EA044827B1 - CONJUGATION OF CYTOTOXIC DRUGS THROUGH BIS-BINDING - Google Patents

Description

Field of technology

The present invention relates to the conjugation of cytotoxic compounds to cell-binding molecules via a bis-linker. It refers to a method of conjugation by bis-linking of cytotoxic drugs/molecules, in particular when the drug contains double amino, hydroxyl, diamino, aminohydroxyl, dihydroxyl, carboxyl, hydrazine, aldehyde and thiol functional groups. The present invention also relates to methods for preparing cell-binding agent-drug (cytotoxic agent) conjugates using a bis-linker in a specific manner.

State of the art

Antibody-drug conjugates (ADCs) have emerged as one of the promising targeted therapies for cancer, as evidenced by the clinical success of brentuximab vedotin (Adcetris) for relapsed/refractory Hodgkin lymphoma (Okeley, N., et al., Hematol Oncol. Clin. North. Am , 2014, 28, 13-25; Gopal, A., et al., Blood 2015, 125, 1236-43) and adotrastuzumab emtansine for recurrent HER2+ breast cancer (Peddi, P. and Hurvitz, S., Ther. Adv Med Oncol 2014, 6(5), 202-9; Lambert, J. and Chari, R., J Med Chem 2014, 57, 6949-64). The three important components, the monoclonal antibody, the cytotoxic payload and the conditional linker of the ADC, as well as the attachment sites of the linker-payload components, are all important factors for the success of the ADC. Three decades have passed since the study of each factor of the ADC components began. However, linker technologies remain limited in scope because conjugate drugs must contain certain reactive functional groups, the liqueurs must provide stability in circulation and facilitate drug release upon antigen binding and intracellular uptake and, importantly, not cause harm to normal tissues once linker-payload components bind to off-target targets during circulation (Ponte, J. et al., Bioconj. Chem., 2016, 27(7), 1588-98; Dovgan, I., et al. Sci. Rep. 2016 , 6, 30835; Ross, P. L. and Wolfe, J. L. J. Pharm. Sci. 105(2), 391-7; Chen, T. et al. J. Pharm. Biomed. Anal., 2016, 117, 304-10).

In early ADCs, linkers, particularly those used for liquid tumor-targeting ADCs, were too labile and resulted in the release of free drug into the bloodstream and subsequent off-target toxicity (Bander, N.H. et al., Clin. Adv Hematol. Oncol., 2012, 10, 1-16). In this generation of ADCs, the linkers are more stable and the cytotoxic agents are significantly more effective (Behrens, C.R. and Liu, B., mAbs, 2014. 6, 46-53). However, off-target toxicity is still a major concern in ADC drug development (Roberts, S.A. et al., Regul. Toxicol. Pharmacol. 2013, 67, 382-91). For example, in clinical practice, adotrastuzumab emtansine (T-DM1, Kadcyla®), which uses a stable (non-cleavable) MCC linker, has shown greater benefit in patients with HER2-positive metastatic breast cancer (mBC - metastatic breast cancer) or in patients who have already been treated for mBC or who have developed a HER2 tumor recurrence within six months of adjuvant therapy (Peddi, P. and Hurvitz, S., Ther. Adv. Med. Oncol. 2014, 6(5), 202 -209; Piwko C, et al., Clin Drug Investig. 2015, 35(8), 487-93; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57, 6949-64). Ho TDM1 failed in clinical trials as a first-line treatment for patients with HER2-positive unresectable locally advanced or metastatic breast cancer and as a second-line treatment for HER2-positive advanced gastric cancer due to little benefit to patients when comparing adverse toxicity- effectiveness (Ellis, P.A., et al., J. Clin. Oncol. 2015, 33, (suppl; abstr 507 of 2015 ASCO Annual Meeting); Shen, K. et al., Sci Rep. 2016; 6: 23262; de Goeij, B. E. and Lambert, J. M. Curr Opin Immunol 2016, 40, 14-23; Barrios, S. N. et al., J Clin Oncol 2016, 34, (suppl; abstr 593 of 2016 ASCO Annual Meeting).

To address off-target toxicity issues, research and development in ADC chemistry and design are now expanding the areas of linker-payload packaging and conjugation chemistry beyond just high-potency payloads, and especially to address the issue of linker-payload ADC activity relative to target/disease targets. (Lambert, J.M. Ther Deliv 2016, 7, 279-82; Zhao, R.Y. et al., 2011, J. Med. Chem. 54, 3606-23). Currently, many drug developers and academic institutions are placing great emphasis on developing new robust specific conjugation linkers and site-specific ADC conjugation methods that appear to have a longer half-life in the bloodstream, higher potency, potentially reduced off-target toxicity, and a narrow range in vivo pharmacokinetic (PK) properties of ADCs, as well as better batch-to-batch uniformity in ADC manufacturing (Hamblett, K.J. et al., Clin. Cancer Res. 2004, 10, 7063-70; Adem, Y.T. et al., Bioconjugan Chem 2014, 25, 656-664; Boylan, N. J. Bioconjugate Chem. 2013, 24, 1008-1016; Strop, P., et al., 2013 Chem. Biol. 20, 16167; Wakankar, A. mAbs, 2011, 3 , 161-172). These specific conjugation methods reported to date include the introduction of recombinant cysteines (Junutula, J.R. et al., Nat. Biotechnol. 2008, 26, 925-32; Junutula, J.R., et al., 2010 Clin. Cancer Res. 16, 4769; US Patents 8309300; 7855275; 7521541; 7723485, WO 2008/141044), selenocysteines (Hofer, T., et al., Biochemistry 2009, 48, 12047-57; Li, X., et al., Methods 2014, 65, 133-8; US Patent 8916159 US National Cancer Institute), cis

- 1,044,827 theines containing a tag with perfluoroaromatic reagents (Zhang, S. et al., Nat. Chem. 2015, 8, 1-9), thiolfucose (Okeley, N.M., et al., 2013, Bioconjugate Chem. 24, 1650) , unnatural amino acids (Axup, J.Y., et al., Proc. Nat. Acad. Sci. USA. 2012, 109, 16101-6; Zimmerman, E.S., et al., 2014, Bioconjug. Chem. 25, 351-361; Wu, P., et al., 2009 Proc. Natl. Acad. Sci. 106, 3000-5; Rabuka, D., et al., Nat. Protoc. 2012, 7, 1052-67; US patent 8,778,631 and patent applications 20100184135, WO 2010/081110 Sutro Biopharma; WO 2006/069246, 2007/059312, US patents 7332571, 7696312 and 7638299 for Ambrx; WO 2007/130453, US patents 7632492 and 7829659 Allozyne), conjugation with reduced intermolecular disulfides by re-bridging dibromomalemides (Jones, M.W. et al., J. Am. Chem. Soc. 2012, 134, 1847-52) bisulfone reagents (Badescu, G. et al., Bioconjug. Chem. 2014, 25, 1124-36; WO 2013 /190272, WO 2014/064424 PolyTherics Ltd), dibromopyridazinediones (Maruani, A. et al., Nat. Commun. 2015, 6, 6645), galactosyl and sialyl transferases (Zhou, Q. et al., Bioconjug. Chem. 2014, 25, 510-520; US patent application 20140294867 Sanofi-Genzyme), formylglycine generating enzyme (FGE) (Drake, P. M. et al., Bioconj. Chem. 2014, 25, 1331-41; Carrico, I. S. et al., US patents 7985783; 8097701; 8349910 and US patent applications 20140141025, 20100210543 Redwood Bioscience), phosphopanthethynyltransferases (PPT ases) (Grunewald, J . et al. Bioconjug. Chem. 2015, 26, 2554-62), sortase A (Beerli, R.R., et al., PLoS One 2015, 10, e0131177), genetically introduced glutamine tag with Streptoverticillium mobaraense transglutaminase (mTG) (Strop , P., Bioconj. Chem., 2014, 25, 855-62; Strop, P., et al., Chem. Biol. 2013, 20, 161-7; US patent 8871908 Rinat-Pfizer) or with microbial transglutaminase ( MTGase) (Dennler, P., et al., 2014, Bioconjug. Chem. 25, 569-78; Siegmund, V. et al., Angew. Chemie - Int. Ed. 2015, 54, 13420-4; patent application US 20130189287 Innate Pharma; US Patent 7893019 Bio-Ker S.r.l. (IT)), enzyme/bacterium-formed isopeptide-peptide bonds that form off the main protein chain (Kang, H. J., et al., Science 2007, 318, 1625-8; Zakeri, B. et al., Proc. Natl. Acad. Sci. USA 2012, 109, E690-7; Zakeri, B. & Howarth, M. J. Am. Chem. Soc. 2010, 132, 4526-7).

We have disclosed several methods for conjugating reusable thiol pairs of reduced interchain disulfide bonds of a native antibody, such as the use of bromomaleimide and dibromomaleimide linkers (WO 2014/009774), 2,3-disubstituted succinic/2-monosubstituted/2,3-disubstituted fumaric or maleic linkers (WO 2015/155753, WO 20160596228), acetylenedicarboxylic linkers (WO 2015/151080, WO 20160596228) or hydrazine linkers (WO 2015/151081). ADCs generated using these linkers and methods have demonstrated better therapeutic index windows than traditional nonselective conjugation via cysteine or lysine residues on the antibody. Herein we disclose the invention of bis-linkers and methods for conjugating a cytotoxic molecule, in particular when the cytotoxic agent contains diamino, aminohydroxyl, dihydroxyl, carboxyl, aldehyde and thiol groups. Immunoconjugates produced by bis-coupling have a longer half-life during targeted delivery and minimize exposure to non-target cells, tissues or organs during circulation, resulting in reduced off-target toxicity.

The essence of the invention

The present invention provides bis-linking of an antibody to a cytotoxic agent, in particular when the cytotoxic agent contains two functional groups of amino, hydroxyl, diamino, aminohydroxyl, dihydroxyl, carboxyl, hydrazine or thiol. It also provides a bis-linker for conjugating a cell-binding molecule to a cytotoxic molecule in a specific manner.

In one aspect of the present invention, the bis bond is represented by formulas (I-e), (I-f), (I-p) and (I-r):

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in which

--- represents a single bond;

—— optionally represents either a single bond or a double bond, or may optionally be absent;

n - integer from 1 to 20;

a cell-binding frame molecule that is a molecule/agent that binds, complexes, or reacts with some portion of the cell population that is desired to be therapeutically or otherwise biologically modified; the molecule/agent that binds/binds to the cell is an antibody; wherein the molecule/agent that binds/binds to the cell contains a disulfide bond, the reduction of which produces a pair of thiols for bis-binding;

the cytotoxic molecule in the box is selected from tubulisin, auristatin, amatoxin, pyrrolobenzodiazepine dimers (PBD);

X and Y represent the same or different and independently a functional group that binds a cytotoxic drug, and X and Y are independently selected from NH; NHNH; N(R1); N(R1)N(Ri); ABOUT; C(O)NH, C(NH)NH;

L ₁ and L2 are the same or different, and are independently selected from O; NH; NHNH;

N(R ₃ ); N(R ₃ )N(R ₃ '); C1-C8 alkyl, amides, amines, imines, hydrazines or hydrazones;

ethers, esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, peptides, amides; 1-8 amino acids; polyethyleneoxy groups of the formula (OCH2CH2) _p OR ₃ , or (OCH ₂ -CH(CH ₃ )) _p OR ₃ , or NH(CH2CH2O) _p R ₃ , or NH(CH2CH(CH ₃ )O) _p R ₃ , or N[ (CH2CH2O)pR3]-[(CH2CH2O)p'R3'], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, where p and p' independently represent an integer selected from 0 to about 5000, or a combination thereof ; R ₃ and R ₃ ' independently represent H or polyethyleneoxy groups of the formula (OCH ₂ CH ₂ ) _p or (OCH ₂ CH (CH ₃ )) _p , where p is an integer from 0 to about 5000, or the above combination;

R1 and R2 are independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl, or C2-C8 esters, ether or amide; or peptides containing 1-8 amino acids; or a polyethyleneoxy group having the formula (OCH ₂ CH ₂ ) _p or (OCH ₂ CH (CH ₃ )) _p , where p is an integer from 0 to about 5000, or a combination of the above groups;

X ₇ and Y7 independently represent CH, CH2, NH, O, S, NHNH, N(R1) and N; a chemical bond between two atoms means that it can connect any adjacent atoms.

The present invention also relates to a method for preparing cell-binding molecule-drug conjugates of formulas (I-e), (I-f), (I-p) and (I-r).

Brief description of graphic materials

In fig. 1 depicts the general synthesis of the bis-linked conjugates of this patent application by double linking a phenyldiamine, phenyldiol, or aminophenol group of a drug at one end and a thiol pair of a cell-binding molecule at the other end, with the wavy line representing the remainder of the drug or bound component drug that is missing (not shown).

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In fig. Figure 2 presents the synthesis of analogues of tyrosine (Tyr) and tubutyrosine (Tut), which contain an amino or nitro group on the benzene ring for double binding to a cell-binding molecule.

In fig. Figure 3 shows the synthesis of the components of tubulisin analogues.

In fig. 4 shows the synthesis of the components of tubulisin analogues.

In fig. 5 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a pair of antibody thiols.

In fig. 6 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 7 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 8 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 9 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 10 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 11 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 12 shows the synthesis of bis-linker components and bis-linking to an analogue of tubutyrosine (Tup), a component of tubulisin.

In fig. 13 shows the synthesis of tubulisin analogues containing a bis-linker and their conjugation to an antibody via a pair of thiols in the antibody.

In fig. 14 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a thiol pair in the antibody.

In fig. 15 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a pair of antibody thiols.

In fig. 16 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a pair of antibody thiols.

In fig. 17 depicts the conjugation of a bis-linker-containing tubulysin analog to an antibody via a pair of antibody thiols, and the synthesis of a tubupenylalline (Tup) analog containing a double-amide-linked bis-linker.

In fig. 18 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a thiol pair in the antibody.

In fig. 19 depicts the conjugation of a bis-linker-containing tubulysin analog to an antibody via a pair of antibody thiols, and the synthesis of a tubupenylalline (Tup) analog containing a double-amide-linked bis-linker.

In fig. 20 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a thiol pair in the antibody.

In fig. 21 shows the synthesis of a tubulisin analogue containing a bis-linker and its conjugation to an antibody via a pair of antibody thiols.

In fig. 22 shows the synthesis of the components of the dimethyl auristatin analogue.

In fig. 23 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 24 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 25 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 26 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 27 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 28 shows the synthesis of bis-linker-containing dimethyl auristatin F analogues and their conjugation to an antibody via a pair of antibody thiols.

In fig. 29 shows the synthesis of an amatoxin analog containing a diamino group on its aromatic ring.

In fig. 30 shows the synthesis of an amatoxin analogue containing a bis-linker and its conjugation to an antibody via a pair of antibody thiols.

In fig. 31 shows the synthesis of the bis-linker and its coupling to an amatoxin analogue.

In fig. 32 shows the synthesis of amatoxin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 33 presents the synthesis of amatoxin analogues containing a bis-linker and their conjugation

- 4 044827 with an antibody via a pair of antibody thiols.

In fig. 34 shows the synthesis of amatoxin analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 35 shows the synthesis of amatoxin analogs and dimethyl auristatin F analogs containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 36 shows the synthesis of tubulisin analogs and bislinker-containing CBI dimer analogs and their conjugation to an antibody via a pair of antibody thiols.

In fig. 37 shows the synthesis of CBI dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 38 shows the synthesis of CBI dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 39 shows the synthesis of CBI dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 40 shows the synthesis of CBI dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 41 shows the synthesis of PBD dimer analogues containing a bis-linker.

In fig. 42 shows the synthesis of PBD dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 43 shows the synthesis of PBD dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 44 shows the synthesis of PBD dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 45 shows the synthesis of PBD dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 46 shows the synthesis of PBD dimer analogues containing a bis-linker and their conjugation to an antibody via a pair of antibody thiols.

In fig. 47 shows a comparison of the antitumor effect of conjugates A-3a, B-6a, B-12a, B15a, B-18a, B-20a, B-21a, B-24a, B-28a, C-3a, D-2a together with T -DM1 and PBS (control) using the cell model of gastric cancer N87, i.v., one injection, dose 3 mg/kg for conjugates A-3a, B-6a, B-12a, B-15a, B-18a, B-20a, B-21a, B-24a, B-28a, T-DM1 and a dose of 1 mg/kg for conjugates C-3a and D-1a. All 12 conjugates tested herein demonstrated antitumor activity. Animals in the B-24a, C-3a, B-20a, B-21a and D-20a conjugate groups demonstrated better antitumor activity than T-DM1. However, animals in the groups of conjugates B-18a, B-15a, A3a, B-6a, B-28a and B-12a demonstrated worse antitumor activity than T-DM1. TDM1 at 3 mg/kg inhibited tumor growth for 28 days but failed to eliminate tumors at any time during the trial. In contrast, conjugates B-20a, B-21a and D-20a completely eliminated the tumors of some animals from 15 to 43 days.

In fig. 48 shows photographs of in vivo tested animals with their purified tumors from PBS, A-3a, B-15a, B-21a and T-DM1 conjugates after the animals were sacrificed. No tumor was detected in five of the eight animals from the B-21a conjugate group (designated as 3s). Five of the eight animals in the conjugate B-15a group died (indicated as ) on day 43 due to too large a tumor.

In fig. 49 presents a study of the stability of the B-21a conjugate in mouse serum compared to a conventional, monolinked T-1a and T-DM1 conjugate. This indicates that the bis-linkage conjugate is more stable than the regular monolinkage conjugates in mouse serum.

Detailed description of the invention definitions

The term alkyl refers to an aliphatic hydrocarbon group or monovalent groups derived from an alkane by the removal of one or two hydrogen atoms from the carbon atoms. It can be straight or branched, having C1-C8 (1 to 8 carbon atoms) in the chain. The term branched means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl chain. Typical alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl and isooctyl. A C1-C8 alkyl group may be unsubstituted or substituted by one or more groups, including, but not limited to, -C1-C8 alkyl, -O-(C1- _C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ , -NHC(O)R ', -SR', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN; wherein each R' is independently selected from -C1-C8 alkyl and aryl.

The term halogen refers to a fluorine, chlorine, bromine or iodine atom; preferably a fluorine atom and

- 5 044827 chlorine.

The term heteroalkyl refers to a C ₂ -C ₈ alkyl in which one to four carbon atoms are independently replaced by a heteroatom from the group consisting of O, S and N.

The term carbocycle refers to a saturated or unsaturated ring containing 3 to 8 carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle. Monocyclic carbocycles contain 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles contain from 7 to 12 ring atoms arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged in a bicyclic [5,6] or [6,6] systems. Typical C ₃ -C ₈ carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1, 3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl and -cyclooctadienyl.

The term C ₃ -C ₈ carbocycle refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic ring. A C ₃ -C ₈ -carbocyclic group may be unsubstituted or substituted by one or more groups, including, but not limited to, -C ₁ -C ₈ alkyl, -O-(C1-C8 alkyl), -aryl, -C(O )R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC( O)R', -SR', -S(O)R', -S(O) ₂ R', -OH, -halogen, -N ₃ , -NH2, -NH(R'), -N(R ') ₂ and -CN; wherein each R' is independently selected from -C1-C ₈ alkyl and aryl.

The term alkenyl refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond, which may be straight or branched, containing from 2 to 8 carbon atoms in the chain. Typical alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3methylbut-2-enyl, n-pentenyl, hexenyl, heptenyl, octenyl.

The term alkynyl refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, which can be straight or branched, containing from 2 to 8 carbon atoms in the chain. Typical alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3methylbutynyl, 5-pentynyl, n-pentynyl, hexylinyl, heptynyl and octinyl.

The term alkylene refers to a saturated, branched or linear or cyclic hydrocarbon radical with 1-18 carbon atoms and contains two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and things like that.

The term alkenylene refers to an unsaturated, branched or linear or cyclic hydrocarbon radical with 2-18 carbon atoms and contains two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkene. Typical alkenylene radicals include, but are not limited to: 1,2-ethylene (-CH=CH-).

The term alkynylene refers to an unsaturated, branched or linear or cyclic hydrocarbon radical with 2-18 carbon atoms and contains two monovalent radical centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical alkynylene radicals include, but are not limited to: acetylene, propargyl and 4-pentynyl.

The term aryl or Ar refers to an aromatic or heteroaromatic group consisting of one or more rings containing from three to fourteen carbon atoms, preferably from six to ten carbon atoms. The term heteroaromatic group refers to one or more carbon atoms in an aromatic group, preferably one, two, three or four carbon atoms are replaced by O, N, Si, Se, P or S, preferably by O, S and N. The term aryl or Ar also refers to an aromatic group in which one or more H atoms are independently replaced by -R', -halogen, -OR', or -SR', -NR'R, -N=NR', -N=R', - NR'R, -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O)2OR', -PR'R, -P(O)R 'R, -P(OR')(OR), -P(O)(OR')(OR) or -OP(O)(OR')(OR), wherein R', R are independently H, alkyl , alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl or pharmaceutical salts.

The term heterocycle refers to a ring system in which one to four carbon atoms in the ring are independently replaced by a heteroatom from the group O, N, S, Se, B, Si and P. The preferred heteroatoms are O, N and S. Heterocycles are also described in The Handbook of Chemistry and Physics, ^78th Edition, CRC Press, Inc., 1997-1998, p. 225-226, the description of which is incorporated herein by reference. Preferred non-aromatic heterocyclyl includes epoxide, aziridinyl, thiiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxalanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanil, as well as condensed systems formed as a result of condensation with a phenyl group.

The term heteroaryl or aromatic heterocycles refers to 3-14, preferably 5-10

- 6 044827 membered aromatic hetero, mono-, bi- or multicyclic rings. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, as well as condensed systems formed as a result of condensation with a phenyl group.

Alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl and the like also refer to the corresponding alkylene, cycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, heterocyclene and the like, which are formed by the removal of two hydrogen atoms.

The term arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, usually the terminal or sp ³ carbon atom, is replaced by an aryl radical. Typical arylalkyl groups include benzyl, 2-phenylethan-1-yl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and things like that.

The term heteroarylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, usually the terminal or sp ³ carbon atom, is replaced by a heteroaryl radical. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl, 2furylethyl.

Examples of the hydroxyl protecting group include methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate esters, pivaloate, benzoate, methanesulfonate and ntolu olsulfonate .

The term leaving group refers to a functional group that can be replaced by another functional group. Such leaving groups are well known in the art, and examples include halide (eg, chloride, bromide and iodide), methanesulfonyl (mesyl), ntoluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate) and trifluoromethylsulfonate. The preferred leaving group is selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflata; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5phenylisoxazolium-3'-sulfonate, simple and mixed anhydrides, for example, acetyl anhydride, formyl anhydride; or an intermediate produced by a condensation reagent for peptide coupling reactions or for Mitsunobu reactions.

The following abbreviations may be used throughout this document with the following definitions: Boc, tert-butoxycarbonyl; BroP, bromotripyrrolidinophosphonium hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethyl phosphorocyanidate; DMA, N,N-dimethylacetamide; DMAP, 4-(K,K-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC, 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; MS-EI, electrospray mass spectrometry; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate buffered saline (pH 7.0~7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine.

The term amino acid(s) may refer to natural and/or non-natural amino acids, preferably alpha amino acids. Natural amino acids are acids that are encoded by the genetic code: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine Unnatural amino acids are derived forms of proteogenic amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid (neurotransmitter), ornithine, citrulline, beta-alanine (3-aminopropanoic acid), gamma-carboxyglutamate, selenocysteine (also present in many non-eukaryotes as well as many eukaryotes, but is not directly encoded by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formylmethionine (which is often the starting amino acid of proteins in bacteria, mitochondria and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L- 3,4-dihydroxyphenylalanine (DOPA) and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. Analogues are compounds having the same general H ₂ N(R)CHCO ₂ H structure of a natural amino acid, except that the R group is not found among natural amino acids. Examples of analogues include homoserine, norleucine, methionine sulfoxide and methionine methylsulfonium.

- 7 044827

Preferably, the amino acid mimetic is a compound that has a structure different from, but functions similar to, the general chemical structure of an alpha amino acid. The term unnatural amino acid is intended to denote the stereochemical form D, and natural amino acids have the L form. In the case where 1~8 amino acids are used in the present patent application, the amino acid sequence is preferably a recognition sequence for protease cleavage. Many recognition sequences for protease cleavage are known in the art. See, for example, Matayoshi et al., Science 247: 954 (1990); Dunn et al., Meth. Enzymol. 241:254 (1994); Seidah et al., Meth. Enzymol. 244:175 (1994); Thornberry, Meth. Enzymol. 244:615 (1994); Weber et al., Meth. Enzymol. 244:595 (1994); Smith et al., Meth. Enzymol. 244:412 (1994); and Bouvier et al., Meth. Enzymol. 248:614 (1995); the disclosure of which is incorporated herein by reference. Specifically, the sequence is selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit- Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser and Glu.

The term glycoside describes a molecule in which a sugar group, through its anomeric carbon, is linked to another group through a glycosidic bond. Glycosides can be linked through an O-(Oglycoside), N-(glycosylamine), S-(thioglycoside), or C-(C-glycoside) glycosidic bond. They have a structural empirical formula C _m (H ₂ O) _n (where m may differ from n, am and n<36), the glycoside in this context includes glucose (dextrose), fructose (levulose), allose, altrose, mannose, gulose , iodose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-O-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, maltose, sucrose, lactose , maltose, trehalose, maltodextrins, raffinose, glucuronic acid (glucuronide) and stachyose. It may be in the D form or the L form, the 5 atom cyclic form of furanose, the 6 atom cyclic form of pyranose, or the acyclic form, the α-isomer (-OH anomeric carbon is below the plane of carbon atoms of the Haworth projection) or β-isomer (-OH anomeric the carbon atom is above the plane of carbon atoms in the Haworth projection). A carbohydrate, as used herein, can be either a monosaccharide, disaccharide, polyol, or oligosaccharide containing 3-6 sugar units.

The term pharmaceutically or pharmaceutically acceptable refers to molecular substances and compositions that do not cause an adverse, allergic or other adverse reaction when administered to an animal or human, as appropriate.

The terms pharmaceutically acceptable solvate or solvate refer to the association of one or more solvent molecules and the disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The term pharmaceutically acceptable excipient includes any carriers, diluents, adjuvants or carriers such as preservatives or antioxidants, excipients, disintegrants, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional carrier or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Additional active ingredients may also be included in the compositions in suitable therapeutic combinations.

As used herein, the term pharmaceutical salts refers to derivatives of the disclosed compounds in which the parent compound is modified by producing acidic or basic salts thereof. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric acid and the like; and salts derived from organic acids such as acetic, propionic, succinic, tartaric, citric, methanesulfonic, benzenesulfonic, glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic acid and the like. Additional addition salts include ammonium salts such as tromethamine, meglumine, epolamine, etc., metal salts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Lists of suitable salts can be found

- 8 044827 found in Remington's Pharmaceutical Sciences, ^17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

The term administration refers to any method of carrying, delivering, administering or transporting a pharmaceutical drug or other agent to a subject. Such routes include oral administration, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. The present invention also provides for the use of a device or instrument for administering the agent. Such a device may use active or passive transport and may be a slow or rapid release delivery device.

The novel conjugates described herein use bridging linkers. Examples of some suitable linkers and their synthesis are presented in FIG. 1-34.

A cell-binding agent conjugate is a cytotoxic molecule linked through bis-linking.

