-5-isopropyl-16-oxide-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16 lambda~5~-phosphicosan-20-yl, CP-1

[1014] [ka]

[1015] Intermediate-1 (15.0 mg, 0.013 mmol) was dissolved in 1,4-dioxane (0.29 mL) and treated with 4 M HCl in 1,4-dioxane (0.15 mL, 0.6 mmol) at room temperature for 1 h. The volatiles were removed and dried under vacuum for 1 h to give 4-A as a crude product, which was used directly in the next step. Separately, a vial was charged with Intermediate-3 (17.6 mg, 0.013 mmol), DMAP (3.3 mg, 0.027 mmol), TEA (0.015 mL, 0.11 mmol), and DCM (0.35 mL). To this mixture was slowly added a solution of 4-A in DMF (0.42 mL). The reaction was stirred at room temperature for 15 min and concentrated to a small volume (approximately 0.5 mL). The crude compound was purified by preparative HPLC (Phenomenex 5 micron C5 21.2 x 250 mm column, eluting with solvent A [10 mM ammonium acetate 99% / 1% acetonitrile] / solvent B [10 mM ammonium acetate 5% / 95% acetonitrile] at a flow rate of 20 mL / min, from 60% B to 100% B in 16 min) to give CP-1 (11.0 mg, 40.3%) as the ammonium salt. LCMS (AA): m / z = 1972.8 (M+H). 1 H NMR (MeO D) δ8.55(br,s,1H),8.28(br,s,1H),7.75(br,s,1H)7.52-7.42(m,2H),7.24-7.17(m,3H),6.43 -6.36(m,2H),5.60(dd,J=4.0and52.0Hz,1H),5.17-5.12(m,1H),5.10-5.00(m,1H),4.99(s,2H) ), 4.95-4.89 (m, 1H), 4.78-4.76 (hidden within the MeOD residual water peak, 1H), 4.48-4.28 (m, 6H), 4.23 (m, 1H), 4.13-4.09 (m, 2H), 3.96-3.82 (m, 5H), 3.42-3.33 (m, 4H), 2.91 (s, 3H), 2.73-2.64 (m, 2H), 2.31-2.16 (m,8H),2.11-2.03(m,1H),1.99-1.92(m,2H),1.89-1.82(m,2H),1.57-1.47(m,4H) ,1.49(d,J=8.0Hz,3H),1.27-1.28(br,56H),0.93-0.91(m,6H),0.85-0.81(m,6H). 31 P-NMR(MeOD)δ57.00(s,1P),52.26(s,1P),-0.20(s,1P). 19 F-NMR (MeOD) δ -165.24 to -165.46 (s, br, 1F), -202.98 to -203.24 (m, 1F). HRMS (C 88 H 138 F2N 13 O 25 P3S2) Expected value: 1972.8622; Measured value: 1972.8674

[1016] Example A5 2-[(tert-butoxycarbon...

Claims

1. Formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, wherein a is an integer from 1 to 20; b is an integer from 1 to 20; m is 0, 1, 2, 3, or 4; n is 0 or 1; D-NH- is part of an amino-substituted compound, said amino-substituted compound having the formula D-NH ２ having Each R １ is independent, C １ -C ４ Alkyl, O-C １ -C ４ alkyl, and halogen; R ２ is C １ -C ４ Alkyl and -(CH ２ CH ２ O) ｓ -CH ３ where s is an integer from 1 to 10; R ３ and R ３’ are each independently hydrogen and C １ -C ３ alkyl; L is a cleavable linker; Ab is an antibody, an antibody fragment, or an antigen-binding fragment, or a pharma- ceutically acceptable salt thereof.

2. During the ceremony, a is an integer from 1 to 4; b is an integer from 1 to 10; 2. The compound of claim 1, wherein m is 0, or a pharma- ceutically acceptable salt thereof.

3. During the ceremony, m is 0; n is 0; R ３ and R ３’ 3. The compound of claim 1 or 2, or a pharma- ceutically acceptable salt thereof, wherein each is hydrogen.