The bis-coupling conjugate is represented by formula (I)

Where

---- represents a single bond;

----- is optionally either a single bond or a double bond, or may optionally be absent; n and m1 are independently equal from 1 to 20;

the cell-binding agent/molecule in the framework that binds to Z1 and _Z2 may be of any kind now known or to become known, a molecule that binds, complexes, or reacts with some portion of the cell population that required to be therapeutically or otherwise biologically modified. Preferably, the cell binding agent/molecule is an immunotherapeutic protein, antibody, single chain antibody; an antibody fragment that binds to a target cell; monoclonal antibody; single chain monoclonal antibody; or a fragment of a monoclonal antibody that binds to a target cell; chimeric antibody; a chimeric antibody fragment that binds to a target cell; antibody domain; an antibody domain fragment that binds to a target cell; adnectins, which mimic antibodies; DARPins (darpins); lymphokine; hormone; vitamin; growth factor; colony-stimulating factor; or a molecule that transports nutrients into the cell (transferrin); binding peptides containing more than four amino acids, or protein, or antibody, or small molecule, cell binding or ligand attached to albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles or (viral) capsids;

the cytotoxic molecule/agent in the framework is a therapeutic drug/molecule/agent or an immunotherapeutic protein/molecule, or a functional molecule for enhancing binding or stabilizing a cell binding agent or a ligand binding to a cell surface receptor, or for inhibiting cell proliferation, or to observe, detect, or study the action of a cell-binding molecule. It may also be an analogue, or prodrug, or pharmaceutically acceptable salt, hydrate or hydrated salt, or crystal structure, or optical isomer, racemate, diastereomer or enantiomer, of an immunotherapeutic compound, chemotherapeutic compound, antibody (proantibody) or fragment of an antibody (proantibody), or siRNA, or DNA molecule, or ligand that binds to the cell surface;

preferably, the cytotoxic molecule is any of a variety of small molecule drugs, including, but not limited to, tubulisins, calicheamicins, auristatins, maytansinoids, CC-1065 analogues, morpholino doxorubicins, taxanes, cryptophycins, amatoxins (e.g., amanitins), epothilones, eribulins, geldanamycins, duocarmycins, daunomycins, methotrexates, vindesines, vincristines and benzodiazepine dimers (eg pyrrolobenzodiazepine dimers (PBD), tomaimycin, indolinobenzodiazepines, imidazobenzothiadiazepines or oxazolidinobenzodiazepines);

X and Y represent the same or different, and independently functional group that binds a cytotoxic drug via a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary or quaternary) , imine, cycloheteroalkyane, heteroaromatic, alkoxime or amide linkage; preferably X and Y are independently selected from NH; NHNH; N(R1); N(R1)N(R ₂ ); ABOUT; S; S.S., O-N.H.

- 9 044827

ON(Ri), CH2-NH. CH ₂ -N(Ri), CH=NH. CH=N(Ri), S(O), S(O2), P(O)(OH), S(O)NH, S(O2)NH, P(O)(OH)NH, NHS(O) NH, NHS(O2)NH, NHP(O)(OH)NH, N(R1)S(O)N(R2), N(R1)S(O ₂ )N(R ₂ ), N(R1)P (O)(OH)N(R2), OS(O)NH, OS(O2)NH, OP(O)(OH)NH, C(O), C(NH), C(NRi), C(O )NH, C(NH)NH, C(NRi)NH, OC(O)NH, OC(NH)NH; OC(NR ₁ )NH, NHC(O)NH; NHC(Nh)NH; NHC(NR1)NH, C(O)NH, C(NH)NH, C(NR ₁ )NH, OC(O)N(R ₁ ), OC(NH)N(R ₁ ), OC(NR ₁ ) N(R ₁ ), NHC(O)N(R ₁ ), NHC(NH)N(R ₁ ), NHC(NR ₁ )N(R ₁ ), N(R ₁ )C(O)N(R ₁ ), N(R ₁ )C(NH)N(R ₁ ), N(R ₁ )C(NR ₁ )N(R ₁ ); or C ₁ -C ₆ alkyl; C ₂ -C ₈ alkenyl, heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl;

Z ₁ and Z 2 are the same or different and independently represent a functional group that is associated with a cell-binding molecule to form a disulfide, ether, ester, thioether, thioester, peptide, hydrazone, carbamate, carbonate, amine (secondary, tertiary or quaternary), imine, cycloheteroalkyane, heteroaromatic, alkyloxime or amide linkage; preferably Z1 and Z2 independently have the following structures: C(O)CH, C(O)C, C(O) _CH2 , ArCH2, C(O), NH; NHNH; N(R ₁ ); N(R ₁ )N(R ₂ ); ABOUT; S; S.S., O-N.H. ON(R1), SSH-NH. CH2-N(R1), CH=NH. CH=N(R1), S(O), S(O2), P(O)(OH), S(O)NH, S(O2)NH, P(O)(OH)NH, NHS(O) NH, NHS(O ₂ )NH, NHP(O)(OH)NH, N(R ₁ )S(O)N(R ₂ ), N(R ₁ )S(O ₂ )N(R ₂ ), N (R1)P(O)(OH)N(R ₂ ), OS(O)NH, OS(O ₂ )NH, OP(O)(OH)NH, C(O), C(NH), C( NR1), C(O)NH, C(NH)NH, C(NR ₁ )NH, OC(O)NH, OC(NH)NH; OC(NR ₁ )NH, NHC(O)NH; NHC(NH)NH; NHC(Nr ₁ )NH, C(o)nH, C(NH)NH, C(NR ₁ )NH, OC(O)N(R ₁ ), OC(NH)N(R ₁ ), OC(NR ₁ )N(R ₁ ), NHC(O)N(R ₁ ), NHC(NH)N(R ₁ ), NHC(NR ₁ )N(R ₁ ), N(R ₁ )C(O)N(R ₁ ), N(R ₁ )C(NH)N(R ₁ ), N(R ₁ )C(NR ₁ )N(R ₁ ); or C ₁ -C ₈ alkyl; C ₂ -C ₈ heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl;

preferably Z ₁ and Z ₂ are associated with thiol pairs of the cell binding agent/molecule. Thiols are preferably pairs of sulfur atoms reduced from disulfide bonds of the internal chain of a cell-binding agent via a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP) ), 2-mercaptoethylamine (β-MEA), and/or beta mercaptoethanol (β-ME, 2-ME);

L ₁ and L2 are a chain of atoms selected from C, N, O, S, Si and P, containing 0~500 atoms, which is covalently bonded to X and Z ₁ , and to Y and Z ₂ . The atoms used in the formation of L ₁ and L2 can be combined in all chemically significant ways, preferably they are C ₁ -C ₂₀ alkylene, alkenylene and alkynylene, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones , amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxyamines, hydroxamic acids or the above combination. More preferably, L ₁ and L2 are the same or different, independently selected from O, NH, S, NHNH, N(R ₃ ), N(R ₃ )N(R ₃ ), C ₁ -C ₈ alkyl, amide , amines, imines, hydrazines, hydrazones; C ₂ -C ₈ heteroalkyl, alkylcycloalkyl, ethers, esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes, amino acids, peptides, acyloxyamines, hydroxamic acids or heterocycloalkyls; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl; polyethyleneoxy groups of the formula (OCH ₂ CH ₂ ) _p OR ₃ , or (OCH ₂ _CH(CH ₃ )) _p OR ₃ , or NH(CH ₂ CH ₂ O) _p R ₃ , or NH(CH ₂ CH(CH ₃ )O ) _p R ₃ , or N[(CH ₂ CH ₂ O) _p R ₃ ]-[(CH ₂ CH ₂ O) _p R ₃ .], or (OCH ₂ CH ₂ ) _p COOR ₃ , or CH ₂ CH ₂ (OCH ₂ CH ₂ ) _p COOR ₃ where p and p' are independently an integer selected from 0 to about 5000, or a combination thereof; where R ₃ and R ₃ ' independently represent H; C ₁ -C ₈ alkyl; C ₂ -C ₈ heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl; or C ₂ -C ₈ esters, ether or amide; or 1~8 amino acids; or a polyethyleneoxy group of the formula (OSH ₂ CH ₂ ) _p or (OSH ₂ CH (CH ₃ )) _p , where p is an integer from 0 to about 5000 or the above combination;

optionally, L ₁ and L2 may independently consist of one or more linker components 6maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PAB), 4-thiopentanoate (SPP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC), (4-acetyl)aminobenzoate (SIAB), 4-thiobutyrate (SPDB), 4-thio-2- hydroxysulfonyl butyrate (2-sulfo-SPDB) or natural or non-natural peptides containing 1~8 natural or non-natural amino acids. The natural amino acid is preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, alanine;

L1 and L2 may also independently contain a self-cleaving or non-self-cleaving component, peptide units, a hydrazone bond, a disulfide bond, an ester bond, an oxime bond, an amide bond, or a thioester bond. The self-cleaving moiety includes, but is not limited to, aromatic compounds that are electronically similar to para-aminobenzylcarbamoyl (PAB) groups, such as 2-aminoimidazole-5-methanol derivatives, heterocyclic analogues of PAB, beta-glucuronide, and ortho- or para- aminobenzylacetals;

- 10 044827 preferably, the self-cleaving linker component has one of the following structures:

where (*) the atom represents the point of attachment of additional spacer or releasable linker moieties or a cytotoxic agent and/or binding molecule (CBA); X ¹ , Y ¹ , Z ² and Z ³ are independently NH, O or S; Z ¹ independently represents H, NHR1, OR1, SR1, COX1R1 wherein X1 and R1 are as defined above; v is 0 or 1; U ¹ independently represents H, OH, CrC ₆ alkyl, (OCH2CH2)n, F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5,NR5R5', NO2, SOR5R5', SO2R5 , SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5') or OCH2PO(OR5(OR5'), where R5 and R5' are independently selected from H, C1~C ₈ alkyl; C ₂ ~ C ₈ alkenyl, alkynyl, heteroalkyl, or amino acid; C ₃ ~C ₈ aryl, heterocyclyl, carbocyclyl, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl or glycoside; or pharmaceutical cationic salts;

The non-self-cleaving linker component has one of the following structures:

(CH ₂ ) _n CO(OCH ₂ CH ₂ ) _r OCH ₃ (CH ₂ ) _n CON(CH ₂ CH ₂ O) _r COCH ₃ *(CH ₂ CH ₂ O) _G * *£< _n * ,^ _HA .

9 9 (CH ₂ ) _P (OSN ₂ CH ₂ ) _G OSOSN ₃ (CH ₂ ) _P CO (OSN ₂ CH ₂ ) _G OSOSN ₃ 4/^ _N . _Ν Λ *CH* *CH* 't N .

r ₅

- 11 044827

BUT HE

ABOUT

ABOUT

ABOUT

O p-OH 'on ^COOH

UNS

COOH

UN

NHAc where (*) the atom represents the point of attachment of additional spacer or releasable linkers, cytotoxic agents and/or binding molecules; X ¹ , Y ¹ , U ¹ , R5 , R5' are as defined above; r is 0~100; m and n are independently equal to 0-6;

Moreover, preferably, L1 and L2 may independently represent a releasable linker. The term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions, such as a pH-labile, acid-labile, alkali-labile, oxidation-labile, metabolically labile, biochemically labile, or enzyme-labile bond. It will be understood that such physiological conditions leading to bond cleavage do not necessarily involve a biological or metabolic process and may instead involve a standard chemical reaction such as a hydrolysis or displacement reaction, such as an endosome having a lower pH than the cytosolic pH and/or reaction disulfide bond exchange with intracellular thiol, such as millimolar range of excess glutathione within malignant cells;

Examples of releasable L1 or L2 linkers include, but are not limited to:

-(C5RR6) _m (Aa)r(CR7R8)n(OCH2CH2) _t -, -(CR5R6) _m (CR ₇ R8)n(Aa)r(OCH2CH2)t-, -(Aa)r(CR5R6) _m ( CR7R8)n(OCH2CH2) _t -, -(CR5R6)m(CR7R8)n(OCH2CH2)r(Aa)t-, -(CR5R6)m(CR7=CR8)(CR9R10)n(Aa)t(OCH2CH2)r -, -(CR5R6) _m (NRnCO)(Aa\(C^^

-(CR5R6)m(Aa)t(NR11CO)(CR9R10)n(OCH2CH2)r-, -(CR5R6)m(OCO)(Aa)t(CR9R10)n-(OCH2CH2)r-,

-(CR5R6)m(OCNR7)(Aa)t(CR9R10)n(OCH2CH2)r-, -(CR5R6)m(CO)(Aa)t-(CR9R10)n(OCH2CH2)r-,

-(CR5R6)m(NR11CO)(Aa)t(CR9R10)n(OCH2CH2)r-, -(CR5R6)m-(OCO)(Aa)t(CR9R10)n-(OCH2CH2)r-,

-(CR5R6)m(OCNR7)(Aa)t(CR9R10)n(OCH2CH2)r-, -(CR5R6)m(CO)(Aa)t(CR9R10)n-(OCH2CH2)r-, -(CR5R6V phenyl- CO(Aa)t(CR7R8)n-, -(CR5R6)m-furyl-CO(Aa)t(CR7R8)n-, -(CR5R6)m-oxazolyl-CO(Aa)t(CR7R8)n-, - (CR5R6)m-thiazolyl-CO(Aa)t(CCR7R8)n-, -(CR5R6)t-thienyl-CO(CR7R8)n-, -(CR5R6)t-imidazolyl-CO(CR7R8)n-, -( CR5R6)t-morpholino-CO(Aa)t-(CR7R8)n-, -(CR5R)gπuperαzino-CO(AaHCR7R8)n-, -(CRsRX-Nmethylπiperazine-CO(Aa) _t -(CR ₇ R ₈ ) _n -, -(CR ₅ R) _m -(Aa) _t -phenyl-, -(CR ₅ R ₆ ) _m -(Aa) _t -furyl-, -(CR ₅ R ₆ ) _m -oxa- 12 044827 zolyl( Λa) _t -, -(CR ₅ R ₆ ) _m- thiazolyl(Aa) _t -, -(CR ₅ R ₆ ) _m- thienyl-(Aa) _t -, -(CR ₅ R ₆ ) _m- imidazolyl(Λa ) _t -,

-(CR ₅ R ₆ ) _m -morpholino-(Aa) _r , -(CR ₅ R ₆ ) _m -piperazino-(Λa) _t -, -(CR ₅ R ₆ ) _m -N-methylpiperazine-(Λa) _t -,

-K(CR5R6)m(Aa)r(CR7R8)n(OCH2CH2)t-, (CR5R ₆ )m(CR ₇ R ₈ ) _n (OCH ₂ CH ₂ )t-,

-K(CR5R6)m(CR7R ₈ )n(Aa)r(OCH ₂ CH ₂ )t-,

-K(CR5R6)m(CR7R8)n(OCH2CH2)r(Aa)t-, (CR7=CR8)(CR9Rio)n(Aa)t(OCH2CH2)r-,

-K(CR5R6)m(Aa)t(NR _n CO)(CR9Rio)n(OCH2CH2)r-,

-K(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-,

-K(CR5R6)m(NR _n CO)(Aa)t(CR9Rio)n(OCH2CH2)r-,

-K(Aa)rK(CR5R6)mK(CR5R6)m(NR _n CO)(Aa)t(CR9Rio)n(OCH2CH2)rK(CR5R6)m(OCO)(Aa)t(CR9Rio)n-(OCH2CH2) rK(CR5R6)m(CO)(Aa)t-(CR9Rio)n(OCH2CH2)rK(CR5R6)m-(OCO)(Aa)t(CR9Rio)n(OCH2CH2)r-

-K(CR5R6)m(OCNR7)(Aa)t(CR9Rio)n(OCH2CH2)r-, -K-(CR5R6)m(CO)(Aa)t(CR9R _w )n(OCH2CH2)r-, -K ^R^phenyl-CO(La)t(CR7R8)n-, -K-(CR5R6)m-furyl-CO(La)t-(CR7R8)n-, -K(CR5R6)m-oxazolyl-CO(La )t(CR7R8)n-, -K(CR ₅ R ₆ ) _m -τiazolyl-CO(Aa) _t -(CR ₇ R ₈ ) _n -, -K(CR ₅ R ₆ ) _t -thienyl-CO(CR ₇ R ₈ ) _n -, -K(CR ₅ R ₆ ) _t -imidazolyl-CO(CR^-, -K(CR5R6)t-morpholino-CO(Λa)t(CR7R8)n-, -K(CR5R6) t-πipazino-CO(Λa)t-(CR7R8)n-,

-K(CR ₅ R ₆ ) _t -N-methylpiperazine-CO(La) _t (CR ₇ R ₈ ) _n -, -K(CR ₅ R) _m (La) _t -phenyl, -K-(CR ₅ R ₆ ) _m -(La) _t -furyl-, -K(CR ₅ R ₆ ) _m -oxazolyl-(La) _t -, -K(CR ₅ R ₆ ) _m -τiazolyl(La) _t -, -K( CR ₅ R ₆ ) _m -thienyl-(La) _t -, -K(CR5R ₆ ) _m imidazolyl(La) _t -, -K(CR ₅ R ₆ ) _m- morpholino(La) _t -, -K(CR ₅ R ₆ ) _m -piperazino-(Λa) _t G, -K(CR5R ₆ ) _m -Nmethylpiperazine(Λa) _t -;

where m, Aa, m and n are as described above; t and r are independently equal to o - 1oo; R ₃ , R ₄ , R5 , R ₆ , R ₇ , and R ₈ are independently selected from H; halide; C1~C ₈ alkyl; C ₂ ~C ₈ aryl, alkenyl, alkynyl, ether, ester, amine or amide, which is optionally substituted with one or more halide, CN, NR1R2, CF3, OR1, aryl, heterocycle, S(O)R1, SO2R1, CO2H , -SO3H, -OR1, -CO2R1, -CONR1, -PO2R1R2, PO3H or P(O)R ₁ R ₂ R ₃ ; K represents NR _b -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=NNH-, - C(=O)NH-NH-, O, S, Se, B, Het (heterocyclic or heteroaromatic ring having C ₃ C ₈ ), or peptides containing 1-2o amino acids;

In addition, L ₁ and L ₂ may independently contain one of the following hydrophilic structures: c /% r ₃ A Λ Λ ^{0 0} * ^NN^s ^_ _x F _Xs ^

where * represents the binding site;

X ₂ , X ₃ , X ₄ , X ₅ or X ₆ are independently selected from NH; NHNH; N(R ₃ ); N(R ₃ )N(R ₃ ·); ABOUT; S; C ₁ -C ₆ alkyl; C ₂ C ₆ heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, ΛΓ-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 amino acids;

- 13 044827 where R3 and R3' independently represent H; C1-C8 alkyl; C ₂ -C ₈ heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl; or C ₂ -C ₈ esters, ethers or amides; or polyethyleneoxy groups having the formula (OCH ₂ -CH ₂ ) _p or (OCH ₂ CH (CH ₃ )) _p , where p is an integer from 0 to about 5000, or combinations thereof;

More preferably, R1, L1 or L2 is independently a linear alkyl containing 1 to 6 carbon atoms, or a polyethyleneoxy group having the formula (OCH ₂ CH ₂ ) _p , p=1~5000, or a peptide containing 1~4 amino acid units (L or D shape) or the above combination.

Additionally, X, Y, L1, L2, Z1 or Z2 may independently consist of one or more of the following components, as shown below:

6-maleimidocaproyl (MC),

(MR),

thioaminooxobutanoic acid, maleimidopropanoyl

nin-phenylalanine (ala-phe),

4-thiopentanoate (SPP), thiomaleido, minooxobutenoic acid, lysine-alanine (lys-ala),

valine-citrulline (val-cit), alysine-phenylalanine (lys-phe),

4-(N(ME), maleimidoethyl para-aminobenzyloxycarbonyl (PAB),

4-thiobutyrate (SPDB), maleimidomethyl)cyclohexane-1-carboxylate (MCC),

4-thio-2-hydroxysulfonylbutyrate (2-sulfo-SPDB),

arylthiol (4-acetyl)aminobenzoate (SIAB),

(PySS)

dioxylbenzylthio, oxybenzylthio,

diaminobenzylthio, aminobenzylthio,

(EO), phosphondiamide, aminooxylbenzylthio,

alkoxyamino (AOA),

ethyleneoxy

4-methyl-4-dithiopentanoyl (MPDP),

He . . and P alkylsulfonyl, θ O 1, p I

HE

alkylsulfonamide, alkylphosphonamide, I V I

N-methylphosphonamidic acid, triazole, n fl and

ABOUT

dithio, sulfone bisamide,

phosphinic acid,

N,N'-dimethylphosphonamidic acid,

- 14 044827 θ N -4-Ν. ΗΝ. G

N,N'-dimethylphosphondiamide,

oxime,

acetylacetohydrazide, hydrazine,

aminoethylamycetimidamide;

on, aminoethylaminoethylamine and L- or D-, natural or non-natural peptides containing 1-20 amino acids; where a bonding bond between atoms means that it can connect any adjacent carbon bonds; a wavy line is a place with which another connection may be connected;

Alternatively, X, Y, L1, _L2 , Z1, or _Z2 may be independently absent, but L1 and Z1, or L2 and _Z2 cannot be absent simultaneously.

Preferably, the bis-coupling conjugate is further represented by the formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j) , (I-k), (I-m), (I-n), (I-o), (I-p), (I-q), (I-r), (I-s), (I-t), (I-u), (I-v) and (Iw) below:

<

Cytotoxic ।.

.agentJ

Cell binding molecule

Molecule that binds to the cell _____________ _χ o o (Cytotoxic (GT (1-c)

Cell-binding molecule (Id) (Cytotoxic^ ; ^J

La re nt J L

Cell binding molecule (1st) (Cytotoxic

Tsagent__________ x jj

Molecule, cell (I-f)

Eitotoxic gent

Cell binding molecule (I-g)

- 15 044827

[Cytotoxic

[.agent_____________

Cell binding molecule (I-h)

Cell binding molecule (I-i)

ABOUT

Cell binding molecule (i-j)

Cell binding molecule (I-k)

Cell binding molecule (I-l)

Cell binding molecule (I-m)

Cell binding molecule (I-n)

- 16 044827

(I-P)

(I-q)

(Cytotoxic!

L agent____________

Cell binding molecule (I-t)

About /-s' but-<

(Iu) (Iv) (Iw) where X ₇ and Y ₇ independently represent CH, CH2, NH, O, S, NHNH, N(R ₁ ) and N; a chemical bond between two atoms means that it can connect any adjacent atoms;

, X, Y, R1, n, L1 and

- 17 044827

L2 are as described above; the cytotoxic agent is the same cytotoxic molecule described above. In a more preferred aspect, X and Y independently represent an amino, hydroxy, diamino, aminohydroxy, dihydroxy, carboxy, aldehyde, hydrazino, thiol, phosphate or sulfonyl group on an aromatic ring.

Preparation of drug conjugates with cell-binding molecules using bis-coupling.

The preparation of drug conjugates with cell binding molecules according to the present invention and the synthetic routes for preparing the conjugates by bis-coupling are presented in FIG. 1-46.

In one aspect, this invention relates to a reactive bis-linker containing a cytotoxic molecule of formula (II) below, in which two or more residues of the cell binding molecule can be simultaneously or sequentially brought into contact therewith to form formula (I)

where represents a single bond;

----- is optionally either a single bond, a double bond, or a triple bond, or may optionally be absent;

Provided that when ----- represents a triple bond, both Lv ₁ and Lv2 are absent;

The cytotoxic molecule in the framework, m _b X, Y, L _b L2, Z _b and Z2 are the same as defined in formula (I);

Lv ₁ and Lv 2 represent the same or different leaving group, which can react with a thiol, amino, carboxyl, selenol, phenolic or hydroxyl group in the cell-binding molecule. Lv ₁ and Lv2 are independently selected from OH; F; Cl; Br; I; nitrophenol; Nhydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflata; imidazole; dichlorophenol; tetrachlorophenol; 1hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, simple and mixed anhydrides, for example acetic anhydride, formyl anhydride; or an intermediate produced by a condensation reagent for peptide coupling reactions or for Mitsunobu reactions. Examples of condensation reagents are: EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexylcarbodiimide), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2morpholino-ethyl)carbodiimide meso p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N ,N',N'tetramethyl-O-( 1 H-benzotriazol-1 -yl)uronium hexafluorophosphate (HBTU), (benzotriazol-1 -yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium -3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridinium-1-ium 3-oxide hexafluorophosphate (HDMA) , 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N', N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S- (1-oxido-2-pyridyl)N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl)-cyanomethyleneamino]-N,N,N',N'tetramethyluronium hexafluorophosphate (HOTU), (1-cyano -2-ethoxy-2-oxoethylideneamineoxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), N-benzyl-N '-cyclohexylcarbodiimide (polymer bound or not), dipyrrolidino (N-succinimidyloxy)carbenium hexafluorophosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCH2), 2-chloro-1,3-dimethylimidazolidine tetrafluorobirate (CIB), (benzotriazol-1-yloxy) dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)phosphonium hexafluorophosphate (BgoP)propylPhosphonic anhydride PPACA, T3P®), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (VER), O-[(ethoxycarbonyl) cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3benzotriazin-3-yl)-N ,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicar- 18 044827 bonyl)dipiperidine (ADD), di-(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate ( DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD). Besides,

Lv1 and Lv ₂ can be a simple or mixed anhydride of C1~ _C8 acids;

preferably, Lv ₁ and Lv ₂ are independently selected from halide (eg, fluoride, chloride, bromide and iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyl hydroxyl (NHS) phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3'-sulfonyl, phenyloxadiazolyl (-sulfone-ODA), 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazolyl (ODA), oxadiazolyl, unsaturated carbon atom (double or triple bond between carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus , sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen or carbon-oxygen) or one of the following structures:

haloacetyl;

disulfide;

N-hydroxysuccinimide ester;

maleimide;

disubstituted maleimide;

disubstituted succinimide; -CHO aldehyde;

ABOUT

2-(tosyloxy)acetyl;

Ts' ryloyl);

2-(nitrophenoxy)acetyl;

acyl halide (acid halide);

maleimide;

monosubstituted monosubstituted succinimide;

acrylic (ac2-(mesyloxy)acetyl;

2-(fluorophenoxy)acetyl;

ethenesulfonyl;

ABOUT

2-(dinitrophenoxy)acetyl;

2-(difluorophenoxy)acetyl;

ketone or aldehyde,

F

x ₂ '·

F

F F

2-(((trifluoromethyl)sulfonyl)oxy)acetyl;

N-]MeOjS-^. 2-(pentafluorophenoxy)acetyl;

lstep ° ^x g' ash (ODA); ' acid anhydride, 'methylsulfonephenyloxadialkyloxyamino; ³ azidos,

alkynyl or ^H 2 ^NH hydrazide, where X1' represents F, Cl, Br, I or Lv ₃ ; X ₂ ' represents O, NH, N(R1), or CH ₂ ; R ₃ independently represents H, an aromatic, heteroaromatic or aromatic group, where one or more H atoms are independently replaced by -R ₁ , -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R ₁ , -S(O) ₂ R ₁ , or -COOR1; Lv ₃ represents a leaving group selected from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, triflate; imidazole; dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5phenylisoxazolium-3'-sulfonate, simple and mixed anhydrides, for example acetic anhydride, formyl anhydride; or an intermediate obtained using a condensation reagent for peptide coupling reactions or for Mitsunobu reactions;

R1 and R2 are independently selected from H, C1-C8 alkyl, _C2 - _C8 alkenyl, heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl or C ₂ -C ₈ esters, ether or amide; or peptides containing 1-8 amino acids; or a polyethyleneoxy group having the formula (OCH ₂ CH ₂ )p or (OCH ₂ CH(CH ₃ )) _p , where p is an integer from 0 to about 5000, or a combination of

- 19 044827 of the above groups.