4. In the formula, L is 【Chemistry 2】 and During the ceremony, 【Chemistry 3】 is the point of attachment to the nitrogen atom; 【Chemistry 4】 is the point of attachment to the Ab; t is an integer from 1 to 10; W is absent or is a self-immolative group; Z is absent or is a peptide of 2 to 5 amino acids; U and U' are independently absent or a spacer; Q is a heterobifunctional group; 4. A compound according to any one of claims 1 to 3, or a pharma- ceutically acceptable salt thereof, with the proviso that at least one of W and Z is present.

5. W 【Chemistry 5】 is a self-immolative group selected from During the ceremony, 【Chemistry 6】 is the point of attachment to the carbonyl group; 【Chemistry 7】 is the point of attachment to Z, or a pharma- ceutically acceptable salt thereof.

6. W 【Chemistry 8】 6. The compound of claim 5, selected from:

7. 7. The compound according to any one of claims 4 to 6, or a pharma- ceutically acceptable salt thereof, wherein Z is an enzymatically cleavable peptide.

8. 8. The compound of claim 7, or a pharma- ceutically acceptable salt thereof, wherein Z is cleavable by a cathepsin.

9. 9. The compound of any one of claims 4 to 8, or a pharma- ceutically acceptable salt thereof, wherein Z is a two amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe.

10. U and U' are 【Chemistry 9】 are independently selected from During the ceremony, 【Chemistry 10】 is the point of attachment to Z; 【Chemistry 11】 is the point of attachment to Q; p is an integer from 1 to 6; q is an integer from 1 to 20; x is O or -CH ２ - and 10. The compound of any one of claims 4 to 9, wherein each r is independently 0 or 1, or a pharma- ceutically acceptable salt thereof.

11. 11. The compound of any one of claims 4 to 10, or a pharma- ceutically acceptable salt thereof, wherein Q is a heterobifunctional group that is attached to Ab via a chemical or enzyme-mediated bond.

12. Q 【Chemistry 12】 is selected from During the ceremony, 【Chemistry 13】 is the point of attachment to U, or, if U is not present, is the point of attachment to Z; 【Chemistry 14】 12. The compound of claim 11, or a pharma- ceutically acceptable salt thereof, wherein: is the point of attachment to U', or, if U' is absent, is the point of attachment to Ab.

13. t is 1; W is absent or is a self-immolative group; 5. The compound of claim 4, wherein Z is absent or is a peptide of two amino acids.

14. R ２ Ga-CH ３ , or -(CH ２ CH ２ O) ｓ -CH ３ and s is an integer from 1 to 10.

15. D-NH ２ The compound according to any one of claims 1 to 14, which is an amino-substituted compound that modulates STING activity.

16. The compound of claim 15, wherein the amino-substituted compound that modulates STING activity comprises a guanine derivative or an adenine derivative.

17. The amino-substituted compound that modulates STING activity has the formula (II): 【Chemistry 15】 or a pharma- ceutically acceptable salt thereof, During the ceremony, X １０ is SH or OH; X ２０ is SH or OH; Y ａ is O, S, or CH ２ and Y ｂ is O, S, NH or NR ａ In this case, R ａ is C １ -C ４ alkyl; R １０ is hydrogen, fluoro, OH, NH ２ , OR ｂ , or N.H.R. ｂ and R ２０ is hydrogen or fluoro; R ３０ is hydrogen; R ４０ is hydrogen, fluoro, OH, NH ２ , OR ｂ , or N.H.R. ｂ or R ３０ and R ４０ Together, CH ２ Forming O; R ５０ is hydrogen or fluoro; R ｂ is C １ -C ６ Alkyl, halo (C １ -C ６ ) alkyl, or C ３ -C ６ is cycloalkyl; Ring A １０ is an optionally substituted 5- or 6-membered monocyclic heteroaryl ring containing 1 to 4 heteroatoms selected from N, O, or S, or an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 1 to 5 heteroatoms selected from N, O, or S, wherein ring A １０ contains at least one N atom in the ring, and Y ｂ is ring A １０ is attached to a carbon atom of Ring B １０ is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O, or S, wherein ring B １０ contains at least two N atoms in the ring; However, ring A １０ Or ring B １０ is attached to the carbonyl group of formula (I) via an -NH- group.

18. The amino-substituted compound that modulates STING activity is 【Chemistry 16】 or a pharma- ceutically acceptable salt thereof; During the ceremony, 【Chemistry 17】 is the point of attachment to the carbonyl group of formula (I). The compound according to any one of claims 15 to 17.