In addition, functional groups X or Y that mediate drug or cytotoxic agent binding preferably include groups that mediate binding via a disulfide, thioether, thioester, peptide, hydrazone, ester, carbamate, carbonate, alkoxime, or amide bond. Such functional groups include, but are not limited to, thiol, disulfide, amino, carboxyl, aldehydes, ketone, maleamido, haloacetyl, hydrazines, alkoxyamino and/or hydroxy;

Preferably, the bis-coupling conjugate is further represented by the formulas (I-a), (II-b), (II-c), (II-d), (II-e), (II-f), (II-g), (II-h), (II-i), (II-j), (II-k), (II-m), (II-n), (II-o), (II-q), (II -r), (II-s), (II-t), (II-u), (II-v), (II-w), (II-x), (II-y), (II-z ), (II-a1), (II-a2), (P-a3) and (P-a4):

(Π-b),

(Π-h),

- 20 044827

agent

- 21 044827

where X ₇ and Y7 independently represent CH, CH2, NH, O, S, NHNH, N(R1) and N; X, Y, R1, n, -----, L1 and L2 are as described above; a chemical bond between two atoms means that it can bond to any of the adjacent two atoms; R1, X, Y, n, L1, L2, Lv1 and Lv2 are as described above. Preferably, Lv1 and Lv2 are independently selected from Cl, Br, I, methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate and nitrophenoxyl. In another aspect, this invention provides a reactive bis-linker conjugated to a cell-binding agent/molecule of formula (III) below, wherein two or more functional groups of the cytotoxic molecule can contact it simultaneously or sequentially to form formula (I):

- 22 044827 where m _b n, , cell binding agent/molecule, L1, L2, Z1, and Z2 are as defined>7

Molecule/Agent,

Z ₂ —

Jn cell binding (III) is divided in formula (I);

X' and Y' represent a functional group that can independently or sequentially contact groups of cytotoxic drug residues to form X and Y, respectively, X and Y being as defined in formula (I);

X' and Y' are preferably independently disulfide substituent, maleimido, haloacetyl, alkoxyamine, azido, ketone, aldehyde, hydrazine, amino, hydroxyl, carboxylate, imidazole, thiol or alkyne; or N-hydroxysuccinimide ester, para-nitrophenyl ester, dinitrophenyl ester, pentafluorophenyl ester, pentachlorophenyl ester; tetrafluorophenyl ester; difluorophenyl ester; monofluorophenyl ester; or pentachlorophenyl ester, dichlorophenyl ester, tetrachlorophenyl ester or 1-hydroxybenzotriazole ester; triflate, mesylate or tosylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate; pyridyl disulfide or nitropyridyl disulfide; maleimide, haloacetate, acetylenedicarboxyl group or carboxylic acid halogenate (fluoride, chloride, bromide or iodide). Preferably X and Y have one of the following structures:

N-hydroxysuccinimide ester;

maleimide; ^R s

haloacetyl;

acyl halide (acid halide);

acrylic (acryloyl);

2-(tosyloxy)acetyl;

(mesyloxy)acetyl;

(dinitrophenoxy)acetyl;

(difluorophenoxy)acetyl;

and F

Disulfide;

ethenesulfonyl;

2-(nitrophenoxy)acetyl;

2-(fluorophenoxy)acetyl;

222ke2-(((trifluoromethyl)sulfonyl)oxy)acetyl;

F tone or aldehyde, ^{1 1}

NN MeO ₂ S2-(pentafluorophenoxy)acetyl;

fonphenyloxadiazole (ODA);

η ₂ ν· acid anhydride, 'methylsulalkyloxyamino; ^J azido,

alkynyl or

hydrazide, where X1' represents F, Cl, Br,

I or Lv ₃ ;

X ₂ ' represents O, NH, N(R ₁ ), or CH ₂ ; _R3 and R5 are H, R1, aromatic, heteroaromatic, or an aromatic group where one or more H atoms are independently replaced by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1 , -S(O)2R1, or -COOR1; Lv ₃ represents a leaving group selected from methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethanesulfonate, nitrophenoxyl; N-hydroxysuccinimidyl (NHS); phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazol-1-yl; chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazolyl)oxyl, 2-ethyl-5-phenylisoxazolyl, phenyloxadiazolyl (ODA), oxadiazolyl, or an intermediate obtained by the condensation reagent for Mitsunobu reactions, where R1 and R2 are as defined higher;

preferably, the bis-linker for obtaining the conjugate is additionally represented by the formulas (IIIa), (III-b), (III-c), (III-d), (III-e), (III-f), (III-g), (Ш-h), (Ш-i), (Ш-j), (III-k), (III-l), (III-m), (III-n), (III-o), (III -p),

- 23 044827 (III-r), (III-s), (III-t), (III-u), (III-v) and (III-w) below:

Cell-binding molecule -----«

Molecule binding to cell binding to cell

Cell-binding molecule (Ш-1)

Cell-binding molecule (C-t)

Cell binding molecule

Cell-binding molecule (Ш-п) (Ш-о)

- 24 044827

where X ₇ and Y ₇ independently represent CH, CH2, NH, O, S, NHNH, N(R ₁ ), and N; a chemical bond between two atoms means that it can bond to any of the adjacent two atoms; R1, X', Y', n, L1 and L2 are as described above.

In another aspect, the present invention provides a reactive bis-linker of formula (IV) below, in which a cytotoxic molecule and a cell binding molecule can independently, simultaneously or sequentially contact it to form formula (I)

where -----, m1, L1, L2, Z1 and Z2 are as defined in formula (I); Lv1 and Lv2 are as defined in formula (II), and X' and Y' are as defined in formula (III);

preferably, the bis-linker to obtain the conjugate is further represented by the formulas (IVa), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), (IV-g), (IV-h), (IV-i), (IV-j), (IV-k), (IV-m), (IV-n), (IV-o), (IV-p), (IVq ), (IV-r) and (IV-s)

- 25 044827

- 26 044827

o (IV-a2)

where X ₇ and Y7 independently represent CH, CH2, NH, O, S, NHNH, N(R ₁ ) and N; a chemical bond between two atoms means that it can bond any of the adjacent two atoms;

-----, R1, X', Y', n, L1 and L2 are as described above.

Examples of functional groups X' or Y' that provide interaction with the terminal amino or hydroxyl group of the drug/cytotoxic agent include, but are not limited to, N-hydroxysuccinimide esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters esters, carboxylic acid chlorides or carboxylic acid anhydrides; the terminal thiol of the cytotoxic agent may include, but is not limited to, pyridyl disulfides, nitropyridyl disulfides, maleimides, haloacetates, methylsulfonephenyloxadiazole (ODA), carboxylic acid chlorides and carboxylic acid anhydride; the terminal ketone or aldehyde may be, but is not limited to, amines, alkoxyamines, hydrazines, acyloxyamines or hydrazide; azide-terminated may be, but is not limited to, an alkyne.

Preparation of conjugates.

Conjugates of formula (I) can be prepared via intermediates of formulas (II), (III) or (IV), respectively. Some examples of formula (II) are shown structurally in FIG. 1-40. To synthesize a conjugate of formula (I), typically two functional groups on the drug or cytotoxic molecule are first brought into contact sequentially or simultaneously with the X' group and Y' group of the linker of formula (IV) in a chemical solvent or aqueous medium containing 0 ,1-99.5% organic solvents, or in 100% aqueous medium to form a compound of formula (II). The compound of formula (II) may then optionally be first isolated or may be immediately or simultaneously or sequentially brought into contact with two or more residues of the cell-binding molecule, preferably a pair of free thiols resulting from the reduction of the disulfide bonds of the cell-binding molecule , at 0-60°C, pH 5~9, in aqueous medium with or without adding 0~30% water-miscible (miscible) organic solvents, such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol or ethylene diol to form a conjugate of formula (I).

Alternatively, conjugates of formula (I) can also be prepared by first reacting linkers of formula (IV) with two or more residues of a cell-binding molecule, preferably with a pair of free thiols resulting from the reduction of disulfide bonds of the cell-binding molecule at 0 -60°C, pH 5-9, in an aqueous medium with or without the addition of 0~30% water-miscible (miscible) organic solvents to form a modified cell-binding molecule of formula (III). Thiol pairs are preferred disulfide bond pairs reduced from the disulfide bonds of the internal chain of a cell-binding molecule by a reducing agent which may be selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2- carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and/or beta-mercaptoethanol (β-ME, 2-ME) at pH 4~9 in an aqueous medium with or without the addition of 0~30% water miscible (miscible) organic solvents. Reactive groups X' and Y' of formula (III), which may be independently disulfide, thiol, thioester, maleimido, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, triflate, carbonylimidazole, tosylate, mesylate, 2-ethyl -5-phenylisoxazolium-3'sulfonate or esters of carboxylic acids and nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluoro

- 27 044827 phenol, pentachlorophenol, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, anhydrides or hydrazides, or other acid ester derivatives, which can then be brought into contact with two drug/cytotoxic agent groups, simultaneously or sequentially at 0-60°C , pH 4~9.5 in an aqueous medium with or without the addition of 0~30% water-miscible (miscible) organic solvents to obtain the conjugate of formula (I) after column purification or dialysis. The reactive groups of the drug/cytotoxic agent are brought into contact with the modified cell-binding molecule of formula (III) respectively in various ways. For example, the disulfide bond-containing bond in cell-binding agent-drug conjugates of formula (I) is achieved by disulfide exchange between the disulfide bond in the modified cell-binding agent of formula (III) and the drug containing a free thiol group; the thioether bond-containing linkage in cell-binding agent-drug conjugates of formula (I) is obtained by reacting a modified maleimido or haloacetyl or ethylsulfonyl cell-binding agent of formula (III) and a drug containing a free thiol group; the bond containing the acid labile hydrazone bond in the conjugates can be prepared by reacting the carbonyl group of the drug or compound of formula (III) with the hydrazide moiety of the compound of formula (III) or drug, respectively, by methods known in the art (see, for example, P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32: 548-55, 1959; P. Trail et al., Cancer Res. , 57; 100-5, 1997); the triazole linkage in the conjugates can be obtained by reacting the 1-yne group of the drug or compound of formula (III) with the azide moiety of another analogue, respectively, by click chemistry (Husgen cycloaddition) (Lutz, J.-F. et al ., 2008, Adv. Drug Del. Rev. 60, 958-70; Sletten, E. M. et al., 2011, AccChem. Research 44, 666-76). The bond containing the oxime bond in cell-binding agent-drug conjugates linked through the oxime is obtained by reacting a keto or aldehyde group in a modified cell-binding agent of formula (III) or drug with an oxyamine group in the drug or modified agent, cell-binding, formula (III), respectively. The thiol-containing drug can react with a modified cell-binding molecule linker of formula (III) containing a maleimido or haloacetyl or ethylsulfonyl substituent at pH 5.5-9.0 in an aqueous buffer to form a thioether bond in the conjugate binding agent with a cell-drug of formula (I). The thiol-containing drug can undergo a disulfide exchange with a modified linker of formula (III) containing a pyridyl dithio moiety to form a conjugate containing a disulfide bond. A drug containing a hydroxyl group or a thiol group can be reacted with a modified bridging linker of formula (III) containing a halogen, in particular an alpha-halocarboxylate, in the presence of a mild base, for example pH 8.0-9.5, to obtain a modified drug a product containing an ether or thioether linkage. The hydroxyl group in the drug can be condensed with a cross-linker of formula (IV) bearing a carboxyl group in the presence of a dehydrating agent such as EDC or DCC to form an ester linkage, and then the desired drug-modified bridging linker of formula (III) is conjugated to a molecule that binds to a cell. A drug containing an amino group can condense with the carboxyl ester group of NHS, imidazole, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflata; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate molecule binding to the cell linker of formula (III) to form a conjugate through an amide bond.

The synthetic conjugate can be purified by standard biochemical methods, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography and ion exchange or dialysis. In some cases, a small molecule as a cell binding agent (eg, folic acid, melanocyte stimulating hormone, EGF, etc.) conjugated to small molecule drugs can be purified by chromatography such as HPLC, medium pressure column chromatography or ion exchange chromatography.

To achieve higher yields in the conjugation reaction of the cytotoxic molecule-bis-linker complex of formula (II) with the free thiol pair of a cell-binding molecule, preferably an antibody, it may be necessary to add a small percentage of water-miscible organic solvents or phase transfer agents to the reaction mixture. The crosslinking reagent (linker) of formula (II) can first be dissolved in a polar organic solvent that is miscible with water, for example, various alcohols such as methanol, ethanol and propanol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4- dioxane, dimethylformamide (DMF), dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) in high concentration, for example 1-500 mM. At the same time, a cell-binding molecule, such as an antibody, dissolved in an aqueous buffer with a pH of 4- 28 044827

9.5, preferably pH 6-8.5, at a concentration of 1-50 mg/ml, treat with 0.5-20 equivalents of TCEP or DTT for 20 minutes to 48 hours. After reduction, DTT can be removed by SEC chromatographic purification. TCEP may also optionally be removed by SEC chromatography or may remain in the reaction mixture until the next reaction step without further purification. In addition, reconstitution of antibodies or other cell-binding agents by TCEP can be accomplished in conjunction with an existing drug-linker molecule of formula (II), for which cross-conjugation of cell-binding molecules can be achieved simultaneously with reconstitution of TCEP.

Aqueous solutions for modifying the cell binding molecule are buffered between pH 4 and 9, preferably between 6.0 and 7.5, and may contain any non-nucleophilic buffer salts suitable for these pH ranges. Typical buffers include phosphate, acetate, triethanolamine HCl, HEPES and MOPS buffers, which may contain additional components such as cyclodextrins, hydroxypropyl-β-cyclodextrin, polyethylene glycols, sucrose and salts such as NaCl and KCl. After adding the linker drug of formula (II) to the solution containing the reduced cell-binding molecules, the reaction mixture is incubated at a temperature of 4 to 45°C, preferably at 15°C ambient temperature. The progress of the reaction can be monitored by measuring a decrease in absorbance at a specific UV wavelength, such as 254 nm, or an increase in absorbance at a specific UV wavelength, such as 280 nm, or other suitable wavelength. After completion of the reaction, isolation of the modified cell-binding agent can be carried out in the usual manner, using, for example, gel filtration chromatography, ion exchange chromatography, adsorption chromatography or silica gel or alumina column chromatography, crystallization, preparative thin layer chromatography, ion exchange chromatography or HPLC.

The degree of modification can be assessed by measuring the absorbance of the released nitropyridinedithione, dinitropyridinedithione, pyridinethione, carboxylamidopyridinedithione and dicarboxylamidopyridinedithione groups using UV spectra. For conjugation without a chromophore group, the modification or conjugation reaction can be monitored using LCMS, preferably UHPLC-QTOF mass spectrometry (QTOF) or capillary electrophoresis-mass spectrometry (CE-MS). The bridging cross-linkers described herein have a variety of functional groups that can react with any drugs, preferably cytotoxic agents, that have a suitable substituent. For example, modified cell binding molecules bearing an amino or hydroxyl substituent can be contacted with drugs containing N-hydroxysuccinimide ester (NHS), modified cell binding molecules bearing a thiol substituent can be contacted with drugs containing a maleimide or haloacetyl group. In addition, modified cell-binding molecules containing a carbonyl (ketone or aldehyde) substituent can react with drugs containing hydrazide or alkoxyamine. One skilled in the art can readily determine which linker to use based on the known reactivity of the available functional group of the linker.

Cell binding agents.

The cell binding molecule, Cb, which contains the conjugates and modified cell binding agents of the present invention may be of any type now known or to become known, a molecule that binds, complexes or reacts with some part a population of cells that needs to be therapeutically or otherwise biologically modified.

Cell binding agents include, but are not limited to, high molecular weight proteins such as, for example, antibody, antibody-like protein, full length antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (eg , bispecific antibodies, trispecific antibodies or tetraspecific antibodies); single chain antibodies; antibody fragments such as Fab, Fab', F(ab')2, _Fv , [Parham, J. Immunol. 131, 2895-902 (1983)] , fragments derived from a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs, diabody, tribody, tetrabody, mini-antibody, pro-antibody, pro-antibody fragment, small immune proteins (SIPs), and epitope-binding fragments of any of the above that are immuno- specifically bind to cancer cell antigens, viral antigens, microbial antigens, or a protein generated by the immune system that is capable of recognizing, binding to a specific antigen, or exhibiting a desired biological activity (Miller et al. (2003) J. of Immunology 170: 4854-61); interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, interferon-gamma (IFN-γ); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte stimulating hormone), steroid hormones such as androgens and estrogens, melanocyte stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF) such as TGFa, TGFe, insulin and insulin-like growth factors (IGF-I, IGF-II ) G-CSF, M-CSF and GM-CSF [Burgess, Immunology Today, 5, 155-8 (1984)]; vaccinia growth factors (VGF); fibroblast growth factors (FGF); lower molecular weight proteins, polypeptides, peptides and peptide hormones such as bombesin, gastrin, gastrin-releasing peptide; platelet-derived growth factors; interleukin and cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factors, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folate; apoproteins and glycoproteins such as transferrin [O'Keefe et al., 260 J. Biol. Chem. 932-7 (1985)]; sugar binding proteins or lipoproteins such as lectins; molecules that transport nutrients into the cell; and small molecule inhibitors such as prostate-specific membrane antigen (PSMA) inhibitors and small molecule tyrosine kinase inhibitors (TKIs), non-peptides or any other cell-binding molecules or substances such as bioactive polymers (Dhar, et al., Proc. Natl. Acad. Sci. 2008, 105, 17356-61); bioactive dendrimers (Lee, et al., Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al., Proc. Natl. Acad. Sci. 2009, 106, 685-90); nanoparticles (Liong, et al., ACS Nano, 2008, 2, 1309-12; Medarova, et al., Nat. Med. 2007, 13, 372-7; Javier, et al., Bioconjugate Chem. 2008, 19, 1309-12); liposomes (Medinai, et al., Curr. Phar. Des. 2004, 10, 2981-9); viral capsids (Flenniken, et al., Viruses Nanotechnol. 2009, 327, 71-93).

Generally, a monoclonal antibody is preferred as the cell surface receptor binding agent if a suitable monoclonal antibody is available. And the antibody may be murine, human, humanized, chimeric, or derived from other species.

The production of antibodies used in the present invention involves in vivo or in vitro procedures, or combinations thereof. Methods for producing polyclonal antireceptor peptide antibodies are well known in the art, for example in US patent No. 4493795 (Nestor et al.). A monoclonal antibody is typically produced by fusing myeloma cells with spleen cells from a mouse that has been immunized with the desired antigen (Kohler, G.; Milstein, S. (1975). Nature 256: 495-7). Detailed procedures are described in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988), which is incorporated herein by reference. In particular, monoclonal antibodies are produced by immunizing mice, rats, hamsters or any other mammal with an antigen of interest, such as an intact target cell, antigens isolated from a target cell, whole virus, attenuated whole virus and viral proteins. Splenocytes are typically fused to myeloma cells using polyethylene glycol (PEG) 6000. The fusions are selected for their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas producing a monoclonal antibody suitable for use in the practice of this invention are identified by their ability to immunoreactively react with the specified receptors or inhibit the activity of the receptor on target cells.

The monoclonal antibody used in the present invention can be produced by initiating a monoclonal hybridoma culture containing a culture medium containing the hybridoma that secretes antibody molecules of appropriate antigen specificity. The culture is maintained under conditions and for a period of time sufficient to allow the hybridoma to secrete antibody molecules into the medium. The medium containing the antibody is then collected. Antibody molecules can then be isolated by well known methods such as Protein A affinity chromatography; anionic, cationic, hydrophobic or size exclusion chromatography (in particular, by affinity for a specific antigen after protein A and column chromatography that separates molecules by size); centrifugation, differential solubility, or any other standard protein purification technique.

Media suitable for the preparation of these compositions are well known in the art and are commercially available and include synthetic culture media. A typical synthetic medium is Dulbecco's minimum essential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959)) supplemented with 4.5 g/L glucose, 0-20 mM glutamine, 0-20% fetal calf serum, several m.ch. heavy metals such as Cu, Mn, Fe or Zn, etc., and/or other heavy metals added in the form of their salts and with an antifoaming agent such as polyoxyethylene-polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be generated using methods other than fusion, such as direct transformation of B cells with oncogenic DNA or transfection with an oncovirus such as Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4) )) or Kaposi's sarcoma-associated herpesvirus (KSHV). See, US patents No. 4341761; 4399121; 4427783; 4444887; 4451570; 4466917; 4472500; 4491632; 4493890. A monoclonal antibody can also be produced through an antireceptor peptide or peptides containing a carboxyl terminus, which is well known in the art. See Niman et al., Proc. Natl. Acad. Sci.

- 30 044827

USA, 80: 4949-53 (1983); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-82 (1985); Lei et al. Biochemistry

34(20): 6675-88, (1995). Typically, an antireceptor peptide or peptide analog is used, either alone or as a conjugate with an immunogenic carrier, as an immunogen to produce antireceptor peptide monoclonal antibodies.

There are also a number of other well known methods for producing monoclonal antibodies as binding molecules in this invention. Methods for producing fully human antibodies are particularly useful. One method is phage display technology, which can be used to select a range of human antibodies that specifically bind to an antigen using affinity enrichment methods. Phage display has been described in detail in the literature, and the construction and screening of phage display libraries are well known in the art, see, for example, Dente et al., Gene. 148(1):7-13 (1994); Little et al., Biotechnol Adv. 12(3): 539-55 (1994); Clackson et al., Nature 352: 264-8 (1991); Huse et al., Science 246: 1275-81 (1989).

Monoclonal antibodies produced by hybridoma from a non-human species, such as a mouse, can be humanized to avoid the production of anti-mouse human antibodies when introduced into the human body. Among the most common methods for humanizing antibodies are complementarity-determining region grafting and retransplantation. These methods have been described in detail, see, for example, US patent No. 5859205 and 6797492; Liu et al., Immunol Rev. 222:9-27 (2008); Almagro et al., Front Biosci. 13: 1619-33 (2008); Lazar et al., Mol Immunol. 44(8): 1986-98 (2007); Li et al., Proc. Natl. Acad. Sci. USA. 103(10): 3557-62 (2006), each of which is incorporated herein by reference. Fully human antibodies can also be produced by immunizing transgenic mice, rabbits, monkeys, or other mammals with an immunogen that carry large portions of the heavy and light chains of human immunoglobulin. Examples of such mice are: Xenomouse. (Abgenix/Amgen) 5569825. In human therapy, variable mouse and human constant regions can also be combined to create so-called chimeric antibodies, which are significantly less immunogenic in humans than mouse mAbs (Kipriyanov et al., Mol Biotechnol. 26: 39-60 (2004); Houdebine, Curr Opin Biotechnol 13: 625-9 (2002), each of which is incorporated herein by reference). In addition, site-directed mutagenesis in the variable region of an antibody can result in an antibody with higher affinity and specificity for its antigen (Brannigan et al., Nat Rev Mol Cell Biol. 3: 964-70, (2002)); Adams et al., J Immunol Methods. 231: 249-60 (1999)) and exchange of constant regions of the mAb can improve its ability to mediate effector binding functions and cytotoxicity.

Antibodies that are immunospecific for a cancer cell antigen can also be produced commercially or produced by any method known to one skilled in the art, such as, for example, chemical synthesis or recombinant expression methods. The nucleotide sequence encoding antibodies immunospecific to a cancer cell antigen can be obtained commercially, for example from GenBank or a similar database, literature publications, or by conventional cloning and sequencing.

In addition to an antibody, a peptide or protein that binds/blocks/targets or otherwise interacts with epitopes or corresponding receptors on a target cell can be used as a binding molecule. These peptides or proteins can be any random peptide or proteins that have affinity for epitopes or corresponding receptors, and they do not necessarily belong to the immunoglobulin family. These peptides can be isolated using the same technique used for antibodies in phage display (Szardenings, J Recept Signal Transduct Res. 2003, 23(4): 307-49). The use of peptides from such random peptide libraries can be similar to antibodies and antibody fragments. Peptide or protein binding molecules can be conjugated or linked to large molecules or materials, such as, but not limited to, albumin, polymer, liposome, nanoparticle, dendrimer, provided that such attachment allows the peptide or protein to retain its antigen-binding specificity.

Examples of antibodies used to conjugate drugs through linkers of the present invention for the treatment of cancer, autoimmune disease and/or infectious disease include, but are not limited to, 3F8 (anti-GD2), abagovomab (anti-CA-125), abciximab (anti-CD41 (integrin alpha-IIb), adalimumab (anti-TNF-a), adekatumumab (anti-EpCAM, CD326), afelimomab (anti-TNF-a); afutuzumab (anti-CD20), alazizumab pegol (anti-VEGFR2), ALD518 (anti-[L-6), alemtuzumab (capmas, mabcampas, anti-CD52), altumomab (anti-CEA), anatumomab (anti-TAG-72), anrukinzumab (IMA-638, anti-[L-13), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA), aselizumab (anti-G-selectin (CD62L), atlizumab (tocilizumab, actemra, roactemra, anti-[L-6 receptor), atorolimumab (anti-Rh factor ), bapineuzumab (anti-amyloid beta), basiliximab (simulect, anti-CD25 (IL-2 receptor α chain)), bavituximab (anti-phosphatidylserine), bektumomab (lymphoscan, anti-CD22), belimu- 31 044827 mab (benlysta , lymphostat-B, anti-BAFF), benralizumab (aHTu-CD125), bertilimumab (anti-CCLll (eotaxin-1)), besilesomab (scintimune, anti-CEA-related antigen), bevacizumab (Avastin, anti-VEGFA), biciromab (fibrisint, anti-fibrin II beta chain), bivatuzumab (anti-CD44 v6), blinatumomab (BiTe anti-CD19), brentuximab (cAC10, anti-CD30 TNFRSF8), briakinumab (anti-[L-12, IL- 23) canakinumab (ilaris, anti-[L-1), cantuzumab (C242, anti-CanAg), capromab, catumaxomab (removab, anti-ErCAM, anti-CD3), CC49 (anti-TAG-72), cedelizumab (anti -CD4), certolizumab pegol (cimzia anti-TNF-a), cetuximab (erbitux, IMC-C225, anti-EGFR), cituzumab bogatox (anti-ErCAM), cixutuzumab (anti-IGF-I), clenoliximab (anti-CD4 ), clivatuzumab (anti-MUC1), conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), dacetuzumab (anti-CD40), daclizumab (Zenapax, anti-CD25 (IL-2 receptor achain)), daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase), denosumab (prolia, anti-RANKL), detumomab (anti-B cell lymphoma), dorlimomab, dorlixizumab, ecromeximab (anti-GD3 ganglioside), eculizumab (soliris, anti- C5), edobacomab (anti-endotoxin), edrecolomab (ranorex, MAb17-1A, anti-EpCAM), efalizumab (raptiva, anti-LFA-1 (CDlla)), efungumab (mycograb, anti-Hsp90), elotuzumab (anti-SLAMFV) , elsilimomab (anti-[L-6), enlimomab pegol (anti-ICAM-l (CD54)), epitumomab (anti-episialin), epratuzumab (anti-CD22), erlizumab (anti-[TGB2 (CD18)), ertumaxomab (rexomune, anti-HER2/neu, CD3), etaracizumab (abegrin, anti-integrin α _¥ β ₃ ), exbivirumab (hepatitis B surface antigen), fanolesomab (neutrospec, anti-CD15), faralimomab (anti-interferon receptor), farletuzumab (antifolate receptor 1), felvizumab (anti-respiratory syncytial virus), fezakinumab (anti-IL-22), figitumumab (anti-IGF-1 receptor), fonolizumab (anti-IFN-γ), foravirumab (anti-rabies virus glycoprotein), frezolimumab (anti-TGF- e), galiximab (anti-CD80), gantenumab (anti-amyloid beta), gavilimomab (anti-CD147 (basigin)), gemtuzumab (anti-CD33), girentuximab (anti-carbonic anhydrase 9), glembatumumab (CR 011, anti-GPNMB) , golimumab (simponi, anti-TNF-a), gomiliximab (anti-CD23 (IgE receptor)), ibalizumab (anti-CD4), ibritumomab (anti-CD20), igovomab (indimacis-125, anti-CA-125) , imciromab (mayosucint, anticardiac myosin), infliximab (remicade, anti-TNF-a), intetumumab (anti-CD51), inolimomab (anti-CD25 (IL-2 receptor a chain)), inotuzumab (anti-CD22), ipilimumab ( anti-CD152), iratumumab (anti-CD30 (TNFRSF8)), keliximab (anti-CD4), labetuzumab (CEA-seed, anti-CEA), lebrikizumab (anti-IL-13), lemalesomab (anti-NCA-90 ( granulocyte antigen)), lerdelimumab (anti-TGF beta 2), lexatumumab (anti-TRA[L-R2), libivirumab (hepatitis B surface antigen), lintuzumab (anti-CD33), lucatumumab (anti-CD40), lumiximab (anti -CD23 (IgE receptor), mapatumumab (anti-TRAIL-Rl), maslimomab (anti-T cell receptor), matuzumab (anti-EGFR), mepolizumab (Bosatria, anti-GE-5), metelimumab (anti-TGF beta l), milatuzumab (anti-CD74), minretumomab (anti-TAG-72), mitumomab (BEC-2, anti-GD3 ganglioside), morolimumab (anti-Rh factor), motavizumab (Numax, anti-respiratory syncytial virus ), muromonab-CD3 (orthoclone OKT3, anti-CD3), nacolomab (anti-C242), naptumomab (anti-5T4), natalizumab (tysabri, anti-integrin α ₄ ), nebacumab (anti-endotoxin), necitumumab (anti-EGFR) , nerelimomab (anti-TNF-a), nimotuzumab (teracim, teraloc, anti-EGFR), nofetumomab, ocrelizumab (anti-CD20), odulimomab (afolimomab, anti-LFA-l (CDlla)), ofatumumab (arzera, anti-CD20) , olaratumab (anti-PDGF-Ra), omalizumab (xolair, anti-IgE Fc region), oportuzumab (anti-EpCAM), oregovomab (ovarex, anti-CA-125), otelixizumab (anti-CD3), pagibaximab (antilipoteichoic acid), palivizumab (Synagis, Abbosynajis, anti-respiratory syncytial virus), panitumumab (Vectibix, ABX-EGF, anti-EGFR), panobakumab (anti-Pseudomonas aeruginosa), pascolizumab (anti-IL-4), pemtumomab (Teragin, anti-MUCl ), pertuzumab (omnitarg, 2C4, anti-HER2/neu), pexelizumab (anti-C5), pintumomab (anti-adenocarcinoma antigen), priliximab (anti-CD4), pritumumab (anti-vimentin), PRO 140 (anti-CCR5 ), racotumomab (1E10, anti-(I-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), rafivirumab (anti-rabies glycoprotein), ramukirumab (anti-VEGFR2), ranibizumab (Lucentis, anti-VEGF-A), raxibacumab (anti-anthrax toxin, protective antigen), regavirumab (anti-cytomegalovirus glycoprotein B), reslizumab (anti-IL-5), rilotumumab (anti-HGF), rituximab (mabthera, rituxanmab, anti-CD20), robatumumab (anti-CD20), -IGF-l receptor), rontalizumab (anti-IFN-a), rovelizumab (leucarest, anti-CDll, CD18), ruplizumab (antova, anti-CD154 (CD40L)), satumomab (anti-TAG-72), sevirumab ( anti-cytomegalovirus), sibrotuzumab (anti-FAP), sifalimumab (anti-IFN-a), siltuximab (anti-GE-6), siplizumab (anti-CD2)), (smart) MI95 (anti-CD33), solanezumab ( anti-beta-amyloid), sonepcizumab (anti-sphingosine-1-phosphate), sontuzumab (anti-episialin), stamulumab (anti-myostatin), sulesomab (leukoscan, (anti-NCA-90 (granulocyte antigen), takatuzumab (anti-myostatin), alpha-fetoprotein), tadocizumab (anti-integrin a _IIb e ₃ ), talizumab (anti-IgE), tanezumab (anti-NGF), taplitumomab (anti-CD19), tefibazumab (aurexis, (anti-agglutinating factor A), telimomab, tenatumomab (anti-tenascin C), teneliximab (anti-CO40), teplizumab (anti-CD3), TGN1412 (anti-CD28), ticilimumab (tremelimumab, (anti-CTLA-4)), tigatuzumab (anti-TRAIL-R2) , TNX-650 (anti-IL-13), tocilizumab (atlizumab, actemra, roactemra, (anti-IL-6 receptor)), toralizumab (anti-CD154 (CD40L)), tositumomab (anti-CD20), trastuzumab (Herceptin , (anti-HER2/neu), tremelimumab (anti-CTLA-4), tucotuzumab celmoleukin (anti-EpCAM), tuvirumab (anti-hepatitis B virus), urtoxazumab (anti-Escherichia coli), ustekinumab (stelara, anti-IL-12, IL -23), vapalik

- 32 044827 simab (anti-AOSD (VAP-1)), vedolizumab ((anti-integrin α ₄ β ₇ ), veltuzumab (aHTu-CD20), vepalimomab (anti-AOSD (VAP-1)), visilizumab (Nuvion, anti- CD3), Vitaxin (anti-vascular integrin avb3), volociximab (anti-integrin α ₅ β1, votumumab (humaspect, antitumor antigen CTAA16.88), zalutumumab (humax-EGFr, (anti-EGFR), zanolimumab (humax-CD4, anti-CD4) , ziramumab (anti-CD147 (Bazigin)), zolimomab (anti-CD5), etanercept (Enbrel®), alefacept (Ameviv®), abatacept (Orencia®), rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron -regulatory protein 2)], 14G2a (anti-GD2 ganglioside, from the National Cancer Institute for Melanoma and Solid Tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancer), 225.28S [anti-HMW-MAA (high molecular weight melanoma associated antigen), Sorin Radiofarmaci SRL (Milan, Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from the US National Cancer Institute for colorectal and gastric cancer), CYT-356 (Oncoltad®, for prostate cancer), HNK20 (Ora Vax Inc. for respiratory syncytial virus), immuraitis (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharma for oncology), MAK195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2 ), from Abbott/Knoll for septic toxic shock], MEDI-500 [T10B9, anti-CD3, TRae (T cell receptor alpha/beta), complex, from MedImmune Inc. for graft-versus-host disease, RING SCAN [anti-TAG 72 (tumor-associated glycoprotein 72), from Neoprob Corp. for breast, colon and rectal cancer], avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTDl (tumor-associated calcium signal transducer 1), anti-GA733-2 (gastric tumor-associated protein -intestinal tract 2), anti-EGP-2 (epithelial glycoprotein 2); KSA; KS 1/4 antigen; M4S; tumor antigen 17-1A; CD326 from NeoRix Corp. for colon, ovarian, prostate and NHL cancer ]; lymphocide (Immunomedics, New Jersey), smart ID10 (Protein Design Labs), oncolym (Tekniclon Inc., California), allomune (BioTransplant, California), anti-VEGF (Genentech, California); CEAcide (Immunomedics, New Jersey), IMC -1C11 (ImClone, New Jersey) and cetuximab (ImClone, New Jersey).

Other antibodies as cell binding molecules/ligands include, but are not limited to, antibodies to the following antigens: aminopeptidase N (CD13), Annex A1, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15 -3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha-fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostate -specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung cancer, breast cancer , ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases ), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma and liquid tumor, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumors), CD227 ( breast cancer, ovarian cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast, colorectal and lung cancer), DLL3 (delta-like-3), DLL4 (delta-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hemo-oncology, solid tumors), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, prostate, NHL and ovarian cancers), ERBB2 (epidermal growth factor receptor 2 ; lung, breast, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), ganglioside GD2 (cancer), G-28 (cell surface glycolipid antigen, melanoma), idiotype GD3 (cancer), proteins heat shock (cancer), HER1 (lung cancer, stomach cancer), HER2 (breast, lung and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma ), IGF1R (insulin-like growth factor receptor 1, solid tumors, blood cancer), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman disease, IL6-dependent tumors), integrins (α _¥ β ₃ , α ₅ β1, α ₆ β ₄ , αιιβ ₃ , α ₅ β ₅ , α _γ β ₅ , for various types of cancer), MAGE-1 (carcinomas), MAGE- 2 (carcinomas), MAGE3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domain member 1 subfamily A, small cell lung cancer, NHL), nucleolin , oncogenic product Neu (carcinomas), P21 (carcinomas), paratope anti-(N-glycolylneuraminic acid, breast cancer, melanoma), testicular PLAP-like alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumors), PSA (prostate cancer), ROBO4, TAG 72 (tumor associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), Tcellular transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (member of the receptor super family

- ZZ 044827 tumor necrosis factor 10B, cancer), TNFRSF13B (tumor necrosis factor 13B receptor super family member, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various types of cancer). Several other tumor-associated antigens recognized by antibodies have been reviewed (Gerber, et al., mAbs 1:3, 247-53 (2009); Novellino et al., Cancer Immunol Immunother. 54(3), 187-207 (2005). Franke, et al., Cancer Biother Radiopharm. 2000, 15, 459-76).

Cell binding agents, more preferably antibodies, can be any agent that is capable of binding to tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells or melanocytes. More specifically, cell binding agents can be any agents/molecules capable of binding to any of the following antigens or receptors:

CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8,

CD8a, CD8b, CD9, CD10, CDlla, CDllb, CDllc, CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55,CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CDwll3, CD114, CD115, CD116, CD117, CD118, CD119, CDwll9, CD120a, CD120b, CD121a, CD121b, CDwl21b, CD122, CD123, CDwl23, CD124, CD125, CDwl25, CD126, CD127, CD128, CDwl28, CD129, CD1 30, CD131, CDwl31, CD132, CD133, CD134, CD135, CD136, CDwl36, CD137, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CDwl45, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CDwl56c, CD157,

- 34 044827

CD158a, CD158b, CD159a, CD159b, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD1 73, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CDwl86, CD187, CD188, CD189, CD190, Cdl91, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CDwl98, CD199, CDwl99, CD200, CD200a, CD200b, CD201, CD202, CD202b, CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210, CD212, CD213al, CD213a2, CDw21 7, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235ab, CD235b, CD236, CD236R, CD238, CD239, CD240, CD24 0CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271, CD2 73, CD274, CD275, CD276 (B7-H3), CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD30 0e , CD301, CD302, CD3O3, CD304, CD3O5, CD306, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD 331, CD332 , CD333, CD334, CD335, CD336, CD337, CDw338, CD339, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt-activated inhibitory factor 1 or WAIF1), adenocarcinomaantigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, alpha intergrin, alpha v beta 6, amino peptidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, annexation A1, toxin-protective anthrax antigen, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracisanthrax, BAFF (B-cell activating factor)), B-lymphoma cell, bcr-abl, bombesin, BORIS, C5, C242 antigen , CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC motif, chemokine 11), CCR4 (receptor C -C chemokine type 4, CD194), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (claudin-18) , agglutinating factor A, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor, CD115), CSF2 (colony-stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor antigen CTAA16.88, CXCR4 (CD184), C-X-C chemokine receptor type 4, cyclic ADP-ribose hydrolase, cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3 (delta-like ligand 3), DLL4 (delta -like ligand 4), DPP4 (dipeptidyl peptidase 4) DR5 (death receptor 5), E. coli toxin type 1, E. coli toxin type 2, ED-B, EGFL7 (EGF-like domain containing protein 7 ), EGFR, EGFRII, EGFRvIII, endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II, beta chain, fibronectin-B accessory domain, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-associated antigen 1 , respiratory syncytial virus F protein, burn-associated receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C, guanylyl cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase receptor C, heat-stable enterotoxin receptor (hSTAR), heat shock proteins, hemagglutinin, hepatitis surface antigen B, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/scatter factor), HHGFR, HIV-1 complex histones, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, scatter receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1) , idiotype, IGF1R (IGF-1, insulin-like growth factor receptor 1), IGHE, IFN-γ, influenza virus hemagglutinin, IgE, IgE Fc region, IGHE, interleukins (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, IL12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27 or IL-28), IL31RA, ILGF2

- 35 044827 (insulin-like growth factor 2), integrins (α ₄ , α _π1 , β ₃ , α _ν β ₃ , α ₄ β ₇ , α ₅ β ₁ , α ₆ β ₄ , α ₇ β ₇ , α ₁₁ β ₃ , α ₅ β ₅ , α _ν β ₅ ), interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor (GIF)), MS4A1 (membrane spanning 4 domain subfamily A member 1), MSLN (mesothelin), MUC1 (Mucin 1 associated with cell surface (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), Melan-A/MART1, ML-IAP, MPG, MS4A1 (membrane spanning protein 4 domain subfamily A), MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22ME), NGF, neuronal apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, nucleolin, oncogenic product Neu, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, non-mutant p53, P97, Page4, PAP, paratope anti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell protein 1, CD279), PDGF-Ra (platelet-derived growth factor receptor alpha), PDGFR-β, PDL-1 , PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor beta, sodium phosphate cotransporter, PMEL 17, polysialic acid d, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate carcinoma cells, Pseudomonas aeruginosa , PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rh factor, RANKL, RANTES receptors (CCR1, CCR3, CCR5), RhoC, Ras mutant, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation initiation points, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syndecan 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, ST3AP1 (six transmembrane epithelial prostate antigen 1), ST3AP2, STn, TAG-72 (tumor associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-a, TGF-β (transforming growth factor beta), TGF-βΓ TGF-e2 (transforming growth factor beta 2), Tie (CD202b), Tie2, TIM -1 (CDX-014), Tn, TNF, TNF-a, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 ( tumor necrosis-inducing apoptosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signaling transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TROP-2, TRP-2, tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2 or vimentin, WT1, XAGE 1 or cells expressing any insulin growth factor receptors or any epidermal growth factor receptors.

In another specific embodiment, the cell-binding ligand-drug conjugates linked through bridging linkers of the present invention are used for targeted treatment of cancer. Target cancers include, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor (adult, brainstem glioma, pediatric, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumor, optic and hypothalamic glioma), breast cancer, carcinoid tumor, gastrointestinal cancer, carcinoma of unknown origin, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family tumors (PNET ), extracranial germinal cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germinal cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal carcinoma, islet cell carcinoma, renal cancer ( renal cell cancer), laryngeal cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell), lymphoma (AIDS associated, central nervous system , T-cell lymphoma of the skin, Hodgkin's disease, non-Hodgkin's disease), malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the neck with occult primary, multiple myeloma and other plasmatic neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, embryonal cell tumor, borderline ovarian tumor), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid cancer, genital cancer penis, pheochromocytoma cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer (kidney cancer), renal pelvis and ureter cancer (transitional cell), salivary gland cancer, Cesar's syndrome, skin cancer, cancer skin (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), unusual childhood cancer, vaginal cancer, vulvar cancer, tumor

Wilms.

In another specific embodiment, the cell-binding agent conjugates of the present invention are used in accordance with compositions and methods for treating or preventing an autoimmune disease. Autoimmune diseases include, but are not limited to, achlorhydria in autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome , antisynthetase syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome types I, II and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo's disease/Balot's concentric sclerosis, Bechets' syndrome, Berger's disease, Bickerstaff's encephalitis, Blau's syndrome, bullous pemphigoid, Castleman's disease, Chaga's disease, chronic fatigue and immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal ostomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Schurg-Strauss syndrome, cicatricial pemphigoid disease, celiac disease, Cogan's syndrome, cold agglutinin disease, complement component deficiency 2, temporal arteritis, CREST syndrome, Crohn's disease (a type of idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dresser's syndrome, discoid lupus erythematosus, eczema, endometriosis, arthritis enthesitis-related, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibromyalgia, fibromyositis, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulone frith, Goodpasture's syndrome, Graves' disease, syndrome Guillain-Barre, Hashimoto encephalitis, Hashimoto thyroiditis, hemolytic anemia, Genoche-Schonlein purpura, gestational herpes, hidradenitis suppurativa, Hughes syndrome (see. antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (see autoimmune thrombocytopenic purpura), IgA nephropathy (also Berger's disease), including body myositis, inflammatory demyelinating polyneopathy, interstitial cystitis, irritable bowel syndrome , juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosis, linear IgA disease (LAD), Lou Gehrig's disease (also amyotrophic lateral sclerosis), lupoid hepatitis, lupus erythematosus, syndrome Majida, Menier's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Haberman disease, Muckle-Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, optic neuromyelitis (Devik's disease), neuromyotonia, ocular cicatricial pemphigoid, opso-myoclonal syndrome, atrophic form of autoimmune thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonnage-Turner syndrome, parsplanitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, true erythrocytic aplasia, Rasmunsen's encephalitis, Raynad's phenomenon , relapsing polychondritis, Reiter's syndrome, restless legs syndrome, fibrous periureteritis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthropathy, sticky blood syndrome, Still's syndrome, stiff person syndrome, subacute bacterial infective endocarditis, Susac syndrome, Sweet syndrome, Sydenham disease, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis (giant cell arteritis), Toloza-Hunt syndrome, transverse myelitis, ulcerative colitis (a type of idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome.

In another specific embodiment, the binding molecule used for the bis-linker conjugate of the present invention for the treatment or prevention of an autoimmune disease may be, but is not limited to, an anti-elastin antibody; Abys antibody against epithelial cells; antibody against basement membrane collagen type IV protein; antinuclear antibody; antibody to double-stranded native DNA; antibody to single-stranded native DNA, anti-cardiolipin antibody IgM, IgG; anti-celiac antibody; antiphospholipid antibody IgK, IgG; anti-SM antibody; anti-mitochondrial antibody; antithyroid antibody; microsomal antibody, T cell antibody; anti-thyroglobulin antibody; anti-SCL-70; anti-Jo; anti-U.sub.1RNP; anti-La/SSB; anti-SSA; anti-SSB; antibody to perital cells; anti-histone antibody; anti-RNP; C-ANCA; P-ANCA; anti-centromere antibody; anti-fibrillarin and anti-GBM antibody, anti-ganglioside antibody; anti-desmogein 3 antibody; anti-p62 antibody; anti-sp100 antibody; anti-mitochondrial (M2) antibody; anti-rheumatoid factor antibody; anti-MCV antibody; anti-topoisomerase antibody; antineutrophil cytoplasmic antibody (cANCA).

In some preferred embodiments, the binding molecule for the conjugate of the present invention can bind to both a receptor and a receptor complex expressed on the surface of an activated lymphocyte that is associated with an autoimmune disease. The receptor or receptor complex may contain a member of the immunoglobulin super family of genes (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD137, CD138, CD147, CD152/CTLA-4, PD-1 or ICOS), a member of the TNF receptor super family (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INFR1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), integrin, cytokine receptor, chemokine receptor, major histocompatibility protein, lectin (C -type, S-type or 1-type) or complement control protein.

In another specific embodiment, beneficial cell-binding ligands that are immunospecific for a viral or microbial antigen are humanized or human monoclonal antibodies. 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 neuramidase, influenza virus hemagglutinin, HTLV tax, simplex virus glycoprotein herpes (eg gB, gC, gD and gE) and hepatitis B surface antigen) capable of inducing 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, for example, LPS and capsular polysaccharide 5/8), which is capable of inducing an immune response. Examples of antibodies available for viral or microbial infection include, but are not limited to, palivizumab, which is a humanized monoclonal antibody against respiratory syncytial virus for the treatment of RSV infection; PRO542, which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir, which is a human antibody for the treatment of hepatitis B virus; PROTVIR, which is a humanized IgG 1 antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.

The cell-binding molecule-drug conjugates linked through bilinkers of the present invention can be used to treat infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infections, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amoebiasis, anaplasmosis, anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, aspergillosis , astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, bacteroides infection, balantidiasis, baylisascaris infection, BC virus infection, nigra piedra, blastocystis humanis infection, blastomycosis, Bolivian hemoragic fever, borrelia infection, botulism (and infant botulism ), Brazilian hemoragic fever, brucilosis, Burkholderia infection, Buruli ulcer, calicivirus infection (norovirus and sapovirus), campylobacteriosis, candidiasis (moniliasis; thrush), cat scratch disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, chicken pox, chlamydia , Chlamydophila pneumoniae infection, cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile infection, coxidiodomycosis, American mountain tick fever, colds (acute viral nasopharyngitis; acute rhinitis), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migrans, cyclosporiasis, cystikercosis, cytomegalovirus infection, Dengue fever, dienthamoebiasis, diphtheria, diphylobosriasis, dracunculiasis, Ebola hemoragic fever, ekinocoxosis, ehrlichiosis, enterobiasis (pinworm), enterocol infection, enterovirus infection, epidemic typhoid, erytime infection (fifth illness), sudden exentem, fasciolopsiasis, fasciolosis, fatal family insomnia, phylariasis, food poisoning of Clostridium Perfringens, and freely living an ammes usobacterium, gas gangrene ( Clostridial myonecrosis), geotrichosis, Gerstmann-Strüssler-Scheinker syndrome, giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale

- 38 044827 (Donovanosis), group A streptococcal infection, group B streptococcal infection, hemophilic infection, enteroviral vesicular stomatitis (HFMD), hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic-uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis Hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ehrlichiosis, human granulocytic anaplasmosis, metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, hymenolepasis, infectious mononucleosis (mono) Epstein-Barr virus, influenza, isosporiasis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, Lassa fever, legionellosis (Legionnaires' disease), legionellosis (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (borreliosis Lyme), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, meliodosis (Whitmore disease), meningitis, meningococcal disease, metagonimiasis, microsporidiosis, molluscum contagiosum, mumps, murine typhus (endemic typhus), mycoplasma pneumonia , mycetoma, myiasis, neonatal conjunctivitis (ophthalmia of the newborn), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness), paracoccidioidosis (South American blastomycosis), paragonimiasis, pasteurellosis, Pediculosis capitis (head lice) , Pediculosis corporis (body lice), Pediculosis pubis (pubic lice, crab lice), pelvic inflammatory disease, pertusitis (whooping cough), plague, pneumococcal infection, Pneumocystis pneumonia, pneumonia, polio, Prevotella infection, primary amoebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsial smallpox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, ARVI (o severe respiratory syndrome ), scabies, schistosomiasis, sepsis, shigellosis (bacillary dysentery), herpes zoster (Herpes zoster), natural ocny(Variola), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, syphilis, teniasis, tetanus, dermatomycosis of the beard and mustache (pseudofollica lit beard hair), fungal infection of the scalp (ringworm of the scalp), ringworm of the smooth skin (ringworm of the body), ringworm inguinal (itching of the groin), tinea (ringworm of the hands), tinea nigra, fungal disease of the feet (athlete's foot), dermatophytic onychomycosis (onychomycosis), pityriasis versicolor (Pityriasor versicolor), toxocariasis (eye larva migrans), toxocariasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomoniasis, trichuriasis (whipworm), tuberculosis, tularemia, Ureaplasma urealyticum infection, Venezuela equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white piedra (Tinea blanca), Yersinia pseudotuberculosis infection, yersiniosis, yellow fever, zygomycosis.

Cell-binding molecules, which are more preferably antibodies described in this patent against pathogenic strains which include, but are not limited to, antibodies against the pathogens Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei , HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus cereus, various bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, genus Borrelia, Clostridium botulinum, Sabia, genus Brucella, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, family Caliciviridae, genus Campylobacter, usually Candida albicans and other Candida species , Bartonella henselae, group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides po sadasii, Colorado tick-borne virus fevers, rhinoviruses, coronoviruses, CJD prion, Congo-Crimean hemorrhagic fever virus, Cryptococcus neoformans, genus Cryptosporidium, Ancylostoma braziliense; various parasites, Cyclospora cayetanensis, Taenia solium, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) - flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, ebolavirus, genus Echinococcus, Ehrlichia genus, Enterobius vermicularis, genus Enterococcus, genus Enterovirus, Rickettsia prowazekii, parvovirus B19, human herpevirus 6 and human herpevirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, genus Fusobacterium, Clostridium perfringens; other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, enteroviruses, mainly coxsackie A virus and enterovirus 71, Sin virus Nombre, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, human bocavirus, Ehrlichia ewingii, Ana

- 39 044827 plasma phagocytophilum, human metapneumovirus, Ehrlichia chaffeensis, human papilloma virus, human parainfluenza virus, Hymenolepis papa and Hymenolepis diminuta, Epstein-Barr virus, family Orthomy-xoviridae, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebs iella rhinoscleromotis , Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, lymphocytic choriomeningitis virus (LCMV), Plasmodium genus , Marburg virus, measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Microsporidia phylum, molluscum contagiosum virus (MCV), mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, various representatives of bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic larvae of dipterans insects, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, genus Pasteurella, Pediculus humanus capitis, Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis, Yersinia pestis , Streptococcus pneumoniae, Pneumocystis jirovecii, poliovirus, genus Prevotella, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, rabies virus, Streptobacillus moniliformis and Spirillum minus, respiratory syncytial virus, Rhinosporidium seeberi, rhinovirus, genus Rickettsia, Rickettsia akari, valley fever virus Rift, Rickettsia rickettsii, rotavirus, measles rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigella genus, chickenpox virus, Variola major or Variola minor, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes , Strongyloides Stercoralis, TrePonema Pallidum, genus Taenia, Clostridium Tetani, genus Trichophyton, Trichophyton Tonsurans, genus Trichophyton, Epidermophyton Floccosum, Trichophy Ton Rubrum, and Trichophyton Mentagrophytes, Trichophyton Rubrum, Hortaea Werneckii, genus Trichophyton, genus Malassezia, Toxocara Canis or Toxocara Cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio colerae, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, yellow virus oh fever, Mucorales Order (Mucormycosis) and Entomophthorales Order (Entomophthora-Mycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) Fetus, Aeromonas Hydrophila, EDwardsella Tanda, EDwardsiella Tanda, Yersinia Pestis, Shigella Dysenteriae, Shigella Flexneri, Shigella Sonnei, Salmonella Typhimurium, Treponyma Pertenue, TrePonema Carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or helminths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

Other antibodies as cell binding ligands used in this invention for the treatment of viral diseases include, but are not limited to, antibodies against antigens of pathogenic viruses, including but not limited to: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, measles rubella, Arboviridae, Rhabdoviridae, Arenaviridae, non-A/non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, oncovirus [ such as, HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpevirus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma), MCPyV ( Merkel cell cancer), SV40 (simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult T-cell leukemia/lymphoma)], immune disorders caused by virus: [such as human immunodeficiency virus (AIDS)]; central nervous system virus: [such as, JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing leukoencephalitis), LCV (lymphocytic choriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (possibly) (Encephalitis lethargica), RV (rabies), Chandipura virus, herpes virus meningitis, Ramsay-Hunt syndrome type II; polyvirus (poliomyelitis, post-polio syndrome), HTLV-I (tropical spastic paraparesis)]; cytomegalovirus (Cytomegalovirus retinitis, HSV (herpetic keratitis)); cardiovascular virus [such as CBV (pericarditis, myocarditis)]; respiratory systemic/acute viral nasopharyngeal/viral pneumonia: [Epstein-Barr virus (EBV infection/infectious mononucleosis), cytomegalovirus; SARS coronavirus (severe acute respiratory syndrome) Orthomyxoviridae: influenza A/B/C virus (influenza/avian influenza), paramyxovirus: human parainfluenza viruses (parainfluenza), RSV (human respiratory syncytial virus), hMPV]; digestive system virus [MuV (mumps), cytomegalovirus (cytomegalovirus esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus; HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus)]; urogenital virus [eg, BK virus, MuV (mumps)].

- 40 044827

In accordance with yet another object, the present invention also relates to pharmaceutical compositions containing a conjugate of the invention together with a pharmaceutically acceptable carrier, diluent or excipient for the treatment of cancer, infections or autoimmune disorders. The method for treating cancers, infections and autoimmune disorders can be practiced in vitro, in vivo or ex vivo. Examples of in vitro applications include treating cell cultures to kill all cells except desired variants that do not express the target antigen; or eliminating variants that express an undesired antigen. Examples of ex vivo uses include treating hematopoietic stem cells (HSCs) prior to performing a transplant (HSCT) on the same patient to kill diseased or malignant cells. For example, ex vivo clinical treatment to remove tumor cells or lymphoid cells from bone marrow prior to autologous transplantation in the treatment of cancer or in the treatment of an autoimmune disease, or to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplantation in order to prevent graft-versus-host disease can be accomplished as follows. Bone marrow is collected from a patient or other individual and then incubated in a medium containing serum to which a conjugate of the invention is added, at a concentration range of about 1 pM to 0.1 mM, for about 30 minutes to about 48 hours at about 37°C. The exact concentration conditions and incubation time (=dose) are easily determined by qualified physicians. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known techniques. In cases where the patient receives other treatment, such as a course of ablative chemotherapy or whole body irradiation, between the time of bone marrow collection and reinfusion of the treated cells, the treated bone marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

Drugs/cytotoxic agents for conjugation.