19. The amino-substituted compound that modulates STING activity has the formula (III): 【Chemistry 18】 or a pharma- ceutically acceptable salt thereof, During the ceremony, X １０ is SH or OH; X ２０ is SH or OH; Y ｃ is O, S, or CH ２ and Y ｄ is O, S, or CH ２ and B １００ is the formula (B １ -A) or formula (B １ -B), 【Chemistry 19】 R １３ , R １４ , R １５ , R １６ , and R １７ are each independently a hydrogen atom or a substituent; R １０００ is hydrogen or a bond to the carbonyl group of formula (I); Y １１ , Y １２ , Y １３ , Y １４ , Y １５ , and Y １６ each independently represents N or CR １ａ In this case, R １ａ is hydrogen or a substituent; Z １１ , Z １２ , Z １３ , Z １４ , Z １５ , and Z １６ are each independently N or C; R １０５ is a hydrogen atom or a substituent; B ２００ is the formula (B ２ -A) or formula (B ２ -B), 【Chemistry 20】 R ２３ , R ２４ , R ２５ , R ２６ , and R ２７ are each independently a hydrogen atom or a substituent; R １００’ is hydrogen or a bond to the carbonyl group of formula (I); Y ２１ , Y ２２ , Y ２３ , Y ２４ , Y ２５ , and Y ２６ each independently represents N or CR ２ａ In this case, R ２ａ is hydrogen or a substituent; Z ２１ , Z ２２ , Z ２３ , Z ２４ , Z ２５ , and Z ２６ are each independently N or C; R ２０５ is a hydrogen atom or a substituent, where R １０５ and R ２０５ are each independently bonded to the 2- or 3-position of the 5-membered ring to which they are attached; however, B １００ Or B ２００ is attached to the carbonyl group of formula (I) via an -NH- group.

20. Formula (IIIa): 【Chemistry 21】 or a pharma- ceutically acceptable salt thereof, During the ceremony, B １００ is the formula (B １ -A) or formula (B １ -B) is a group represented by 【Chemistry 22】 ； R １３ , R １４ , R １５ , R １６ , and R １７ are each independently a hydrogen atom or a substituent; R １０００ is hydrogen or a bond to the carbonyl group of formula (I); Y １１ , Y １２ , Y １３ , Y １４ , Y １５ , and Y １６ each independently represents N or CR １ａ In this case, R １ａ is hydrogen or a substituent; Z １１ , Z １２ , Z １３ , Z １４ , Z １５ , and Z １６ are each independently N or C; R １０５ is a hydrogen atom or a substituent; B ２００ is the formula (B ２ -A) or formula (B ２ -B) is a group represented by 【Chemistry 23】 ； R ２３ , R ２４ , R ２５ , R ２６ , and R ２７ are each independently a hydrogen atom or a substituent; R １００’ is hydrogen or a bond to the carbonyl group of formula (I); Y ２１ , Y ２２ , Y ２３ , Y ２４ , Y ２５ , and Y ２６ each independently represents N or CR ２ａ In this case, R ２ａ is hydrogen or a substituent; Z ２１ , Z ２２ , Z ２３ , Z ２４ , Z ２５ , and Z ２６ are each independently N or C; R ２０５ is a hydrogen atom or a substituent, where R １０５ and R ２０５ are each independently bonded to the 2- or 3-position of the 5-membered ring to which they are attached; however, B １００ Or B ２００ On the other hand, 【Chemistry 24】 and During the ceremony: R １８ is hydrogen or C １－６ is alkyl; R １９ is a halogen atom; and the other is attached to the carbonyl group of formula (I) via an -NH- group, or a pharma- ceutically acceptable salt thereof; a compound of formula (IIIa), or a pharma- ceutically acceptable salt thereof, according to claim 19.

21. The amino-substituted compound that modulates STING activity is 【Chemistry 25】 or a pharma- ceutically acceptable salt thereof; During the ceremony, 【Chemistry 26】 21. The compound of claim 19 or 20, wherein is the point of attachment to the carbonyl group of formula (I).