Drugs that can be conjugated to the cell-binding molecule of the present invention are small molecule drugs including cytotoxic agents that can be associated with or after being modified to bind to the cell-binding agent. The term small molecule drug is broadly used herein to mean an organic, inorganic or organometallic compound which may have a molecular weight of, for example, 100 to 2500, more suitably 200 to 2000. Small molecule drugs are well characterized in the art, e.g. in WO 05058367A2 and in US patent No. 4956303, among others, and are incorporated by reference in their entirety. Medicines include known drugs and those that may become known drugs.

Medicines that are known include, but are not limited to:

1) Chemotherapeutic agents: a), alkylating agents: such as nitrogen mustards: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobromane, novembiquine, fenester in, prednimustine , thiotepa, trofosfamide, uracil mustard; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); duocarmycin (including synthetic analogues, dimers KW-2189, CBI-TMI and CBI); benzodiazepine dimers (eg, pyrrolobenzodiazepine (PBD) or tomaimicin dimers, indolinobenzodiazepines, imidazobenzothiadiazepines or oxazolidinobenzodiazepines); nitrosoureas: (carmustine, lomustine, chlorosotocin, fotemustine, nimustine, ranimustine); alkylsulfonates: (busulfan, treosulfan, improsulfan and piposulfan); triazenes: (dacarbazine); platinum-containing compounds: (carboplatin, cisplatin, oxaliplatin); aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine]; b) plant alkaloids: such as vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine, navelbine); taxoids: (paclitaxel, docetaxol) and their analogs, maytansinoids (DM1, DM2, DM3, DM4, maytansine and ansamitocins) and their analogs, cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); epothilones, eleutherobine, discodermolide, bryostatins, dolostatins, auristatins, tubulisins, cephalostatins; pancratistatin; sarcodictin; spongistatin; c), DNA topoisomerase inhibitors: such as [epipodophyllins: (9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novanthrone, retinoic acids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycins: (Mitomycin C) and its analogues]; d). antimetabolites: such as {[anti-folates: DHFR inhibitors: (methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteric acid) or other folic acid analogues); IMPdehydrogenase inhibitors: (mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors: (hydroxyurea, deferoxamine)]; [pyrimidine analogs: uracil analogs: (ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxycuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (tomudex)); cytosine analogues: (cytarabine, cytosine arabinoside, fludarabine); purine analogues: (azathioprine, fludarabine, mercaptopurine, thiami

- 41 044827 prin, thioguanine)]; a folic acid replenisher such as frolinic acid}; e). hormonal therapies: such as {receptor antagonists: [antiestrogens: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goskrklin, leuprolide acetate); antiandrogens: (bicalutamide, flutamide, calusterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mepithiostane, nilutamide, testolactone, trilostane and other androgen inhibitors)]; retinoids/deltoids: [vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); photodynamic therapy: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrellin A); cytokines: (interferon-alpha, interferon-gamma, tumor necrosis factor (TNF), human proteins containing the TNF domain)]}; f). kinase inhibitors such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2), mumbritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab , cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib; g). poly(ADFribose) polymerase (PARP) inhibitors such as olaparib, niraparib, iniparib, talazoparib, veliparib, veliparib, CEP 9722 (Cefalon), E7016 (Eisai), BGB-290 (BeiGen), 3-aminobenzamide; h) antibiotics such as enediyne antibiotics (eg calicheamicins, especially calicheamicin γ1, δ1, α1 and β1, see, for example, J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Intl ED. English. 33: 183-186 (1994); Dynamic, including dimeicin A and deoxydinemicin; Espericin, Kedarcidine, S-1027, Maduropeptin, as well as neurzinostine chromoophers and related chromoprotein antiobiotics chromophores), aclacinomisins, acticians, acticians, acticians, acticians, components. n, authramitsin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6diazo-5-oxo-b-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin, epirubicin, ezorubicin, idarubicin, marcellomycin, nitomycins, myco phenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; i) others: such as polyketides (acetogenins), especially bulatacin and bulatacinone; gemcitabine, epoxomycins (eg, carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, Stimuvax, Allovectin-7, Xegeva, Provenge, Yervoy, isoprenylation inhibitors (such as lovastatin), dopaminergic neurotoxin s ( such as 1-methyl-4phenylpyridinium ion), cell cycle inhibitors (such as staurosporine), actinomycins (such as actinomycin D, dactinomycin), bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), anthracyclines (such as daunorubicin, doxorubicin ( adriamycin), idarubicin, epirubicin, eribulin, pyrarubicin, zorubicin, methoxantrone, MDR inhibitors (such as verapamil), Ca ²⁺ -ATPase inhibitors (such as thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valprucic acid, mocetinostat (MGCD0103) ), belinostat, PCI-24781, entinostat, SB939, resminostat, givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A.; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside, bestrabucil; bisanthrene; edatraxate; defofamine; demecolcine; diaziquon; eflornithine (DFMO), elfomitin; elliptinium acetate, ethoglucide; gallium nitrate; gacytosine, hydroxyurea; ibandronate, lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and anguidine); urethane, siRNA, antisense drugs and nucleolytic enzyme.

2) Anti-autoimmune disease agent includes, but is not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonides, betamethasone, budesonides, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA (DHEA), enanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus.

3) Agent against infectious disease includes, but is not limited to, a) aminoglycosides: amikacin, astromycin, gentamicin (netilmicin, sisomycin, isepamycin), hygromycin B, kanamycin (amikacin, arbekacin, bekanamycin, dibekacin, tobramycin), neomycin ( framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, verdamycin; b) amphenicols: azidamphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycins: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; f) cephems: carbacefem (loracarbef), cefacetril, cefaclor, cefradine, cefadroxil, cephalonium, cephaloridine, cephalothin or cephalosin, cephalexin, cephaloglycin, cefamandole, cephapirin, cefatrizine, cefazaflur, cephasedone, cefazolin, cefbuperazone, cefcapen, cefaldoxime, cefepime, cefminox , cefoxitin, cefprozil, cefroxadine, ceftezol, cefuroxime, cefixime, cefdinir, cefdito

- 42 044827 ren, cefepime, cefetamet, cefmenoxime, cefodizim, cefonicide, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole, cefpyramide, cefpirome, cefpodoxime, cefprozil, cefx inom, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolen, ceftizoxime , ceftobiprole, ceftriaxone, cefuroxime, cefusonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacefem (flomoxif, latamoxif); f) glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g) glycylcyclines: for example tigecycline; g) β-lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulanic acid); i) lincosamides: clindamycin, lincomycin; j) lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDAs); k) macrolides: azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin, flurithromycin, josamycin, ketolide (telithromycin, cethromycin), midecamycin, myocamycin, oleandomycin, rifamycin (rifampicin rifampin, rifabutin, rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; l) monobactams: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, hetacyclin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin, procaine benzylpenicillin, carbenicillin llin (carindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin , mecillinam (pivmecillinam), mezlocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, phenethylcillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) polypeptides: bacitracin, colistin, polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, cano trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin sacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin , trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin, quinupristin/dalfopristin); r) sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (cotrimoxazole); s) steroidal antibacterial agents: for example, fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, clomocycline, demeclocycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimecycline, roletetracycline, tetracycline, glycylcyclines (for example, tigecycline); u) other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors (bacitracin), DADAL/AR inhibitors (cycloserine), dictyostatin, discodermolide, eleuterobine, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin , mycolactone, NAM synthesis inhibitors (eg, fosfomycin), nitrofurantoin, paclitaxel, platensimicin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin; 4). Antiviral drugs: a) entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b) integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) maturation inhibitors: bevirimat, vivecon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; f) Nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'- fluoro-substituted 2',3'-dideoxynucleoside analogues (e.g. 3'-fluoro-2',3'dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, ganciclovir, idoxuridine , lamivudine (3TC), 1-nucleosides (for example, β-1-thymidine and β-1-2'-deoxycytidine), penciclovir, rativir, ribavirin, stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alpha, loviride, lodenosine, methizazone, nevirapine, NOV-205, peginterferon alpha podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other types of antiviral drugs: abzyme, arbidol, calanolide A, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, combination hybrids inhibitors ribavirin, selicilib.

5) The drugs used for the bis-linker conjugates of the present invention also include radioisotopes. Examples of radioisotopes (radionuclides) are ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ³² P, 35 S, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁹ Tc, ^m In, ¹²³ I, ^{124 I, 125 I} , ¹³¹ I , ¹³³ Xe, ¹⁷⁷ Lu, ²¹¹ At or ²¹³ Bi. Radiolabeled antibodies are useful in receptor targeting experiments, or can be used for targeted treatments, such as the antibody-drug conjugates of the invention (Wu et al. (2005) Nature Biotechnology 23(9): 1137-46). Molecules that bind to a cell, such as an antibody, can be reagent-labeled ligands through bridging

- 43 044827 linkers of the present patent that bind, chelate or otherwise complex a radioactive metal isotope using the techniques described in Current Protocols in Immunology,

Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, Pubs. (1991). Chelating ligands that can complex with the metal ion include DOTA, DOTP, DOTMA, DTPA and THETA (Macrocyclics, Dallas, TX, USA).

6) Pharmaceutically acceptable salts, acids, derivatives, hydrated or hydrated salts; or crystal structure; or an optical isomer, racemate, diastereomer or enantiomer of any of the above drugs.

In another embodiment, the drug molecule/cytotoxic molecule in formula (I) and/or (II) may be a chromophore molecule such that the conjugate can be used to detect, monitor, or study the interaction of a cell-binding molecule with a target cell. Chromophore molecules are a compound that has the ability to absorb types of light such as UV light, fluorescent light, IR light, near-IR light, visible light; a chromatophore molecule includes the class or subclass of xanthophores, erythrophores, iridophores, leucophores, melanophores and cyanophores; a class or subclass of fluorophore molecules, which are fluorescent chemical compounds that re-emit light after irradiation; a class or subclass of visible phototransduction molecules; a class or subclass of photophore molecules; a class or subclass of luminescent molecules; and a class or subclass of luciferin compounds.

The chromophore molecule may be selected from, but not limited to, non-protein organic fluorophores such as: xanthene derivatives (fluorescein, rhodamine, Oregon green, eosin and Texas red); cyanine derivatives: (cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine); squarine derivatives and ring-substituted squarine derivatives, including Seta, SeTau and Square dyes; naphthalene derivatives (dansil and sold derivatives); coumarin derivatives; oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); anthracene derivatives (anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); pyrene derivatives (cascade blue, etc.); oxazine derivatives (Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (auramine, crystal violet, malachite green), tetrapyrrole derivatives (porphin, phthalocyanine, bilirubin).

Or the chromophore molecule can be selected from any of the analogs and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (BioStatus), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), Abberior stains (Abberior), DY and MegaStokes stains (Dyomics), Sulfo Cy stains (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square stains (SETA BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), SureLight dyes (APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech).

Examples of commonly used fluorophore compounds that are reactive or conjugated with the linkers of the invention are: allophycocyanin (APC), aminocoumarin, APC-Cy7 conjugates, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, hydroxycoumarin, IR-783, lysamine rhodamine B, luciferin yellow, methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP, Rphycoerythrin (PE), red 613, Seta-555-azide, Seta-555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta-680NHS, Seta-780-NHS, Seta-APC-780, Seta-PerCP-680, Seta -R-PE-670, SeTau-380-NHS, SeTau-405-maleimide, SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, Texas Red, TRITC, TruRed, X-rhodamine.

Fluorophore compounds that can be linked to the linkers of the invention for the study of nucleic acids or proteins are selected from the following compounds or derivatives thereof: 7-AAD (7-aminoactinomycin D, CG-selective), acridine orange, chromomycin A3, CyTRAK orange (Biostatus , red excitation light dark), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS 751, mithramycin, propidium iodide (PI), SYTOX Blue, SYTOX Green, SYTOX Orange, thiazole orange, TO-PRO: cyanine monomer, TOTO-1, TO-PRO-I, TOTO-3, TO-PRO-3, YOSeta-1, YOYO-1. Fluorophore compounds that can be associated with the linkers of the invention for cell research are selected from the following compounds or derivatives thereof: DCFH (2'7'-dichorodihydrofluorescein, oxidized form), DHR (dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo -3 (AM ester: pH>6), Fluo-4 (AM ester pH 7.2), Indo-1 (AM ester, low/high calcium (Ca ²⁺ )) and SNARF (pH 6/9) . Preferred fluorophore compounds that can be linked to the linkers of the invention for protein/antibody studies are selected from the following compounds or derivatives thereof: Allophycocyanin (APC), AmCyanl (tetramer, Clontech), AsRed2 (tetramer, Clontech), Azami Green (monomer, MBL), azurite, Bphycoerythrin (BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 (RFP, Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65T mutation), GfP (Y66F mutation), GFP (Y66H mutation), GFP (Y66W mutation), GFPuv , HcRed1, J-Red, Katusha, Kusabira Orange

- 44 044827 (monomer, MBL), mCFP, mCherry, mCitrine, Midoriishi Cyan (dimer, MBL), mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer, Tsien lab), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome complex), Peridinin Chlorophyll (PerCP), Rphycoerythrin (RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP ( dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP ( dimer, Evrogen), Venus, wild-type GFP, YPet, ZsGreenl (tetramer, Clontech), ZsYellowl (tetramer, Clontech).

Examples of the structure of antibody-chromophore molecule conjugates connected through a bridging linker are the following: Ac01, Ac02, Ac03, Ac04, Ac05, Ac06 and Ac07:

Ac06 (IR800CW conjugate)

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X ₁ and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O )S, OC(O)N(R ₁ ), N(R ₁ )C(O)N(R ₁ ), CH, C(O)NHNHC(O) and C(O)NRi;

The mAb is an antibody, preferably a monoclonal antibody;

n and m1 are independently equal to 1-20;

R ₁₂ and R ₁₂ ' independently represent OH, NH ₂ , NHR1, NHNH ₂ , NHNHCOOH, O-R1-COOH, NHR1-COOH, NH-(Aa)nCOOH, O(CH2CH2O)pCH2CH2OH, O(CH2CH2O)pCH2CH2NH2,

NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NH ₂ , O(CH ₂ CH ₂ O) _p CH ₂ CH ₂ COOH, NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ COOH,

O(CH2CH2O)pCH2CH2NHSO3H, NH(CH2CH2O)pCH2CH2NHSO3H R1-NHSO3H, NH-R1-NHSO3H, O(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHPO ₃ H ₂ , NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHPO ₃ H ₂ , R1-NHPO ₃ H ₂ , R1-OPO ₃ H ₂ , O(CH2CH2O)pCH2CH2OROzH2, NH(CH2CH2O)pCH2CH2NHPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, NH-ArCOOH, NH-Ar -NH ₂ where p=0-5000, Aa represents an amino acid;

- 45 044827

R1, m1, n, L1, and _L2 are as defined in formula (I).

In another embodiment, the drug in formulas (I) and (II) may be polyalkylene glycols, which are used to increase the half-life of the cell-binding molecule when administered to a mammal. Polyalkylene glycols include, but are not limited to, poly(ethylene glycols) (PEG), poly(propylene glycol) and copolymers of ethylene oxide and propylene oxide; particularly preferred are PEGs, and more particularly preferred are monofunctionally activated hydroxyPEGs (e.g., hydroxyl PEGs activated at one end, including reactive hydroxyPEG monocarboxylic acid esters, hydroxyPEG monoaldehydes, hydroxyPEG monoamines, hydroxyPEG monohydrazides, hydroxyPEG monocarbazates, hydroxyPEG -monoiodoacetamides, hydroxyPEG-monomaleimides, hydroxyPEG-monoorthopyridyl disulfides, hydroxyPEG-monooximes, hydroxyPEG-monophenyl carbonates, hydroxyPEG-monophenylglyoxals, hydroxyPEG-monothiazolidin-2-ones, hydroxyPEG-monothioesters, hydroxyPEG-monotis ols, hydroxyPEG monotriazines and hydroxyPEG monovinyl sulfones).

In some such embodiments, the polyalkylene glycol has a molecular weight of from about 10 Da to about 200 kDa, preferably from about 88 Da to about 40 kDa; two branches, each with a molecular weight from about 88 Da to about 40 kDa; and more preferably two branches, each from about 88 kDa to about 20 kDa. In one specific embodiment, the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa or about 40 kDa. In specific embodiments, the PEG is 10 kDa PEG (linear or branched), 20 kDa PEG (linear or branched), or 40 kDa PEG (linear or branched). A number of US patents disclose the preparation of linear or branched non-antigenic PEG polymers and their derivatives or conjugates, for example, US patent No. 5428128; 5621039; 5622986; 5643575; 5728560; 5730990; 5738846; 5811076;5824701; 5840900;5880131; 5900402; 5902588; 5919455; 5951974;

5965119; 5965566; 5969040; 5981709; 6011042; 6042822; 6113906; 6127355; 6132713; 6177087 and 6180095. The structure of the antibody-polyalkylene glycol conjugates linked through a bridging linker is as follows: Pg01, Pg02 and Pg03:

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR ₅ , S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O )S, OC(O)N(R1), N(R _u C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; mAb is an antibody, preferably a monoclonal antibody ; n and m1 are independently equal to 1-20; p is equal to 1-5000; R1, L1 and L2 are as defined in formula (I). Preferably, R1 and _R3 are independently H, OH, _OCH3 , _CH3 or OS ₂ N ₅ .

In yet another embodiment, preferred cytotoxic agents that are conjugated to a cell-binding molecule via the bridging linker of the present patent are tubulisins, maytansinoids, taxanoids (taxanes), CC-1065 analogues, daunorubicins and doxorubicins, amatoxins (including amanitins), indole carboxamide, benzodiazepine dimers (eg, pyrolobenzodiazepine dimers (PBD), tomayamycin, anthramycin, indolinobenzodiazepines, imidazolobenzothiazepines or oxazolidinobenzodiazepines), calicheamicins and enediine antibiotics, actinomycin, azaserines, bleomycins, epirubicin, eribulin, tamoxifen, idarubicin, up to lastatins, auristatins (for example, monomethyl aur isatin E, MMAE, MMAF, auristatin PYE, auristatin TR, auristatins 2-AQ, 6-AQ, EB (AEB) and EFP (AEFP) and their analogues), duocarmycins, geldanamycins or other HSP90 inhibitors, centanamycin, methotrexates, thiotepa, vindesines , vincristines, hemiasterlins, nasumamides, microginins, radiosumins, streptonigtin, SN38 or other analogues or metabolites of camptothecin, alterobactins, microsclerodermins, theonellamides, esperamycins, PNU-159682; and their analogs or derivatives, pharmaceutically acceptable salts, acids, derivatives, hydrate or hydrated salt; or crystal structure; or an optical isomer, racemate, diastereomer or enantiomer of any of the above drugs.

- 46 044827

The tubulisins that are preferred for conjugation in the present invention are well known in the art and can be isolated from natural sources according to known methods or produced synthetically according to known methods (e.g.

Balasubramanian R., et al. J. Med. Chem., 2009, 52, 238-40; Wipf, P., et al. Org. Lett., 2004, 6, 4057-60; Pando, 0., et al. J. Am. Chem. Soc., 2011, 133, 7692-5; Reddy, J. A., et al. Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33;

Patterson, A. W., et al. J. Org. Chem., 2008, 73, 4362-9; Pando, 0., et al. Org. Lett., 2009, 11 (24), 5567-9; Wipf, P., et al. Org. Lett., 2007, 9 (8), 1605-7; Friestad, G. K., Org. Lett.,2004, 6, 3249-52; Peltier, N. M., et al. J. Am. Chem. Soc., 2006, 128, 16018-9; Chandrasekhar, S., et al J. Org. Chem., 2009, 74, 9531-4; Liu, Y., et al. Mol. Pharmaceutics, 2012, 9, 168-75; Friestad, G. K., et al. Org. Lett., 2009, 11, 1095-8; Kubicek, K., et al., Angew Chem Int Ed Engl, 2010.49: 4809-12; Chai, Y., et al., Chem Biol, 2010, 17: 296-309; Ullrich, A., et al., Angew Chem Int Ed Engl, 2009, 48, 4422-5; Sani, M., et al. Angew Chem Int Ed Engl, 2007, 46, 35269; Domling, A., et al., Angew Chem Int Ed Engl, 2006, 45, 7235-9; Patent applications: Zanda, M., et al, Can. USA Appl. CA 2710693 (2011); Chai, Y., et al. Eur. USA Appl. 2174947 (2010), WO 2010034724; Leamon, C. et al, WO2010033733, WO 2009002993; Ellman, J., et al, PCT WO2009134279; WO 2009012958, US appl. 20110263650, 20110021568; Matschiner, G., et al, WO2009095447; Vlahov, I., et al, WO2009055562, WO 2008112873; Low, P., et al, WO2009026177; Richter, W., WO2008138561; Kjems, J., et al, WO 2008125116; Davis, M.; et al, WO2008076333; Diener, J.; et al, U.S. Pat.Appl. 20070041901, WO2006096754;

Matschiner, G., et al, WO2006056464; Vaghefi, F., et al, WO2006033913; Doemling, A., Ger. Offen. DE102004030227, WO2004005327, WO2004005326, WO2004005269; Stanton, M. et al, U.S. Pat. Appl. Publ. 20040249130; Hoefle, G., et al, Ger. Offen. DE10254439, DE10241152, DE10008089; Leung, D., et al, WO2002077036; Reichenbach, H., et al, Ger.

Offen. DE19638870; Wolfgang, R„ US20120129779; Chen, H„ US Patent Application 20110027274. Preferred tubulysin structures for conjugating cell binding molecules are described in patent application PCT/IB2012/053554.

Examples of the structures of antibody-tubulisin analogue conjugates linked through a bis-linker are T01, T02, T03, T04, T05, T06 T07, T08, T09, T10 and T11:

- 47 044827

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O,

- 48 044827

C(O)NH, OC(O)NH, OS(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R ₁ ), N(R1)C(O)N( R1), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; R12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) _nCOOH ,

O(CH ₂ CH ₂ O)pCH ₂ CH ₂ OH, O(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NH ₂ , NH(CH ₂ CH ₂ O)pCH ₂ CH ₂ NH ₂ , NR1R1', NHOH, NHOR1,

O(CH ₂ CH ₂ O) _p CH ₂ CH ₂ COOH, NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ COOH, NH-Ar-COOH, NH-Ar-NH ₂ , o(cH ₂ CH ₂ o ) _p CH ₂ CH ₂ NHSO ₃ H, NH(CH ₂ CH ₂ o) _p CH ₂ CH ₂ NHSO ₃ H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O)pCH2CH2NHPO3H2, NH(CH2CH2O)pCH2CH2NHPO3H2, OR1 , R1-NHPO3H2, R1-OPO3H2, o(CH ₂ CH ₂ o) _p CH ₂ CH ₂ OPO ₃ H ₂ , OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH ₂ CH ₂ NH)pCH ₂ CH ₂ NH ₂ , NH(CH ₂ CH ₂ S)pCH ₂ CH ₂ NH ₂ , NH(CH ₂ CH ₂ NH)pCH ₂ CH ₂ OH, NH(CH ₂ CH ₂ S) _p CH ₂ CH ₂ OH NH-R1-NH2 , or NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHPO ₃ H ₂ where Aa represents 1-8 amino acids; n and m1 independently represent 1-20; p equals 1-5000; preferably R1, R1', R2, R3, and _R4 are independently H, C1-C8 linear or branched alkyl, amide or amines; C ₂ -C ₈ aryl, alkenyl, alkynyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, heterocycloalkyl or acyloxyamines; or peptides containing 1-8 amino acids, or a polyethyleneoxy group having the formula (OSH ₂ CH ₂ ) _p or (OSH ₂ CH (CH ₃ )) _p , where p is an integer from 1 to about 5000; two Rs: R1R2, R2R3, R1R3 or R ₃ R ₄ can form a 3-8 membered cyclic ring from an alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl group; X ₃ represents H, CH ₃ , CH2CH3 , C ₃ H ₇ , or X1'R1', where X1' represents NH, N(CH ₃ ), NHNH, O or S; R1' represents H or C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl or acyloxyamines; R ₃ ' represents H or C1-C6 linear or branched alkyl; Z ₃ represents H, COOR1, NH2, NHR1, OR1, CONHR1, NHCOR1, OCOR1, OP(O)(OM1)(OM ₂ ), OCH ₂ OP(O)(OM1)(OM ₂ ), OSO3M1, R1, O-glycoside (glucoside, galactoside, mannoside, glucuronoside/glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH ₂ -glycoside; M1 and _M2 independently represent H, Na, K, Ca, Mg, _NH4 , NR1R2R3; L1 and L2 are as defined in formula (I).

The calicheamicins and related enediyne antibiotics, which are preferred for the cell-binding molecule-drug conjugates of the present patent, are described in: Nicolaou, K.S. et al., Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-8), US Patent No. 4970198; 5053394; 5108912; 5264586; 5384412; 5606040; 5712374; 5714586; 5739116; 5770701; 5770710; 5773001; 5877296; 6015562; 6124310; 8153768. Examples of the structures of antibody-calicheamicin analogue conjugates linked through a bis-linker are C01 and C02:

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR ₅ , S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O )S, OC(O)N(Ri), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; p is 1-5000, R1, L1 and L2 are as defined in formula (I).

Maytansinoids that are preferred for use in the present invention include maytansinol and its analogs, and are described in US Pat. Nos. 4,256,746, 4,361,650, 4,307,016, 4,294,757, 4,294,757, 4371,533, 4,424,219, 4,331,598, 4,450,254, 4,364 866, 4313946, 4315929, 4362663 , 4322348, 4371533, 4424219, 5208020, 5416064, 5208020; 5416064; 6333410; 6441163; 6716821, 7276497, 7301019, 7303749, 7368565, 7411063, 7851432 and 8163888. An example of the structure of an antibody-maytansinoid conjugate linked through a bis-linker is My01, My02, My03, My04, My05 and My06:

- 49 044827

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O) S, OC(O)N(R1), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; p is 1-5000, R1, L1 and _L2 are as defined in formula (I).

Taxanes, which include paclitaxel (Taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semisynthetic derivative, and analogs thereof, which are preferred for conjugation, are given in the following examples: K.S. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-20, (1995); Ojima et al., J. Med. Chem. 39: 3889-3896 (1996); 40: 267-78 (1997); 45, 5620-3 (2002); Ojima et al., Proc. Natl. Acad. Sci., 96:4256-61 (1999); Kim et al., Bull. Korean Chem. Soc, 20, 1389-90 (1999); Miller et al. J. Med. Chem., 47, 4802-5(2004); US patents No. 5475011 5728849, 5811452; 6340701; 6372738; 6391913, 6436931; 6589979; 6596757; 6706708; 7008942; 7186851; 7217819; 7276499; 7598290; and 7667054.

Examples of antibody-taxane conjugate structures linked through a linker according to this patent are Tx01, Tx02 and Tx03

- 50 044827

TxO where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O) S, OC(O)N(Ri), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; R1, L1 and L2 are as defined in formula (I).

CC-1065 analogs and docarmycin analogs are also preferred for use in the conjugate containing the bis-bridge linker of the present patent. Examples of CC-1065 analogs and doucarmycin analogs, as well as their synthesis, are described, for example, in

Warpehoski, et al, J. Med. Chern. 31:590-603 (1988); D. Boger et al., J. Org.

Chern; 66; 6654-61, 2001; US Patents No: 4169888, 4391904, 4671958, 4816567, 4912227,

4923990, 4952394, 4975278, 4978757, 4994578, 5037993, 5070092, 5084468, 5101038, 5117006, 5137877, 5138059, 5147786, 5187 186, 5223409, 5225539, 5288514, 5324483, 5332740, 5332837, 5334528, 5403484, 5427908, 5475092, 5495009, 5530101, 5545806, 5547667, 5569825, 5571698, 5573922, 5580717, 5585089, 5585499, 5587161, 5595499, 5606017, 5622929, 5625 126, 5629430, 5633425, 5641780, 5660829, 5661016, 5686237, 5693762, 5703080, 5712374, 5714586, 5739116, 5739350, 5770429, 5773001, 5773435, 5786377 5786486, 5789650, 5814318, 5846545, 5874299, 5877296, 5877397, 5885793, 59395 98, 5962216, 5969108, 5985908, 6060608, 6066742, 6075181, 6103236, 6114598, 6130237, 6132722, 6143901, 6150584 , 6162963, 6172197, 6180370, 6194612, 6214345, 6262271, 6281354, 6310209, 6329497, 6342480, 6486326, 6512101, 6521404, 65 34660, 6544731, 6548530, 6555313, 6555693, 6566336, 6586618, 6593081, 6630579, 6756397, 6759509, 6762179 , 6884869, 6897034, 6946455, 7049316, 7087600, 7091186, 7115573, 7129261, 7214663, 7223837, 7304032, 7329507, 7329760, 73 88026, 7655660, 7655661, 7906545 and 8012978. Examples of the structures of the “antibody-CC-1065 analogues” conjugate connected through the linker according to the present patent are CC01, CC02, CC03 and CC04.

- 51 044827

where mAb is an antibody; Z ₃ represents H, PO(OM1)(OM ₂ ), SO ₃ M1, CH ₂ PO(OM1)(OM ₂ ), CH ₃ N(CH ₂ CH ₂ ) ₂ NC(O)-, O(CH ₂ CH ₂ ) ₂ NC(O)-, R1 or glycoside; - is optionally either a single bond or a double bond, or may optionally be absent; X1, X5, Y1 and Y5 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; R1, L1 and L2 are as defined in formula (I).

Daunorubicin/doxorubicin analogues are also preferred for conjugation via the bislinker according to the present patent. Preferred structures, as well as their synthesis, are given in the following examples:

Hurwitz, E., et al., Cancer

Res. 35, 1175-81 (1975). Yang, N. M., and Reisfeld, R. A., Proc. Natl. Acad. Sci. 85, 1189-93 (1988); Pietersz, S. A., E., et al., E., et al.,” Cancer Res. 48, 926-311 (1988); Trouet, et al., 79, 626-29 (1982); Z. Brich et al., J. Controlled Release, 19, 245-58 (1992); Chen et al., Syn. Comm., 33, 2377-90, 2003; King et al., Bioconj. Chem., 10, 279-88, 1999; King et al., J. Med. Chem., 45, 4336-43, 2002; Kratz et al., J Med Chem. 45, 5523-33, 2002; Kratz et al., Biol Pharm Bull. Jan. 21, 56-61, 1998; Lau et al., Bioorg. Med. Chem. 3, 1305-12, 1995; Scott et al., Bioorg. Med. Chem. Lett. 6, 1491-6, 1996; Watanabe et al., Tokai J. Experimental Clin. Med. 15, 32734, 1990; Zhou et al., J. Am. Chem. Soc. 126, 15656-7, 2004; WO 01/38318; US Patents No. 5,106,951; 5122368; 5146064; 5177016; 5208323; 5824805;6146658;6214345;7569358; 7803903; 8084586; 8053205.

Examples of antibody-CC-1065 analog conjugate structures linked through a linker according to the present patent are Da01, Da02, Da03, Da04, Da05, Da06, Da07 and Da08.

- 52 044827

is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O) S, OC(O)N(R1), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NRi; R12 is OH, NH2, NHR _b NHNH2, NHNHCOOH, OR _r COOH, NH-R _r COOH, NH(Aa) _n COOH, O(CH ₂ CH ₂ O)pCH ₂ CH ₂ OH, O(CH ₂ CH ₂ O)pCH ₂ CH ₂ NH ₂ , NH(CH ₂ CH ₂ O)pCH ₂ CH ₂ NH ₂ , NR1R1', NHOH, NHOR1, O(CH2CH2O)pCH2CH2COOH, NH(CH2CH2O)pCH2CH2COOH, NH-Ar-COOH, NH -Ar-NH2,

- 53 044827

O(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHSO3H, NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NH-SO ₃ H, R1-NHSO3H, NH-R1-NHSO3H, o(cH ₂ CH ₂ o) _p CH ₂ CH ₂ NHPO ₃ H ₂ , NH(CH ₂ CH ₂ O) _p CH ₂ -CH ₂ NHPO ₃ H ₂ , OR1, R1-NHPO3H2, R1-OPO3H2, o(cH ₂ CH ₂ o) _p CH ₂ CH ₂ OPO3H ₂ , OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH ₂ CH ₂ NH) _p CH ₂ CH ₂ NH ₂ , NH(CH ₂ CH ₂ S) _p CH ₂ CH ₂ NH ₂ , NH (CH ₂ CH ₂ NH) _p CH ₂ CH ₂ OH, NH(CH ₂ CH ₂ S) _p CH ₂ CH ₂ OH,NH-R1-NH ₂ , or NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHPO3H ₂ where Aa represents 1-8 amino acids; p equals 1-5000; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; R1, L1 and L2 are as defined in formula (I).

Auristatins and dolastatins are preferred in conjugations involving bislinkers according to the present patent. Auristatins (e.g., auristatin E (AE), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin (MMAF), auristatin F phenylenediamine (AFP)) and the synthetic phenylalanyl variant of MMAE), which are synthetic versions of dolastatin analogues are described in Int. J. Oncol. 15: 367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-32 (2004); US Patent Application No.

11134826, 20060074008, 2006022925. US patents No. 4414205, 4753894, 4764368, 4816444, 4879278, 4943628, 4978744, 5122368, 5165923.51 69774, 5286637, 5410024, 5521284, 5530097, 5554725, 5585089, 5599902, 5629197,5635483, 5654399, 5663149 , 5665860,5708146, 5714586, 5741892, 5767236, 5767237, 5780588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065, 604 8720, 6054297, 6054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 6342219, 6342221, 6407213 , 6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097840, 7098305, 7098308, 7498298, 7375078, 7462352, 75 53816, 7659241, 7662387, 7745394, 7754681, 7829531, 7837980, 7837995,7902338, 7964566, 7964567, 7851437 , 7994135.

Examples of antibody-auristatin conjugate structures linked through the linker of this patent are Au01, Au02, Au03, Au04, Au05, Au06, Au07, Au08, Au09, Au10, Au11, Au12 and Au13

- 54 044827

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O) S, OC(O)N(R ₁ ), N(R ₁ )C(O)N(R ₁ ), CH, C(O)NHNHC(O) and C(O)NRi; R12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa)nCOOH, O(CH2CH2O)pCH2CH2OH, O(CH2CH2O)pCH2CH2NH2, NH(CH2CH2O)pCH2CH2NH2, NR1R1', NHOH, NHOR1, O(CH2CH2O)pCH2CH2COOH, NH(CH2CH2O)pCH2CH2COOH, NH-Ar-COOH, NHAr-NH2, O(CH2CH2O)pCH2CH2NHSO3H, NH(CH2CH2O)pCH2CH2NHSO3H, R1-NHSO3H, NH-R1-NHSO 3H . 2CH2NH2, NH(CH2CH2S)pCH2CH2NH2 , NH(CH2CH2NH)pCH2CH2OH, NH(CH2CH2S)pCH2CH2OH NH-R1-NH2 or NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ NHPO ₃ H ₂ , where Aa represents 1-8 amino acids; p equals 1-5000; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; p equals 1-5000; preferably R1, _R2 , R3 and R4 are independently H, C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether, amide, amines, heterocycloalkyl or acyloxyamines; or peptides containing 1-8 amino acids, or a polyethyleneoxy group having the formula (OSH ₂ CH ₂ ) _p or (OSH ₂ CH (CH ₃ )) _p , where p is an integer from 1 to about 5000; Two Rs: R1R2, R2R3, R1R3 or R ₃ R ₄ can form a 3-8 membered cyclic ring from an alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl group; X3 is H, CH3 or X1'R1', where X1' is NH, N(CH ₃ ), NHNH, O or S; R1' is H or C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkyl

- 55 044827 cycloalkyl or acyloxyamines; R ₃ ' represents H or C1-C6 linear or branched alkyl; Z ₃ ' represents H, COOR1, NH2, NHR1, OR1, CONHR1, NHCOR ₁ , OCOR1, OP(O)(OM ₁ )(OM ₂ ), OCH ₂ OP(O)(OM1)(OM ₂ ), OSO3M1 , R1, O-glycoside (glucoside, galactoside, mannoside, glucuronoside/glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH ₂ -glycoside; M1 and _M2 independently represent H, Na, K, Ca, Mg, NH4, NR1R2R3; Z1, _Z2 , L1 and L2 are as defined in formula (I).

Benzodiazepine dimers (eg, pyrrolobenzodiazepine (PBD) or (tomaimycin) dimmers, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines), which are the preferred cytotoxic agents of the present invention, are described in the literature: US Pat. No.

8163736; 8153627; 8034808; 7834005; 7741319; 7704924; 7691848; 7678787; 7612062;

7608615; 7557099; 7528128; 7528126; 7511032; 7429658; 7407951; 7326700; 7312210;

7265105; 7202239; 7189710; 7173026; 7109193; 7067511; 7064120; 7056913; 7049311;

7022699; 7015215;6979684; 6951853;6884799; 6800622;6747144;6660856; 6608192;

6562806; 6977254; 6951853; 6909006; 6344.451; 5880122; 4935362; 4764616; 4761412;

4723007; 4723003; 4683230; 4663453; 4508647; 4464467; 4427587; 4000304;

US patent applications 20100203007, 20100316656, 20030195196. Examples of antibody-benzodiazepine dimer conjugate structures linked through a linker are PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB08, PB09, PB10, PB1 1, RV12, RV13, RV14, RV15, RV16, RV17, RV18, RV19, RV20, RV21 and RV22:

- 56 044827

- 57 044827

- 58 044827 where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1, and Y1 independently represent O, NH, NHNH, NR ₅ , S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC( O)S, OC(O)N(Ri), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; L1, _L2 , Z1 and _Z2 are as defined in formula (I). R1, _R2 , _R3 , _R1 ', R2' and _R3 ' independently represent H; F; Cl; =O; =S; HE; SH; C1-C ₈ linear or branched alkyl, aryl, alkenyl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester (COOR ₅ or -OC(O)R ₅ ), ether (OR ₅ ), amide (CONR ₅ ), carbamate (OCONR ₅ ), amines (NHR ₅ , NR ₅ R ₅ '), heterocycloalkyl or acyloxyamines (-C(O)NHOH, -ONHC(O)R ₅ ); or peptides containing 1-8 natural or unnatural amino acids, or a polyethyleneoxy group having the formula (OCH ₂ -CH ₂ )p or (OCH ₂ CH(CH ₃ )) _p , where p is an integer from 1 to about 5000. Two Rs: R1R2, R ₂ R ₃ , R1R ₃ , Ri'R ₂ ', R ₂ 'R ₃ ' or R1'R ₃ ' may form a 3-8 membered cyclic ring from an alkyl, aryl, heteroaryl, heteroalkyl or alkylcycloalkyl group; X ₂ and Y2 independently represent N, CH2 or CR5, where R ₅ represents H, OH, NH2, NH(CH ₃ ), NHNH2, COOH, SH, OZ ₃ , SZ3 or C1-C8 linear or branched alkyl, aryl , heteroaryl, heteroalkyl, alkylcycloalkyl or acyloxyamines; Z ₃ is H, OP(O)(OM1)(OM ₂ ), OCH ₂ OP(O)(OM1)(OM ₂ ), OSO3M1 or O-glycoside (glucoside, galactoside, mannoside, glucuronoside/glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH ₂ -glycoside; M1 and _M2 independently represent H, Na, K, Ca, Mg, _NH4 , NR1R2R3.

Amatoxins, which are a subset of at least ten toxic compounds originally found in several genera of poisonous mushrooms, particularly Amanita phalloides and some other species of mushrooms, are also preferred for conjugation according to the present patent. These ten amatoxins, called α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanullin, amanullinic acid, amaninamide, amanin, proamanullin, are rigid bicyclic peptides that are synthesized as proproteins of 35 amino acids, of which the last eight amino acids are cleaved by prolyl oligopeptidase (Litten, W. 1975 Scientific American232 (3): 90-101; N. E. Hallen, et al., 2007 Proc. Nat. Ac. Sci. USA 104, 19097-101; K. Baumann, et al., 1993 Biochemistry 32 (15): 4043-50; Karlson-Stiber C., Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, P. A. et al. 1978 Arch. Microbio. 118 (3 ): 317-9). Amatoxins kill cells by inhibiting RNA polymerase II (Pol II), stopping gene transcription and protein biosynthesis (Brodner, O.G. and Wieland, T. 1976 Biochemistry, 15(16): 3480-4; Fiume, L., Curr Probl Clin Biochem , 1977, 7: 23-8; Karlson-Stiber C, Persson H. 2003, Toxicon 42(4): 339-49; Chafin, D. R., Guo, H. & Price, D. H. 1995 J. Biol. Chem. 270 ( 32): 19114-19; Wieland (1983) Int. J. Pept. Protein Res. 22(3): 257-76.). Amatoxins can be obtained from harvested Amanita phalloides mushrooms (Yocum, R.R. 1978 Biochemistry 17(18): 3786-9; Zhang, P. et al., 2005, FEMS Microbiol. Lett.252(2), 223-8), or by fermentation using a basidiomycete (Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89(1): 73-6) or by fermentation using A. fissa (Guo, X.W., et al., 2006 Wei Sheng Wu Xue Bao 46(3): 373-8), or by culturing Galerina fasciculata or Galerina helvoliceps, a strain belonging to the genus (WO/1990/009799, JP11137291). However, the yields from isolation and fermentation were quite low (less than 5 mg/L of culture). Over the past three decades, several methods for the production of amatoxins and their analogues have been reported.

- 59 044827 (W. E. Savige, A. Fontana,

Chern. Commun. 1976, 600-1; Zanotti, G., et al, Int J Pept Protein Res, 1981. 18(2): 162-8;

Wieland, T., et al., Eur. J. Biochem. 1981, 117, 161-4; P. A. Bartlett, et al, Tetrahedron Lett.

1982, 23, 619-22; Zanotti, G., et al., Biochim Biophys Acta, 1986. 870(3): 454-62; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 323-9; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 450-9; Zanotti, G., et al., Int J Pept Protein Res, 1988. 32(1): 9-20; G. Zanotti, T. et al, Int. J. Peptide Protein Res. 1989, 34, 222-8; Zanotti, G., et al., Int J Pept Protein Res, 1990.

35(3): 263-70; Mullersman, J. E. and J. F. Preston, 3rd, Int J Pept Protein Res, 1991. 37(6): 54451; Mullersman, J.E., et al, Int J Pept Protein Res, 1991. 38(5): 409-16; Zanotti, G., et al, Int J Pept Protein Res, 1992. 40(6): 551-8; Schmitt, W. et al, J. Am. Chern. Soc. 1996, 118, 4380-7; Anderson, M.O., et al, J. Org. Chern., 2005, 70(12): 4578-84; J. P. May, et al, J. Org. Chern.

2005, 70, 8424-30; F. Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J. P. May, D. M. Perrin, Chern. Eur. J. 2008, 14, 3404-9; J. P. May, et al, Chern. Eur. J. 2008, 14, 3410-17;

Q. Wang, et al, Eur. J. Org. Chern. 2002, 834-9; May, J. P. and D. M. Perrin, Biopolymers, 2007. 88(5): 714-24; May, J.P., et al., Chemistry, 2008. 14(11): 3410-7; S. De Lamo Marin, et al, Eur. J. Org. Chern. 2010, 3985-9; Pousse, G., et al., Org Lett, 2010. 12(16): 3582-5; Luo, H., et al., Chern Biol, 2014. 21(12): 1610-7; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5) and most of these methods were partial synthesis. Because of their extremely potent activity and unique mechanism of cytotoxicity, amatoxins have been used as conjugation payloads (Fiume, L., Lancet,

1969. 2 (7625): 853-4; Barbanti-Brodano, G. and L. Fiume, Nat New Biol, 1973. 243(130): 2813; Bonetti, E., M. et al, Arch Toxicol, 1976. 35(1): p. 69-73; Davis, M. T., Preston, J. F. Science 1981, 213, 1385-1388; Preston, J.F., et al, Arch Biochem Biophys, 1981. 209(1): 63-71; H. Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, L.S., et al., Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and L. Fiume, Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al, Toxicon, 1987. 25(9): 981-7; Khalacheva, K et al, Eksp Med Morfol, 1990. 29(3): 26-30; U. Bermbach, H. Faulstich, Biochemistry 1990, 29, 6839-45; Mullersman, J. E. and J. F. Preston, Int. J. Peptide Protein Res. 1991, 37, 544-51; Mullersman, J.E. and J.F. Preston, Biochem Cell Biol, 1991. 69(7): 418-27; J. Anderl, H. Echner, H. Faulstich, Beilstein J. Org. Chern. 2012, 8, 2072-84; Moldenhauer, G., et al, J. Natl. Cancer Inst. 2012, 104, 622-34; A. Moshnikova, et al; Biochemistry 2013, 52, 1171-8; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5; Zhou, B., et al., Biosens Bioelectron, 2015. 68: 189-96; WO2014/043403, US Patent 20150218220, European Patent 1661584).

We have been working on amatoxin conjugation for some time. Examples of antibody-amatoxin conjugate structures linked via a linker are the following structures that are preferred: Am01, Am02, Am03 and Am04:

- 60 044827

where ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR5, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O) S, OC(O)N(Ri), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; R ₇ , R8 and R9 independently represent H, OH, OR1, NH ₂ , NHR1, C1-C6 alkyl or none; Y ₂ represents O, O ₂ , NR1, NH or absent; R ₁₀ represents CH ₂ , O, NH, NR1, NHC(O), NHC(O)NH, NHC(O)O, OC(O)O, C(O), OC(O), OC(O) (NR1), (NR1)C(O)(NR1), C(O)R1 or absent; R11 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa)nCOOH, O(CH2CH2O)pCH2CH2OH, O(CH2CH2O)pCH2CH2NH2, NH(CH2CH2O)pCH2CH2NH2, NR1R1', O(CH2CH2O)pCH2CH2COOH, NH(CH2CH2O)pCH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(CH2CH2O)pCH2CH2NHSO3H, NH(CH2CH2O)pCH2CH2NHSO3H, R1-NHSO3H, NH-R1-NHSO3H, O (CH2CH2O)pCH2CH2NHPO3H2, NH(CH2CH2O)pCH2CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O)pCH2CH2OPOzH2, OR1-NHPO3H2, NH-R1-NHPO3H2 or NH(CH2CH2O)pCH2CH2 NHPO3H2, where Aa represents 1-8 amino acids; n and mi are independently equal to 1-20; p is equal to 1 -5000; R1, L1 and L2 are as defined in formula (I). L1, L2, R1, Z ₁ and Z ₂ are as defined in formula (I).

In yet another embodiment, the immunotoxin may be conjugated to a cell binding molecule via a bis linker according to the present patent. An immunotoxin as used herein is a macromolecular drug which is typically a cytotoxic protein derived from a bacterial or plant protein such as diphtheria toxin (DT), cholera toxin (CT), trichosanthin (TCS), dianthin, Pseudomonas exotoxin A (ETA '), erythrogenic toxins, diphtheria toxin, AB toxins, type III exotoxins, etc. It may also be a highly toxic bacterial pore-forming protoxin that requires proteolytic treatment for activation. An example of such a protoxin is proaerolysin and its genetically modified form, topsalicin. Topsalizin is a modified recombinant protein that has been designed to be selectively activated by an enzyme in the prostate gland, resulting in localized cell death and tissue destruction without damaging adjacent tissue.

- 61 044827 tissues and nerves.

In yet another embodiment, cell-binding ligands or cell receptor agonists can be conjugated to a cell-binding molecule via a bis-linker according to the present patent. Such conjugated cell-binding ligands or cell receptor agonists, particularly antibody-receptor conjugates, can not only act as a targeting vehicle/targeter to deliver the conjugate to malignant cells, but also be used to modulate or co-stimulate a desired immune response or change signaling pathways.

In immunotherapy, cell binding ligands or cell receptor agonists are preferred for conjugation to TCR (T cell receptor) T cell or CAR (chimeric antigen receptor) T cell or B cell receptor (BCR) natural killer cell antibody. NK) or cytotoxic cells. Such antibody is preferably anti-CD3, CD4, CD8, CD16 (FcyRIII), CD27, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57 ^bright , TNFe, Fasligand, MHC class I molecules (HLA-A, B, C ) or NKR-P1. Cell-binding ligands or receptor agonists are selected from, but are not limited to: folate derivatives (binds to the folate receptor, a protein overexpressed in ovarian cancer and other malignancies) (Low, PS et al., 2008, Acc. Chem. Res. 41, 120-9); urea glutamic acid derivatives (bind to specific membrane antigen of the prostate gland, a surface marker of prostate cancer cells) (Hillier, S.Metal, 2009, Cancer Res. 69, 6932-40); somatostatin (also known as growth hormone-inhibiting hormone (GHIH) or somatotropin-releasing-inhibiting factor (SRIF) or somatotropin-releasing-inhibiting hormone) and its analogs such as octreotide (Sandostatin) and lanreotide (Somatulin) (especially for neuroendocrine tumors, GH-producing pituitary adenoma, paraganglioma, non-functional pituitary adenoma, pheochromocytoma) (Ginj, M., et al., 2006, Proc. Natl. Acad. Sci. USA 103, 16436-41). In general, somatostatin and its receptor subtypes (sst1, sst2, sst3, sst4 and sst5) have been found in many types of tumors, such as neuroendocrine tumors, particularly GH-secreting pituitary adenomas (Reubi JC, Landolt AM 1984 J Clin Endocrinol Metab 59: 1148-51; Reubi JC, Landolt AM 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al., J Clin Endocrinol Metab 61: 98-103) and gastroenteropancreatic tumors (Reubi JC, et al. 1987 J Clin Endocrinol Metab 65: 1127-34; Reubi, JC, et al. 1990 Cancer Res 50: 5969-77), pheochromocytomas (Epelbaum J., et al 1995 J Clin Endocrinol Metab 80:1837-44; Reubi JC, et al., 1992 J Clin Endocrinol Metab 74: 1082-9), neuroblastoma (Prevost G., 1996 Neuroendocrinology 63:188-197; Moertel, CL, et al 1994 Am J Clin Path 102:752-756), medullary thyroid cancer (Reubi, JC, et al 1991 Lab Invest 64:567-573), small cell lung cancer (Sagman U, et al., 1990 Cancer 66:2129-2133), non- neuroendocrine tumors, including brain tumors such as meningiomas, medulloblastomas or gliomas (Reubi JC, et al., 1986 J Clin Endocrinol Metab 63: 433-8; Reubi JC, et al., 1987 Cancer Res 47: 5758-64; Fruhwald, MC, et al., 1999 Pediatr Res 45: 697-708), breast carcinoma (Reubi JC, et al., 1990 Int J Cancer 46: 416-20; Srkalovic G., et al., 1990 J Clin Endocrinol Metab 70: 661-669), lymphoma (Reubi JC, et al., 1992, Int J Cancer 50: 895-900), renal cell carcinoma (Reubi JC, et al., 1992, Cancer Res 52: 6074- 6078), mesenchymal tumors (Reubi JC, et al., 1996 Cancer Res 56: 1922-31), prostate tumors (Reubi JC, et al., 1995, J. Clin. Endocrinol Metab 80: 2806-14; et al. ., 1989, Prostate 14:191-208; Halmos G., et al. J. Clin. Endocrinol Metab 85: 2564-71), ovarian (Halmos, G., et al., 2000 J Clin Endocrinol Metab 85: 3509-12; Reubi JC, et al., 1991 Am J Pathol 138:1267-72), stomach (Reubi JC, et al., 1999, Int J Cancer 81: 376-86; Miller, GV, 1992 Br J Cancer 66: 391-95), hepatocellular carcinoma (Kouroumalis E., et al., 1998, Gut 42: 442-7; Reubi JC, et al., 1999 Gut 45: 66-774) and nasopharyngeal carcinomas (Loh KS, et al., 2002 Virchows Arch 441: 444-8); some aromatic sulfonamides specific for carbonic anhydrase IX (a marker of hypoxia and renal cell carcinoma) (Neri, D., et al., Nat. Rev. Drug Discov. 2011, 10, 767-7); peptides that activate pituitary adenylate cyclase (PACAP) (PAC1) for pheochromocytomas and paraganglia; vasoactive intestinal peptides (VIP) and their receptor subtypes (VPAC1, VPAC2) for lung, gastric, colon, rectal, breast, prostate, pancreatic duct, liver, bladder and epithelial tumors; α-melanocyte-stimulating hormone (α-MSH) receptors for various tumors; cholecystokinin (CCK)/gastrin receptors and their receptor subtypes (CCK1 (formerly CCK-A) and CCK2 for small cell lung cancer, medullary thyroid carcinomas, astrocytes, insulin and ovarian cancers; bombesin (Pyr-Gln-Arg-Leu-Gly- Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2)/gastrin-releasing peptide (GRP) and its receptor subtypes (BB1, GRP receptor subtype (BB2), BB3 and BB4) for kidney cells, carcinomas of the breast, lung, stomach and prostate and neuroblastoma (and neuroblastoma (Ohlsson, B., et al., 1999, Scand. J. Gastroenterology 34 (12): 1224-9; Weber, H. C, 2009, Cur Neurotensin receptors and subtypes its receptors (NTR1, NTR2, NTR3) for small cell lung cancer, neuroblastoma, pancreatic cancer, colon cancer and Ewing's sarcoma; substance P receptors and subtypes of its receptors (such as the NK1 receptor for glial tumors

- 62 044827 lei, Hennig IM, et al., 1995 Int. J. Cancer 61, 786-792); neuropeptide Y (NPY) receptors and its receptor subtypes (Y1 - Y6) for breast carcinomas; homing peptides include RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV), which recognize receptors (integrins) on the tumor surface (Laakkonen P, Vuorinen K. 2010 Integr Biol (Camb) 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E., et al., Anti-Cancer Agents Med Chem. 7 (5): 552-558; Kerr, J. S. et al., Anticancer Research, 19(2A), 959-968; Thumshirn, G., et al., 2003 Chem. Eur. J. 9, 2717-2725), and TAASGVRSMH or LTLRWVGLMS (chondroitin sulfate proteoglycan receptor NG2) and F3 peptides (a 31 amino acid peptide that binds to the nucleolin receptor expressed on the cell surface) (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143 , Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; M. A. Burg, 1999 Cancer Res., 59 (12), 2869-2874; K. Porkka, et al., 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9); cell penetrating peptides (CPP) (Nakase I., et al., 2012, J. Control Release. 159(2), 181-188); peptide hormones, such as luteinizing hormone releasing hormone (LHRH) agonists and antagonists and gonadotropin releasing hormone (GnRH) agonist, affect follicle stimulating hormone (FSH) and luteinizing hormone (LH) production, as well as testosterone, such as buserelin (Pyr -His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-ProNHEt), gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ ), goserelin (Pyr-His-Trp-Ser-Tyr-DSer(OtBu)-Leu-Arg-Pro-AzGly-NH ₂ ), histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu -Arg-Pro-NHEt), leuprolide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-ArgPro -Gly-NH ₂ ), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ ), nafarelin, deslorelin, abarelix (Ac-D-2Nal-D-4 -chloro-Phe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropyl-Lys-ProDAla-NH ₂ ), cetrorelix (Ac-D-2Nal-D -4-chloro-Phe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-AlaNH2), degarelix (Ac-D-2Nal-D-4-chloro -Phe-D-3-(3-pyridyl)Ala-Ser-4-amino-Phe(L-hydroorotyl)-D-4amino-Phe(carbamoyl)-Leu-isopropyl-Lys-Pro-D-Ala-NH ₂ ) and ganirelix (Ac-D-2Nal-D-4-chloro-Phe-D-3(3-pyridyl)Ala-Ser-Tyr-D-(N9,N10-diethyl)-homo-Arg-Leu-(N9 ,N10-diethyl)-homo-Arg-Pro-D-Ala-NH ₂ ) (Thundimadathil, J., J. Amino Acids, 2012, 967347, doi:10.H55/2012/967347; Boccon-Gibod, L.; et al., 2011, Therapeutic Advances in Urology 3(3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A. V; Nagy, A. 1999 Eur J Endocrinol 141:1-14; Koppan M, et al., 1999 Prostate 38:151-158); and pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins, and Nodlike receptors (NLRs) (Fukata, M., et al., 2009, Semin. Immunol. 21, 242-253; Maisonneuve , C., et al., 2014, Proc. Natl. Acad. Sci. USA 111, 1-6; Botos, I., et al., 2011, Structure 19, 447-459; Means, T. K., et al. , 2000, Life Sci. 68, 241-258), which range in size from small molecules (imiquimod, guanine and adenosine analogues) to large and complex biomacromolecules such as lipopolysaccharide (LPS), nucleic acids (CpG DNA, polyI:C ) and lipopeptides (Pam3CSK4) (Kasturi, SP, et al., 2011, Nature 470, 543-547; Lane, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C. 2012, Oncoimmunology 1, 227-228; Dudek, AZ, et al., 2007, Clin. Cancer Res. 13, 7119-25); calcitonin receptors, which are a 32 amino acid neuropeptide involved in the regulation of calcium levels, mainly through its effects on osteoclasts and the kidney (Zaidi M., et al., 1990 Crit Rev Clin Lab Sci 28, 109-174; Gorn, A.H. , et al., 1995 J Clin Invest 95:2680-91); and integrin receptors and its receptor subtypes (such as a _v e1, α _¥ β ₃ , a _v e ₅ , ave ₆ , α ₆ β ₄ , α ₇ β1, α _Ε β ₂ , a _IIb e _3, etc. ), which generally play an important role in angiogenesis, are expressed on the surfaces of various cells, in particular osteoclasts, endothelial cells and tumor cells (Ruoslahti, E. et al., 1994 Cell 77, 477-8; Albelda, SM et al., 1990 Cancer Res., 50, 6757-64). Short peptides, GRGDSPK and cyclic RGD pentapeptides, such as cyclo(RGDfV) (L1) and its derivatives [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N( Me)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me)V-)(silengitide)] demonstrated high binding affinity for intergreen receptors (Dechantsreiter, MA et al., 1999 J. Med. Chem. 42, 3033-40, Goodman, SL, et al., 2002 J. Med. Chem. 45, 1045-51).