22. 【Catalogue 27】 or a pharma- ceutically acceptable salt thereof, During the ceremony, a is an integer from 1 to 20; b is an integer from 1 to 20; k is 0, 1, 2, or 3; m is 0, 1, 2, 3, or 4; D-NH- is part of an amino-substituted compound, said amino-substituted compound having the formula D-NH ２ having R ２ is H, C １ -C ４ Alkyl and -(CH ２ CH ２ O) ｓ -CH ３ where s is an integer from 1 to 10; R ４ is selected from hydrogen and any naturally occurring amino acid side chain; R ５ is C １ -C ４ Alkyl, and O-C １ -C ４ alkyl; L is a cleavable linker; Ab is an antibody, an antibody fragment, or an antigen-binding fragment, or a pharma- ceutically acceptable salt thereof.

23. L 【Chemistry 28】 and During the ceremony: 【Chemical 29】 is the point of attachment to the carbonyl group; 【Chemistry 30】 is the point of attachment to the Ab; W is a self-immolative group; Z is absent or a peptide of 2 to 5 amino acids; U and U' are independently absent or a spacer; 23. The compound of claim 22, or a pharma- ceutically acceptable salt thereof, wherein Q is a heterobifunctional group.

24. Formula (V): 【Chemistry 31】 or a pharma- ceutically acceptable salt thereof, In the formula, D-NH- is 【Chemistry 32】 and X is O or CH ２ and f is an integer from 1 to 10; g is an integer from 1 to 20; U and U' are independently absent or a spacer; Q is a heterobifunctional group and Ab is an antibody, antibody fragment, or antigen-binding fragment, or a pharma- ceutically acceptable salt thereof.

25. 25. The compound of claim 24, wherein X is O.

26. Formula (VI): 【Chemical Formula 33】 or a pharma- ceutically acceptable salt thereof, During the ceremony, a is an integer from 1 to 20; m is 0, 1, 2, 3, or 4; n is 0 or 1; D-NH- is part of an amino-substituted compound, said amino-substituted compound having the formula D-NH ２ having Each R １ is independent and C １ -C ４ Alkyl, O-C １ -C ４ alkyl, and halogen; R ２ is C １ -C ４ Alkyl and -(CH ２ CH ２ O) ｓ -CH ３ where s is an integer from 1 to 10; R ３ and R ３’ are each independently hydrogen and C １ -C ３ alkyl; L is a cleavable linker; The compound or a pharma- ceutically acceptable salt thereof, wherein LP is a lipid.

27. 27. The compound of claim 26, wherein the lipid is cholesterol or a phospholipid.

28. 28. The compound of claim 27, wherein the lipid is a phospholipid.

29. 29. The compound of claim 28, wherein the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid, and egg yolk phospholipid.

30. 29. The compound of claim 28, wherein the phospholipid is a phosphatidylethanolamine, and the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine.

31. A lipid complex comprising a compound according to any one of claims 26 to 30.

32. 32. The lipid complex of claim 31 , wherein the lipid complex is in the form of a liposome.

33. 33. The lipid complex of claim 31 or 32, further comprising one or more phospholipids.

34. 34. The lipid complex of claim 33, wherein the one or more phospholipids are selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid, and egg yolk phospholipid.

35. The lipid complex of claim 34, wherein the phospholipid is a phosphatidylcholine, and the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

36. The lipid complex of any one of claims 31 to 35, further comprising a fatty alcohol.

37. The lipid complex of any one of claims 31 to 36, further comprising cholesterol.

38. The lipid complex of any one of claims 31 to 37, further comprising at least one pegylated lipid.

39. 39. The lipid complex of claim 38, wherein the pegylated lipid is selected from a pegylated phospholipid and a pegylated diacylglycerol.

40. The liposome of claim 39, wherein the PEGylated lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

41. 41. A pharmaceutical composition comprising a compound according to any one of claims 1 to 40, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

42. 41. A method of treating cancer in a subject in need thereof, said method comprising administering to said subject a pharma- ceutically acceptable amount of a compound according to any one of claims 1 to 40.

43. 42. A method of stimulating an immune response in a subject in need thereof, comprising administering to the subject a pharma- ceutically acceptable amount of a compound, lipid complex or composition according to any one of claims 1 to 41.

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