Cell-binding ligands or cell receptor agonists can be Ig-based or non-Ig-based protein scaffold molecules. Ig-based scaffolds can be selected from, but are not limited to, nanobody (VHH (camelid Ig) derivative) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-97); domain antibodies (dAb, derivative of the VH or VL domain) (Holt, L.J., et al., 2003, Trends Biotechnol. 21, 484-90); bispecific T cell recruiter (BiTE, bispecific diabody) (Baeuerle, P.A., et al., 2009, Curr. Opin. Mol. Ther. 11, 22-30); dual affinity retargeting antibody (DART, bispecific diabody) (Moore P.A.P., et al. 2011, Blood 117(17), 4542-51); tetravalent tandem antibodies (TandAb, dimerized bispecific diabody) (Cochlovius, V., et al. 2000, Cancer Res. 60(16):4336-4341). Non-Ig-based scaffolds may be selected, but are not limited to, anticalin (a lipocalin derivative) (Skerra A. 2008, FEBS J., 275 (11): 2677-83; Beste G., et al., 1999 Proc. Nat Acad USA 96(5): 1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2): 337-50; Skerra, A. 2007, Curr Opin Biotechnol. 18(4): 295 -304;Skerra, A. 2008, FEBS J. 275(11):2677-83); adnectins (10th FN3 (fibronectin)) (Koide, A., et al., 1998 J. Mol. Biol., 284(4):1141-51; Batori V., 2002, Protein Eng. 15(12): 1015 -20; Tolcher, A.W., 2011, Clin. Cancer Res. 17(2): 363-71; Hackel, B.J., 2010, Protein Eng. Des.

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Sel. 23(4): 211-19); engineered ankyrin repeat proteins (DARPins) (derivatives of ankyrin repeat proteins (AR)) (Boersma, Y.L., et al., 2011 Curr Opin Biotechnol. 22(6): 849-57), such as DARPin C9, DARPin Ec4 and DARPin E69_LZ3_E01 (Winkler J., et al., 2009 Mol Cancer Ther. 8(9), 2674-83; Patricia M.-K.M., et al., Clin Cancer Res. 2011; 17(l):100-10; Boersma Y. L., et al., 2011 J Biol Chem 286 (48), 41273-85); avimers (domain A/low density lipoprotein (LDL) receptor) (Boersma Y.L., 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al., 2005 Nat. Biotechnol., 23(12) : 1556-61).

Examples of cell-binding antibody-ligand conjugate structures or cell receptor agonists or drugs linked via a bis-linker according to the patent application are listed below: LB01 (folate conjugate), LB02 (PMSA ligand conjugate), LB03 (conjugate with PMSA ligand), LB04 (PMSA ligand conjugate), LB05 (somatostatin conjugate), LB06 (somatostatin conjugate), LB07 (octreotide, somatostatin analog conjugate), LB08 (lanreotide, somatostatin analog conjugate), LB09 (vapreotide (sanvar) , somatostatin analog conjugate), LB10 (CAIX ligand conjugate), LB11 (CAIX ligand conjugate), LB12 (gastrin releasing peptide receptor (GRPr), MBA conjugate) LB13 (luteinizing hormone releasing hormone (LHRH) ligand conjugate and GnRH) , LB14 (luteinizing hormone releasing hormone ligand (LH-RH) and GnRH ligand conjugate), LB15 (GnRH antagonist, abarelix conjugate), LB16 (cobalamin, vitamin B12 analogue conjugate), LB17 (cobalamin, vitamin B12 analogue conjugate), LB18 ( pentapeptide cyclic RGD conjugate for integrin α _γ β ₃ receptor), LB19 (hetero-divalent peptide ligand VEGF receptor conjugate), LB20 (neuromedin B conjugate), LB21 (bombesin conjugate for G protein-coupled receptor), LB22 (TLR2 conjugate for Toll-like receptor), LB23 (for androgen receptor), LB24 (cilengitide/cyclo(-RGDfV-) intergreen a _v receptor conjugate), LB23 (fludrocortisone conjugate), LB25 (rifabutin analogue conjugate), LB26 (conjugate with with rifabutin analogue), LB27 (rifabutin analogue conjugate), LB28 (fludrocortisone conjugate), LB29 (dexamethasone conjugate), LB30 (fluticasone propionate conjugate), LB31 (beclomethasone dipropionate conjugate), LB32 (triam conjugate cinolone acetonide) , LB33 (prednisone conjugate), LB34 (prednisolone conjugate), LB35 (methylprednisolone conjugate), LB36 (betamethasone conjugate), LB37 (irinotecan analog conjugate), LB38 (crizotinib analog conjugate), LB39 (conjugate with bortezomib), LB40 (conjugate with carfilzomib analogue), LB41 (conjugate with carfilzomib analogue), LB42 (conjugate with leuprolide analogue), LB43 (conjugate with triptorelin analogue), LB44 (conjugate with clindamycin), LB45 (conjugate with liraglutide analogue), LB46 (semaglutide analogue conjugate), LB47 (retapamulin analogue conjugate), LB48 (inbulin analogue conjugate), LB49 (vinblastine analogue conjugate), LB50 (lixisenatide analogue conjugate), LB51 (osimertinib analogue conjugate), LB52 ( nucleoside analogue conjugate), LB53 (erlotinib analogue conjugate) and LB54 (lapatinib analogue conjugate), which are represented by the following structures:

/o/ -31. ✓x, 1 L II J n ' LB01 (folate conjugate), .noos 1 ' \ y / \ NOOS N . H LB02 (conjugate with PMSA ligand), ' / HOOC L \ HOOC^N^N - \ HH LB03 (conjugate with PMSA ligand), / HOOC 1 θ Xhooc^n^n ^{L 4} HH LB04 (conjugate with PMSA ligand), O °G° O L.-VZ.-s O ] 1 _x mAb kА N COOH ² ] _p ..... ' PC η ^S / ^mAb \ ° L ₂ JZ ₂ -s ^z COOH / _mi J _n

- 64 044827

(conjugate with CAIX ligand),

LB05 (somatostatin conjugate),

LB06 (somatostatin conjugate),

LB07 (octreotide, somatostatin analogue conjugate),

LB08 (lanreotide, somatostatin analogue conjugate),

LB09 (vapreotide(sanvar), somatostatin analogue conjugate),

LB10 (CAIX ligand conjugate),

- 65 044827

LB12 (gastrin releasing peptide receptor (GRPr), MBA conjugate), (luteinizing hormone releasing hormone (LH-RH) ligand conjugate and GnRH),

LB14 (conjugate of luteinizing hormone releasing factor ligand (LH-RH) and GnRH ligand),

LB17 (cobalamin, vitamin B12 analogue conjugate),

LB15 (GnRH antagonist, abarelix conjugate),

LB16 (cobalamin, vitamin B12 analogue conjugate),

- 66 044827

LB18 ( _pentapeptide cyclic RGD conjugate for _ανβ3 integrin receptor),

(VEGF receptor hetero-divalent peptide ligand conjugate),

LB20 (neuromedin B conjugate),

LB21 (bombesin conjugate for G protein-coupled receptor),

LB22 (TLR2 conjugate for Toll-like receptor),

LB23 (for androgen receptor),

LB24 (cilengitide/cyclo(-RGDfV-) intergreen receptor a _v conjugate)

LB25 (rifabutin analogue conjugate),

- 67 044827

LB32 (triamcinolone acetonide conjugate),

LB26 (rifabutin analogue conjugate),

LB27 (rifabutin analogue conjugate),

LB28 (fludrocortisone conjugate),

LB29 (dexamethasone conjugate),

LB30 (fluticasone propionate conjugate),

LB31 (conjugate with beclomethasone dipropionate),

- 68 044827

LB33 (prednisone conjugate),

LB34 (prednisolone conjugate),

LB35 (methylprednisolone conjugate),

LB36 (betamethasone conjugate),

LB37 (conjugate with irinotecan analogue),

LB38 (conjugate with crizotinib analogue),

LB39 (bortezomib analogue conjugate), where Y5 is N, CH, C(C1), C( _CH3 ) or C(COOR1); R1 represents H, C1-C6 alkyl, C ₃ -C ₈ Ar;

- 69 044827

LB43 (conjugate with triptorelin analogue),

LB40 (conjugate with carfilzomib analogue),

LB41 (conjugate with carfilzomib analogue),

LB42 (conjugate with leuprolide analogue),

LB44 (clindamycin conjugate), mAb -HN-HAQG-TF-T-SD ! ¹ H^yK.AAQ.GQLYSSV £ ₂ —L ₂ QFIAWLVRGRG-COOH

Ш] J η

LB45 (conjugate with liraglutide analogue),

η

LB46 (conjugate with semaglutide analogue),

- 70 044827

LB53 (conjugate with erlotinib analogue),

LB47 (conjugate with retapamulin analogue),

LB48 (inbulin analogue conjugate),

LB49 (conjugate with vinblastine analogue),

HOOC-H-G-E-G-T-F-T-S-D-L-S-KLB50 (lixisenatide analogue conjugate),

LB51 (conjugate with osimertinib analogue),

LB52 (neucleoside analogue conjugate),

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LB54 (lapatinib analogue conjugate), wherein ----- is optionally either a single bond or a double bond, or may optionally be absent; X1 and Y1 independently represent O, NH, NHNH, NR ₅ , S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O )S, OC(O)N(R1), N(Ri)C(O)N(Ri), CH, C(O)NHNHC(O) and C(O)NR1; The mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently equal to 1-20; L1, L2, R1, R1', _R2 , _Z1 and _Z2 are as defined in formula (I). X3 represents CH ₂ , O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR3), R ₁ , NHR1, NR _b C(O) R ₁ or absent; X ₄ represents H, CH ₂ , OH, O, C(O), C(O)NH, C(O)N(R1), R ₁ , NHR _b NR _b C(O)R ₁ or C(O )O; X ₅ represents H, CH3, F or Cl; M ₁ and M ₂ independently represent H, Na, K, Ca, Mg, NH4, NR1R2R3; R ₆ is 5'-deoxyadenosyl, Me, OH or CN;

In yet another embodiment, one, two or more DNA, RNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) and PIWI interacting RNA (piRNA) are preferred for conjugation to a cell-binding molecule via a bis-linker according to this patent. It is known that small RNAs (miRNAs, microRNAs, piRNAs) and long non-coding antisense RNAs are responsible for epigenetic changes in cells (Goodchild, J (2011), Methods in molecular biology (Clifton, N.J.). 764: 1-15). DNA, RNA, mRNA, miRNA, microRNA or piRNA as used herein may be single- or double-stranded with nucleotide units ranging from 3 to 1 million, and some of these nucleotides may be non-natural (synthetic), for example, as in a phosphorothioate-linked oligonucleotide, for example, in fomivirsen, or the nucleotides may be linked by phosphorothioate bonds, instead of phosphodiester bonds in natural RNA and DNA, and the sugar moieties are deoxyribose in the middle part of the molecule and 2'-omethoxyethyl modified ribose at the two ends, as in mipomersen, or an oligonucleotide derived from a peptide nucleic acid (PNA), morpholino, phosphorothioate, thiophosphoramidate, or from a 2'-O-methoxyethyl (MOE), 2'-O-methyl, 2'-fluoro, blocked nucleic acid (LNA), or bicyclic nucleic acid ribose sugar acid (BNA), or nucleic acids are modified to remove the 2'-3' carbon bond in the sugar ring (Whitehead, K. A.; et al. (2011), Annual Review of Chemical and Biomolecular Engineering 2: 77-96; Bennett, C. F.; Swayze, E.E. (2010), Annu. Rev. Pharmacol. Toxicol. 50: 259-29). Preferably, the length of the oligonucleotides ranges from about 8 to more than 100 nucleotides. An example of the structure of the conjugates is presented below:

where mAb, m _b n, X _b L _b L ₂ , Z _b Z ₂ , ----- are as defined in formula (I) or above;

is single or double strands of DNA, RNA, mRNA, miRNA, microRNA or piRNA; Y is preferably O, S, NH or CH ₂ .

In yet another embodiment, IgG antibody conjugates conjugated to one, two or more differently functional molecules or drugs are preferably specifically conjugated to a pair of thiols (via reduction of disulfide bonds) between the light chain and the heavy chain, the top disulfide bond between the two heavy chains and a lower disulfide bond between the two heavy chains, as represented by the following structure, ST1, ST2, ST3, ST4, ST5 or ST6:

- 72 044827

where Z1, Z ₂ , X, Y, Li, L ₂ , -----, mi and cytotoxic molecule are defined in the same way as X1 in formula (I) above.

In addition, the cytotoxic molecules and m1 at different conjugation sites of a cell-binding molecule may differ when cytotoxic molecules containing the same or different bis-linkers are sequentially conjugated to a cell-binding molecule, or when different cytotoxic molecules containing the same and different the same or different bis-linkers are added in stages to the conjugation reaction mixture containing the cell-binding molecule.

Production of dosage forms and application.

The patent pending conjugates are formulated in liquid form or are suitable for lyophilization and are subsequently reconstituted to a liquid formulation. A liquid composition containing 0.1 g/L ~ 300 g/L concentration of the active ingredient conjugate for delivery to a patient without high levels of antibody aggregation may include one or more polyols (eg, sugars), a buffering agent with a pH of 4.5 to 7 5 surfactant (eg polysorbate 20 or 80), antioxidant (eg ascorbic acid and/or methionine), tonicity agent (eg mannitol, sorbitol or NaCl), chelating agents such as EDTA; metal complexes (eg Zn-protein complexes);

- 73 044827 biodegradable polymers such as polyesters; a preservative (eg benzyl alcohol) and/or a free amino acid.

Suitable buffering agents for use in the formulations include, but are not limited to, salts of organic acids such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, tromethamine (tris(hydroxymethyl)aminomethane) hydrochloride or phosphate buffer. In addition, amino acid components can also be used as a buffering agent. Such amino acid component includes, but is not limited to, arginine, glycine, glycylglycine and histidine. Arginine buffers are buffers including arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In some embodiments, the arginine buffer includes arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The buffer compositions have a pH of from 4.5 to 7.5, preferably from about 4.5 to about 6.5, more preferably from about 5.0 to about 6.2. In some embodiments, the concentration of organic acid salts in the buffer is from about 10 mM to about 500 mM.

A polyol, which may optionally be included, is a substance with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and/or isotonicizing agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from both physical and chemical degradation. Preferentially excluded cosolvents increase the effective surface tension of the solvent at the protein interface, causing the most energetically favorable structural conformations to be those with the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids. A reducing sugar is one that contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins, and a non-reducing sugar is one that does not have these reducing sugar properties. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol and glycerol. Sugar acids include L-gluconate and its metal salts. Preferably a non-reducing sugar: sucrose or trehalose at a concentration of about 0.01 to 15% is selected in the formulation, where trehalose is preferred over sucrose due to the stability of the trehalose solution.

The surfactant, optionally in the formulations, is selected from polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and the like); poloxamer (for example, poloxamer 188, poly(ethylene oxide) poly(propylene oxide), poloxamer 407 or polyethylene-polypropylene glycol and the like); Triton; sodium dodecyl sulfate (SDS); sodium laurisulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl betaine (eg lauroamidopropyl); myristamidopropyl, palmidopropyl or isostearamidopropyldimethylamine; sodium methylcocoyl or disodium methyloleyl taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate; and MONAQUAT™ series (for example, isostearylethylimidonium ethosulfate); polyethylglycol, polypropylglycol and ethylene-propylene glycol copolymers (eg Pluronics, PF68, etc.); and so on. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, for example polysorbate 20, 40, 60 or 80 (Tween 20, 40, 60 or 80). The surfactant concentration ranges from 0.0001% to about 1.0%. In certain embodiments, the surfactant concentration is from about 0.01% to about 0.1%. In one embodiment, the surfactant concentration is about 0.02%.

A preservative, optionally in formulations, is a compound that significantly reduces the bacterial action therein. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The preservative is included in the composition in an amount of less than 5%. Preferably 0.01 to 1%. In one embodiment, the preservative herein is benzyl alcohol.

Suitable free amino acids for optional use in the formulation include, but are not limited to, arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine,

- 74 044827 glutamic acid or aspartic acid. The inclusion of a basic amino acid is preferred, ie arginine, lysine and/or histidine. If the composition includes histidine, it can act as either a buffering agent or a free amino acid, but when a histidine buffer is used, it typically includes a free non-histidine amino acid, such as a histidine buffer and lysine. The amino acid may be present in its D and/or L form, but the L form is typical. The amino acid may be present as any suitable salt, for example a hydrochloride salt such as arginine HCl. The amino acid concentration ranges from 0.0001 to 15.0%. Preferably from 0.01 to 5%.

The compositions may optionally contain methionine or ascorbic acid as an antioxidant at a concentration of from about 0.01 to 5 mg/ml; The compositions may optionally contain a chelating agent, such as EDTA, EGTA, etc., at a concentration of about 0.01 to 2 mM.

The final formulation can be adjusted to _the preferred pH using an adjusting agent (e.g., an acid such as HCl, _H2SO4 , acetic acid, _{H3PO4 ,} citric acid, etc., or a base such as NaOH, KOH , NH3OH, ethanolamine, diethanolamine or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc.), and the composition should be kept isotonic, meaning that the composition of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing osmometer, for example.

Other excipients that may be used in the liquid or lyophilized formulation according to the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine , imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, dextran (10, 40 and/or 70 kDa), polydextrose, maltodextrin, Ficoll, gelatin, hydroxypropyl methyl, sodium phosphate, potassium phosphate, ZnCl ₂ , zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohols or polyhydric alcohols, hydrogenated forms of carbohydrates with carbonyl a group reduced to a primary or secondary hydroxyl group.

Other contemplated excipients that may be used in the aqueous pharmaceutical compositions according to the patent application include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such as sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the compositions of the present invention are known in the art, for example, as set forth in The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds. , American Pharmaceuticals Association (2003) and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

In some embodiments, the present invention relates to a method of preparing a formulation, comprising the steps of: (a) lyophilizing a formulation containing conjugates, excipients and a buffer system into a powder; and (b) reconstituting the lyophilized mixture from step (a) in a reconstitution medium such that the reconstituted composition is stable. The composition in step (a) may further comprise a stabilizer and one or more excipients selected from the group consisting of filler, salt, surfactant and preservative, as described above. Several dilute organic acids or water can be used as reducing media, i.e. sterile water, bacteriostatic water for injection (BWFI). The reconstitution medium may be selected from water, i.e. sterile water, bacteriostatic water for injection (BWFI) or the group consisting of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic sodium chloride solution, acidic magnesium chloride solution and an acidic solution of arginine, in an amount of from about 10 to about 250 mM.

The liquid pharmaceutical composition of the conjugates according to this patent application must exhibit a variety of predetermined characteristics. One of the major concerns in liquid drug products is stability, as proteins/antibodies tend to form soluble and insoluble aggregates during production and storage. In addition, various chemical reactions (deamidation, oxidation, restriction, isomerization, etc.) can occur in the solution, leading to an increase in the level of degradation products and/or loss of biological activity. Preferably, the conjugate in liquid or lyophilisate form should have a shelf life of more than 18 months at 25°C. More preferably, the conjugate in liquid or lyophilisate form should have a shelf life of more than 24 months at 25°C. The most preferred liquid formulation should exhibit a shelf life of approximately 24-36 months at 2-8°C, and the lyophilisate formulation

- 75 044827 should exhibit a shelf life of approximately preferably up to 60 months at 2-8°C. Both liquid and lyophilized formulations must demonstrate a shelf life of at least two years at -20 or -70°C.

In some embodiments, the composition is stable after freezing (eg, -20 or -70°C) and thawing the composition, for example, after 1, 2, or 3 freeze-thaw cycles. Stability can be assessed qualitatively and/or quantitatively in a variety of ways, including drug/antibody (protein) ratios and aggregate formation (eg, using UV, size exclusion chromatography, turbidity measurements, and/or visual inspection); by assessing charge heterogeneity using cation exchange chromatography, isoelectric capillary image focusing (icIEF), or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS) or HPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody; peptide map analysis (eg tryptic or LYS-C); assessment of the biological activity or antigen-binding function of the antibody; and so on. Instabilities may include one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), excision/hydrolysis/fragmentation (e.g., hinge region fragmentation), formation succinimide, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

A stable conjugate should also retain its biological activity in the pharmaceutical composition if the biological activity of the conjugate at a given time, for example, over 12 months, is within about 20%, preferably about 10% (within assay errors) of the biological activity exhibited at that time. the time at which the pharmaceutical composition was prepared was determined, for example, in an antigen binding assay and/or in an in vitro cytotoxicity assay.

A pharmaceutical container or vessel is used to store the pharmaceutical composition of any of the conjugates according to the patent application. The container is a vial, bottle, prefilled syringe, or prefilled syringe with autoinjector.

For in vivo clinical use, the bis-linked conjugate of the invention will be supplied as solutions or as a lyophilized solid that can be redissolved in sterile water for injection. Examples of suitable conjugate administration protocols are as follows. Conjugates are administered daily, weekly, biweekly, three times weekly, once every four weeks, or monthly for 8 to 54 weeks as an intravenous bolus. Bolus doses are administered in amounts of 50 to 1000 mL of saline, to which human serum albumin may optionally be added (eg, 0.5 to 1 mL of concentrated human serum albumin solution, 100 mg/mL). Doses will range from about 50 mcg to 20 mg/kg body weight per week, IV. (ranging from 10 mcg to 200 mg/kg per injection) 4 ~ 54 weeks after treatment, the patient can undergo a second course of treatment. Clinicians can define specific clinical protocols regarding route of administration, excipients, diluents, dosages, timing, etc.

Examples of medical conditions that can be treated in accordance with methods of destroying selected cell populations in vivo or ex vivo include malignancies of any type, autoimmune diseases, transplant rejection and infections (viral, bacterial or parasitic).

The amount of conjugate required to achieve the desired biological effect will vary depending on a number of factors, including the chemical characteristics, activity and bioavailability of the conjugates, the type of disease, the species to which the patient belongs, the patient's disease state, the route of administration, all factors that dictate required dosage quantities, delivery and mode for administration.

In general, the bis-linker conjugates of the present invention can be provided in an aqueous physiological buffer solution containing 0.1-10% w/v of the conjugates for parenteral administration. Typical dosage ranges are 1 mcg/kg to 0.1 g/kg body weight per day; weekly, biweekly, triweekly or monthly, the preferred dosage range is 0.01 to 20 mg/kg body weight, weekly, biweekly or monthly equivalent human dose. The preferred dosage of drug to be administered is likely to depend on such variables as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative biological effectiveness of the selected compound, the composition of the compound, the route of administration (intravenous, intramuscular, or other) , the pharmacokinetic properties of the conjugates along the chosen delivery route, as well as the rate (bolus or continuous infusion) and schedule of administration (number of repetitions over a certain period of time).

The linker conjugates of the present invention can also be administered in unit dosage forms, where the term unit dose means a single dose that can be administered to a patient and that can be easily processed and packaged while remaining in the form of a physically and chemically stable unit dose containing either the active conjugate itself, or in the form of a pharmaceutically acceptable composition as described below. Thus, typical total daily/weekly/biweekly/monthly doses range from 0.01 to 100 mg/kg body weight. As a general guide, standard doses for humans range from 1 mg to 3000 mg per day or per week, for two weeks (biweekly), three weeks, or per month. Preferably, the unit dosage range is from 1 to 500 mg administered once to four times a month, and even more preferably from 1 to 100 mg administered once a week or once every two weeks or once every three weeks. The conjugates provided herein can be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be formulated for oral administration, especially in the form of tablets, plain capsules or soft gel capsules; or intranasally, especially in the form of powders, nasal drops or aerosols; or cutaneously, for example, topically in the form of ointments, creams, lotions, gels or sprays or through transdermal patches.

In yet another embodiment, a pharmaceutical composition containing a therapeutically effective amount of a conjugate of formula (II) and any of the conjugates described in this patent application may be administered simultaneously with other therapeutic agents, such as a chemotherapeutic agent, radiation therapy, immunotherapeutic agents, autoimmune agents disorders, anti-infective agents or other conjugates for the synergistically effective treatment or prevention of cancer or an autoimmune disease or infectious disease. The synergistic agents are preferably selected from one or more of the following drugs: abatacept (Orencia), abiraterone acetate (Zytiga®), abraxane, acetaminophen/hydrocodone, adalimumab, afatinib dimaleate (Gilotrif®), alectinib (Alecensa), alemtuzumab (Campath®), alitretinoin (Panretin®), ado-trastuzumab emtansine (Kadcyla™), mixed amphetamine salts (amphetamine/dextroamphetamine or Aderal XR), anastrozole (Arimidex®), aripiprazole, atazanavir, atezolizumab (Tecentriq, MPDL3280A), atorvastatin, axitinib (Inlyta® ), AZD9291, belinostat (Beleodaq™), bevacizumab (Avastin®), bortezomib (PS-341; Velcade, Neomib, Bortecad), cabazitaxel (evtana®), cabozantinib (Cometriq™), bexarotene (Targretin®), blinatumomab (Blincyto ™), bortezomib (Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), budesonide, budesonide/formoterol, buprenorphine, capecitabine, carfilzomib (Kyprolis®), celecoxib, ceritinib (LDK378/Zykadia), cetuximab (Erbitux ®), cyclosporine, cinacalcet, crizotinib (Xalkori®), cobimetinib (Cotellic), dabigatran, dabrafenib (Tafinlar®), daratumumab (Darzalex), darbepoetin alfa, darunavir, imatinib mesylate (Gleevec®), dasatinib (Sprycel®), denileukindiftitox (Ontak®), denosumab (Xgeva®), Depakote, dexamethasone, dexlansoprazole, dexmethylphenidate, dinutuximab (Unituxin™), doxycycline, duloxetine, durvalumab (MEDI4736), elotuzumab (Empliciti), emtricitabine/rilpivirine/tenofovir disoproxil fumarate, emtricitabine/tenofov ir/ efavirenz, enoxaparin, enzalutamide (Xtandi®), epoetin alfa, erlotinib (Tarceva®), esomeprazole, eszopiclone, etanercept, everolimus (Afinitor®), exemestane (Aromasin®), everolimus (Afinitor®), ezetimibe, ezetimibe/simvastatin, fenofibrate , filgrastim, fingolimod, fluticasone propionate, fluticasone/salmeterol, fulvestrant (Faslodex®), gefinitib (Iressa®), glatiramer, goserelin acetate (Zoladex), icotinib, imatinib (Gleevec), ibritutomab tiuxetan (Zevalin®), ibrutinib (Imbruvica ™ ), idelalisib (Zydelig®), infliximab, iniparib, insulin aspart, insulin detemir, insulin glargine, insulin lispro, interferon beta-1a, interferon beta-1b, lapatinib (Tykerb®), ipilimumab (Yervoy®), ipratropium bromide/salbutamol , ixazomib (Ninlaro®), lanreotide acetate (Somatuline® Depot), lenaliomide (Revlimid®), lenvatinib (Lenvima™), letrozole (Femara®), levothyroxine, levothyroxine, lidocaine, linezolid, liraglutide, lisdexamfetamine, MEDI4736 (AstraZeneca, Celgene) , memantine, methylphenidate, metoprolol, modafinil, mometasone, necitumumab (Portrazza), nilotinib (Tasigna®), niraparib, nivolumab (Opdivo®), ofatumumab (Arzerra®), obinutuzumab (Gazyva™), olaparib (Lynparza™), olmesartan, olmesartan/hydrochlorothiazide, omalizumab, omega-3 fatty acid ethyl esters, oseltamivir, osimertinib (or mereletinib, Tagrisso), oxycodone, palbociclib (Ibrance®), palivizumab, panitumumab (Vectibix®), panobinostat (Farydak®), pazopanib (Votrient® ), pembrolizumab (Keytruda®), pemetrexed (Alimta), pertuzumab (Perjeta™), pneumococcal conjugate vaccine, pomalidomide (Pomalyst®), pregabalin, propranolol, quetiapine, rabeprazole, radium 223 chloride (Xofigo®), raloxifene, raltegravir, ramucirumab (Cyramza®), ranibizumab, regorafenib (Stivarga®), rituximab (Rituxan®), rivaroxaban, romidepsin (Istodax®), rosuvastatin, ruxolitinib phosphate (Jakafi™), salbutamol, sevelamer, sildenafil, siltuximab (Sylvant™), sitagliptin, sitagliptin/metformin, solifenacin, sonidegib (LDE225, Odomzo), sorafenib (Nexavar®), sunitinib (Sutent®), tadalafil, tamoxifen, telaprevir, talazoparib, temsirolimus (Torisel®), tenofovir/emtricitabine, testosterone gel, thalidomide (Immunoprin, Talidex), tiotropium bromide, toremifene (Fareston®), trametinib (Mekinist®), trastuzumab, trabectidin (ecteinascidin 743, Yondelis), trifluridine/tipiracil (Lonsurf, TAS-102), tretinoin (Vesanoid®), ustekinumab, valsartan, veliparib , vandetanib (Caprelsa®), vemurafenib (Zelboraf®), venetoclax (Venclexta),

- 77 044827 vorinostat (Zolinza®), ziv-aflibercept (Zaltrap®), Zostavax, and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients thereof, or a combination of the above.

The drugs/cytotoxic agents used for conjugation through the bridging linker according to the present patent may be any analogs and/or derivatives of the drugs/molecules described in the present patent. One skilled in the art of drugs/cytotoxic agents will readily appreciate that each of the drugs/cytotoxic agents described herein can be modified such that the resulting compound still retains the specificity and/or activity of the parent compound. One skilled in the art will also appreciate that many of these compounds can be used in place of the drugs/cytotoxic agents described herein. Thus, the drugs/cytotoxic agents of the present invention include analogs and derivatives of the compounds described herein.

All references cited herein and in the following examples are expressly incorporated by reference in their entirety.

Examples

The present invention is further described in the following examples, which are not intended to limit the scope of the present invention. The cell lines described in the following examples were maintained in culture in accordance with the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), or the Shanghai Institute of Cell Culture, Chinese Academy of Science, if not otherwise stated. Cell culture reagents were obtained from Invitrogen Corp. unless otherwise noted. All anhydrous solvents were commercially prepared and stored in sealed bottles under nitrogen. All other reagents and solvents were purchased of the highest quality and used without further purification. Preparative HPLC separation was performed using Varain PreStar HPLC. NMR spectra were recorded on a Varian Mercury 400 MHz instrument. Chemical shifts (delta) are reported in parts per million (ppm) relative to tetramethylsilane at 0.00, and coupling constants (J) are reported in Hz. Mass spectral data were acquired on a Waters Xevo QTOF mass spectrometer equipped with a Waters Acquity UPLC separation module and an Acquity TUV detector.

Example 1. Synthesis of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate

To di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol) in DMF (150 ml) was added NaH (60% in oil, 2.76 g, 68.8 mmol). After stirring at room temperature. over 30 min, tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, additional methanol (3 ml) was added, evaporated, diluted with EtOAc (100 ml) and water (100 ml), separated and the aqueous layer was extracted with EtOAc (2x50 ml). The organic layers were combined, dried over MgSO ₄ , filtered, evaporated and purified by SiO ₂ column chromatography (EtOAc/Hexane 1:5 to 1:3) to give the title compound (12.98 g 82% yield) in in the form of a colorless oil. ESI MS m/z calculated for C ₂₂ H ₄₁ N ₂ O ₈ [M+H]+ 461.28, found 461.40.

Example 2. Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid

To di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) in 1,4-dioxane (40 ml) HCl (12M, 10 ml) was added. The mixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to obtain the crude title product for the next step without further purification (2.15 g, 103% yield, ~93% purity). ESI MS m/z calculated for C ₄ H ₉ N ₂ O ₄ [M+H]+ 149.05, found 149.40.

Example 3. Synthesis of 2,2'-(1,2-bis((benzyloxy)carbonyl)hydrazine-1,2-diyl)diacetic acid

Cbz Cbz

IK ^7)11

To a solution of 2,2'-(hydrazin-1,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in a mixture of THF (200 ml) and NaH ₂ PO ₄ (0.1 M, 250 ml, pH 8.0) Benzylcarbonyl chloride (5.01 g, 29.47 mmol) was added in 4 portions over 2 hours. The mixture was stirred for a further 6 hours, evaporated and purified on a SiO ₂ column, eluting with H ₂ O/CH ₃ CN (1:9) containing 1% formic acid to give the title compound (2.26 g, 73% yield, ~95% purity). ESI MS m/z calculated for C ₁₀ H ₂₁ N ₂ O ₈ [M+H]+ 417.12, found 417.40.

Example 4 Synthesis of dibenzyl 1,2-bis(2-chloro-2-oxoethyl)hydrazine-1,2-dicarboxylate

- 78 044827

(COCl) ₂ (905 mg, 7.13 mmol), then 0.030 ml of DMF was added. After stirring at room temperature for 2 hours, the mixture was diluted with toluene, evaporated and co-evaporated with dichloroethane (2x 20 ml) and toluene (2x 15 ml) to dryness to obtain the title crude product (which is unstable) for the next step without further purification (365 mg, 96% yield). ESI MS m/z calculated for C ₂₀ H ₁₉ Cl ₂ N ₂ O ₆ [M+H] ⁺ 453.05, found 453.50.

Example 5. Synthesis of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate

To a suspension of NaH (0.259 g, 6.48 mmol 3.0 eq.) in anhydrous DMF (2 ml) at room temperature was added di-tert-butylhydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol 1.0 eq.) in anhydrous DMF (8 ml) for 10 min under nitrogen. The mixture was stirred at room temperature for 10 minutes and then cooled to 0°C. Tert-Butyl 2-bromoacetate (1.4 mL, 8.61 mmol, 4.0 eq.) was added dropwise to the mixture. The resulting mixture was left to warm to room temperature and stirred overnight. Saturated ammonium chloride solution (100 ml) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (3x50 ml). The combined organic solution was washed with water and saturated sodium chloride solution, dried over anhydrous Na ₂ SO ₄ , evaporated and purified by SiO ₂ column chromatography (10:1 hexane/EtOAc) to give the title compound as a colorless oil ( 0.94 g, 99.6% yield). ESI MS m/z [M+Na] ⁺ 483.4.

Example 6. Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid

To a solution of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (0.94 g, 2.04 mmol) in DCM (4 ml) at At 0°C TFA (4 ml) was added. The reaction mixture was stirred for 30 minutes and then warmed to room temperature and stirred overnight. The mixture was evaporated, diluted with DCM and evaporated. This operation was repeated three times to obtain a white solid. Triturate with DCM and filter the white solid (0.232 g, 76.8% yield). ESI MS m/z [M+H]+ 149.2.

Example 7. Synthesis of 2,2'-(1,2-bis(2-chloroacetyl)hydrazine-1,2-diyl)diacetic acid

2-Chloroacetyl chloride (0.38 ml, 4.70 mmol 3.0 eq.) for 10 min. The reaction mixture was warmed to room temperature and stirred overnight, and evaporated. The residue was co-evaporated with THF three times to give a white solid (0.472 g, theoretical yield). ESI MS m/z [M+H] ⁺ 301.1.

Example 8 Synthesis of tert-butyl 2,8-dioxo-1,5-oxazocane-5-carboxylate

NOOS^^ VoDGTNG NOOS^^ P ₂ O _S ^

HOOC-x^ ^NH H ₂ O/NaOH * CH ₂ cf

Di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) was added to a solution of 3,3'-azanediyldipropanoic acid (10.00 g, 62.08 mmol) in 1.0 M NaOH (300 ml) at 4°C. in 200 ml THF for 1 hour. After addition, the mixture was stirred for 2 hours at 4°C. The mixture was carefully acidified to pH ~4 with 0.2 M H ₃ PO ₄ , evaporated under reduced pressure, extracted with CH ₂ Cl ₂ , dried over Na ₂ SO ₄ , evaporated and purified by flash chromatography on SiO ₂ , eluting with AcOH/ MeOH/CH ₂ Cl ₂ (0.01:1:5) to give 3,3'-((tert-butoxycarbonyl)azandiyl)dipropanoic acid (13.62 g, 84% yield). ESI MS m/z C _n H ₁₉ NO ₆ [M+H] ⁺ , calc. 262.27, found 262.40.

Phosphorus oxide _{( 8.70} g, 61.30 mmol). The mixture was stirred at 0°C for 2 hours and then at room temperature. for 1 hour, filtered through a small column of SiO ₂ and the column was washed with EtOAc/CH ₂ Cl ₂ (1:6). The filtrate was evaporated and triturated with EtOAc/hexane to give

- 79 044827 title compound (5.64 g, 74% yield). ESI MS m/z C11H17NO5 [M+H] ⁺ , calc. 244.11, found

244.30.

Example 9. Synthesis of tert-butyl 3-((benzyloxy)amino)propanoate

To O-benzylhydroxylamine hydrochloride (10.0 g, 62.7 mmol) in THF (100 ml) was added Et3N (15 ml) and tert-butyl acrylate (12.1 g, 94.5 mmol). The mixture was refluxed overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/hexane (1:4) to give the title compound 3 (13.08 g, 83% yield). ¹ H NMR (CDCl ₃ ) 7.49-7.25 (m, 5H), 4.75 (s, 2H), 3.20 (t, J=6.4 Hz, 2H), 2.54 (t , J=6.4 Hz, 2H), 1.49 (s, 9H); MS ESI m/z ⁺ C^^NNaOs (M+Na), calc. 274.15, found 274.20.

Example 10. Synthesis of tert-butyl 3-(hydroxyamino)propanoate

To tert-butyl 3-((benzyloxy)amino)propanoate (13.0 g, 51.76 mmol) in methanol (100 ml) was added Pd/C (0.85 g, 10% Pd, 50% humidity) in a vessel for hydrogenation. After the system was evacuated under vacuum and connected to hydrogen gas at 2 atm, the reaction mixture was stirred overnight at room temperature. The crude reaction mixture was passed through a pad of celite, washed with ethanol, evaporated and purified on a SiO ₂ column eluting with MeOH/DCM (1:10-1:5) to give the title compound (7.25 g, 87% yield) . 1H NMR (CDCl ₃ ) 3.22 (t, J=6.4 Hz, 2H), 2.55 (t, J=6.4 Hz, 2H), 1.49 (s, 9H); ESI MS m/z ⁺ C ₇ H ₁₅ NNaO ₃ (M+Na), calc. 184.10, found 184.30.

Example 11. Synthesis of tert-butyl 3-((tosyloxy)amino)propanoate _h /o. °

X z ^OH ►

About ΝO N

NN

To tert-butyl 3-(hydroxyamine)propionate (5.10 g, 31.65 mmol) in a mixture of DCM (50 ml) and pyridine (20 ml) was added tosylate chloride (12.05 g, 63.42) at 4°C. After addition, the mixture was stirred at room temperature overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/DCM (1:10-1:6) to give the title compound (8.58 g, 86% yield) . 1H NMR (CDCl ₃ ) 7.81 (s, 2H), 7.46 (s, 2H), 3.22 (t, J=6.4 Hz, 2H), 2.55 (t, J=6.4 Hz , 2H), 2.41 (s, 3H), 1.49 (s, 9H); ESI MS m/z ⁺ C^NNaOsS (M+Na), calc. 338.11, found 338.30.

Example 12. Synthesis of tert-butyl 3,3'-(hydrazine-1,2-diyl)dipropanoate

V 2 a a 2 V

Η Η tert-Butyl 3-aminopropanoate (3.05 g, 21.01 mmol) in THF (80 ml) was added tert-butyl 3((tosyloxy)amino)propanoate (5.10 g, 16.18 mmol). The mixture was stirred at room temperature for 1 hour and then at 45°C for 6 hours. The mixture was evaporated and purified on a SiO ₂ column, eluting with CH ₃ OH/DCM/Et ₃ N (1:12:0.01- 1:8:0.01) to give the title compound (2.89 g, 62% yield). MS ESI m/z ⁺ C ₁₄ H ₂₈ N ₂ NaO ₄ (M+Na), calc. 311.20, found 311.40.

Example 13. Synthesis of di-tert-butyl 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)hydrazine-1, 2-diyl)dipropanoate

To 3-maleidopropanoic acid (1.00 g, 5.91 mmol) in DCM (50 ml) was added oxalyl chloride (2.70 g, 21.25 mmol) and DMF (50 μl). The mixture was stirred at room temperature for 2 hours, evaporated and co-evaporated with DCM/toluene to obtain crude 3-maleidopropanoic acid chloride. Crude 3-maleidopropanoic acid chloride was added to di-tert-butyl 3,3'-(hydrazine-1,2-diyl)dipropanoate (0.51 g, 1.76 mmol) in DCM (35 mL). The mixture was stirred overnight, evaporated, concentrated and purified on a SiO ₂ column eluting with EtOAc/DCM (1:15-1:8) to give the title compound (738 mg, 71% yield). MS ESI m/z ⁺ C ₂₈ H ₃₈ N ₄ NaO ₁₀ (M+Na), calc. 613.26, found 613.40.

Example 14. Synthesis of 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)hydrazine1,2-diyl)dipropanoic acid

- 80 044827

HCl (1 mL conc.) was added to compound 14 (700 mg, 1.18 mmol) in dioxane (4 mL). The mixture was stirred for 30 min, diluted with EtOH (10 ml) and toluene (10 ml), evaporated and co-evaporated with EtOH (10 ml) and toluene (10 ml) to obtain the crude title product (560 mg) for the next step without additional cleaning. MS ESI m/z ^- C ₂₀ H ₂₁ N ₄ O ₁₀ (M-H), calc. 477.13, found 477.20.

Example 15. Synthesis of bis(2,5-dioxopyrrolidin-1-yl)-3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-Spyrrol-1-yl) propanoyl)hydrazine-1,2-diyl)dipropanoate

To the crude compound of 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)hydrazin-1,2-diyl)dipropanoic acid (~560 mg, ~1.17 mmol) NHS (400 mg, 3.47 mmol) and EDC (1.01 g, 5.26 mmol) were added to DMA (8 ml). The mixture was stirred overnight, evaporated, evaporated and purified on a SiO2 column eluting with EtOAc/DCM (1:12~1:7) to give the title compound (520 mg, 65% yield in two steps). ESI MS m/z ⁺ C ₂₈ H ₂₈ N ₆ NaO ₁₄ (M+Na), calc. 695.17, found 695.40.

Example 16. Synthesis of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate

To 350 ml of anhydrous THF, 80 mg (0.0025 mol) of sodium metal and triethylene glycol (150.1 g, 1.00 mol) were added with stirring. After the sodium had completely dissolved, tert-butyl acrylate (24 mL, 0.33 mol) was added. The solution was stirred for 20 h at room temperature and neutralized with 8 ml of 1.0 M HCl. The solvent was removed in vacuo and the residue was suspended in saturated aqueous sodium chloride (250 ml) and extracted with ethyl acetate (3x125 ml). The combined organic layers were washed with saturated aqueous sodium chloride (100 ml), then water (100 ml), dried over sodium sulfate and the solvent was removed. The resulting colorless oil was dried in vacuo to give 69.78 g (76% yield) of the title product. 1H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4 Hz), 3.59-3.72 (m, 14H). MS ESI m/z ^- C ₁₃ H ₂₅ O ₆ (M-H), calc. 277.17, found 277.20.

Example 17. Synthesis of tert-butyl-3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate

A solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (10.0 g, 35.95 mmol) in acetonitrile (50.0 ml) was treated with pyridine (20.0 ml) . A solution of tosyl chloride (7.12 g, 37.3 mmol) in 50 ml of acetonitrile was added dropwise through a dropping funnel over 30 min. After 5 hours, TLC analysis showed completion of the reaction. The resulting pyridine hydrochloride was filtered off and the solvent was removed. The residue was purified on silica gel eluting with 20% ethyl acetate in hexane to pure ethyl acetate to give 11.2 g (76% yield) of the title compound. 1H NMR: 1.40 (s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4 Hz), 3.52-3.68 (m, 14H), 4.11 (t, 2H, J=4.8 Hz), 7.30 (d, 2H, J=8.0 Hz), 7.75 (d, 2H, J=8.0 Hz); MS ESI m/z ⁺ C ₂₀ H ₃₃ O ₈ S (M+H), calc. 433.18, found 433.30.

Example 18 Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate

o.v o δ /x A/

TsOV ii \ y..* jV ' if ° DMF °

To 50 ml of DMF, tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (4.0 g, 9.25 mmol) and sodium azide (0.737 g, 11) were added with stirring .3 mmol). The reaction mixture was heated to 80°C. After 4 hours, TLC analysis showed completion of the reaction. The reaction mixture was cooled to room temperature and water (25 ml) was added. The aqueous layer was separated and extracted with ethyl acetate (3x35 ml). The combined organic layers were dried over anhydrous magnesium sulfate, filtered and the solvent was removed in vacuo. The crude azide (2.24 g, 98% yield, about 93% purity by HPLC) was used in the next step without further purification. 1H NMR (CDCl ₃ ): 1.40 (s, 9H), 2.45 (t, 2H, J=6.4 Hz), 3.33 (t, 2H, J=5.2 Hz), 3. 53-3.66 (m, 12H). MS ESI m/z ⁺ C^N^ (M+H), calc. 304.18, found 304.20.

- 81 044827

Example 19. Synthesis of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid

To tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (2.20 g, 7.25 mmol) in 1,4-dioxane (40 ml) was added HCl (12M, 10 ml ). The mixture was stirred for 40 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to obtain the crude title product for the next step without further purification (1.88 g, 105% yield, ~92% purity by HPLC). ESI MS m/z calculated for C ₉ H ₁₈ N ₃ O ₅ [M+H]+ 248.12, found 248.40.

Example 20. Synthesis of 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester and 13-amino-bis(4,7,10-trioxadodecanoic acid) tert-butyl ester

Crude 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)propanoic acid azide (5.0 g, ~14.84 mmol) was dissolved in ethanol (80 ml) and 300 mg of 10% Pd/ was added C. The system was evacuated and placed under 2 atm of hydrogen gas through a hydrogenation reactor with vigorous stirring. The reaction mixture was then stirred overnight at room temperature; TLC showed complete consumption of the starting materials. The crude reaction mixture was passed through a thin layer of celite, washing with ethanol. The solvent was removed and the amine was purified on silica gel using a mixture of methanol (5% to 15%) and 1% triethylamine in methylene chloride as eluent to give 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (1.83 g, yield 44%, ESI MS m/z ⁺ C13H27NO5 (M+H), calculated 278.19, found 278.30) and 13-amino-bis(4,7,10trioxadodecanoic acid) tert-butyl ester (2.58 g , 32% yield, ESI MS m/z ⁺ C26H ₅₂ NO ₁₀ (M+H), calculated 538.35, found 538.40).

Example 21. Synthesis of HCl salt of 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoic acid

To 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (0.80 g, 2.89 mmol) in 30 ml of dioxane was added 10 ml of HCl (36%) with stirring. After 0.5 h, TLC analysis indicated completion of the reaction and the reaction mixture was evaporated and co-evaporated with EtOH and EtOH/toluene to form the title product as HCl salt (>90% purity, 0.640 g, 86% yield) without further purification. MS ESI m/z ⁺ C ₉ H ₂₀ NO ₅ (M+H), calc. 222.12, found 222.20.

Example 22. 13-Amino-bis(4,7,10-trioxadodecanoic acid, HCl salt

To 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) (1.00 g, 1.85 mmol) in 30 ml dioxane was added 10 ml HCl (36%) with stirring. After 0.5 h TLC analysis indicated that the reaction was complete, the reaction mixture was evaporated and co-evaporated with EtOH and EtOH/toluene to form the title product as HCl salt (>90% purity, 0.71 g, 91% yield) without further purification. MS ESI m/z ⁺ C ₁₈ H ₃₆ NO ₁₀ (M+H), calc. 426.22, found 426.20.

Example 23. Synthesis of tert-butyl 3-(2-(2-(2-hydroethoxy)ethoxy)ethoxy)propanoate

To a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium G). The mixture was stirred until Na was completely consumed, and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight and then quenched with HCl solution (20.0 ml, 1 N) at 0°C. THF was removed by rotary evaporation, saturated aqueous sodium chloride (300 ml) was added and the resulting mixture was extracted with EtOAc (3x100 ml). The organic layers were washed with saturated aqueous sodium chloride (3x300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give a colorless oil (30.20 g, 79.0% yield), which was used without further purification. MS ESI m/z calc. for C ₁₃ H ₂₇ O ₆ [M+H] ⁺ 278.1729, found 278.1730.

Example 24. Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate.

To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217, 0 mmol, 2.0 eq.) in anhydrous DCM (220 ml) at 0°C was added TEA (30.0

- 82 044827 ml, 217.0 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight and then washed with water (3x300 ml) and saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (mixture hexane isomers 3:1/EtOAc) to give a colorless oil (39.4 g, 84.0% yield). ESI MS m/z calculated for C ₂₀ H ₃₃ O ₈ S [M+H] ⁴ 433.1818, found 433.2838.

Example 25. Synthesis of tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate

Ν ₃

To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF (100 ml) NaN ₃ (20.67 g, 316.6 mmol, 3.5 eq.) was added. The mixture was stirred at room temperature overnight. Water (500 ml) was added and extracted with EtOAc (3x300 ml). The combined organic layers were washed with water (3x900 ml) and saturated aqueous sodium chloride (900 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (5:1 hexane/EtOAc isomer mixture) to give light yellow oil (23.8 g, 85.53% yield). MS ESI m/z calc. for C^O^Na [M+Na] ⁺ 326.2, found 326.2.

Example 26. Synthesis of tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate

H ₂ NO ₂ ^z Bu

Raney's Ni (7.5 g, suspended in water) was washed with water (three times) and isopropyl alcohol (three times) and mixed with tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate ( 5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under H ₂ at room temperature for 16 hours and then filtered through a pad of celite, washing with isopropyl alcohol. The filtrate was evaporated and purified by column chromatography (5-25% MeOH/DCM) to give a light yellow oil (2.60 g, 57% yield). MS ESI m/z calc. for C13H25NO5 [M+H] ⁺ 279.19; found 279.19.

Example 27 Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol

Βη ₂ Ν^^°^^ΟΗ

BnBr ( 57.0 ml, 480 mmol, 2.4 eq.). The mixture was heated to reflux overnight. Water (1 L) was added and extracted with EtOAc (3x300 ml). The combined organic layers were washed with saturated aqueous sodium chloride (1000 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (4:1 hexane/EtOAc isomer mixture) to give a colorless oil (50 .97 g, 89.2% yield). MS ESI m/z calculated for C ₁₈ H ₂₃ NO ₂ Na [M+Na] ⁺ 309.1729, found 309.1967.

Example 28. Synthesis of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate

To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 eq.), tert-butyl acrylate (72.0 ml, 495.9 mmol, 3.0 eq. ) and h-Bu4NI (6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 ml) was added sodium hydroxide solution (300 ml, 50%). The mixture was stirred overnight. The organic layer was separated and the aqueous layer was extracted with EtOAc (3x100 ml). The organic layers were washed with water (3x300 ml) and saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (7:1 hexane/EtOAc isomer mixture), yielding a colorless oil (61.08 g, 89.4% yield). ESI MS m/z calculated for C ₂₅ H ₃₆ NO ₄ [M+H] ⁺ 414.2566, found 414.2384.

Example 29. Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate

---°’------(OEVi

To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THF (30 ml) and MeOH (60 ml) add Pd/C (2.00 g, 10% wt, 50% wet) in a hydrogenation flask. The mixture was shaken at a pressure of 1 atm. H ₂ overnight, filtered through celite (filtration aid) and the filtrate was evaporated to give a colorless oil (10.58 g, 93.8% yield). ESI MS m/z calculated for C11H24NO4 [M+H] ⁺ 234.1627, found 234.1810.

Example 30. Synthesis of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate

Sodium (0.1 g) was added to a solution of 2,2'-oxydiethanol (19.7 mL, 206.7 mmol, 3.0 eq.) in anhydrous THF (100 mL). The mixture was stirred until Na was consumed, and then tert-butyl acrylate (10.0 mL, 68.9 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, saturated sodium chloride solution (200 ml) was added and extracted with EtOAc (3x100 ml). The organic layers were washed with saturated aqueous sodium chloride (3x300 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (1:1 mixture of isomers of hexa-83 044827 on/EtOAc) to obtain a colorless oil (8.10 g, yield 49.4%). MS ESI m/z calc. for C11H23O5 [M +H]+ 235.1467, found 235.1667.

Example 31. Synthesis of tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate

T _S o^°'^o^- ^c ° ² ' ^Bu

To a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate (6.24 g, 26.63 mmol, 1.0 eq.) and TsCl (10.15 g, 53.27 mmol, 2. 0 eq.) in anhydrous DCM (50 mL) at 0° C., pyridine (4.3 mL, 53.27 mmol, 2.0 eq.) was added. The mixture was stirred at room temperature overnight and then washed with water (100 ml) and the aqueous layer was extracted with DCM (3x50 ml). The combined organic layers were washed with saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (5:1 hexane/EtOAc isomer mixture) to give a colorless oil (6 .33 g, 61.3% yield). ESI MS m/z calculated for C ₁₈ H ₂₇ O ₇ S [M+H] ⁺ 389.1556, found 389.2809.

Example 32. Synthesis of tert-butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate

NaN ₃ (5 .02 g, 77.22 mmol, 5.0 eq.). The mixture was stirred at room temperature overnight. Water (120 ml) was added and extracted with EtOAc (3x50 ml). The combined organic layers were washed with water (3x150 ml) and saturated aqueous sodium chloride (150 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (5:1 hexane/EtOAc isomer mixture) , yielding a colorless oil (3.73 g, 69.6% yield). MS ESI m/z calc. for C^O^Na [M+H]+ 260.1532, found 260.2259.

Example 33. Synthesis of tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate

----°---^\θ/χ _4/ (() ₂ 'Βυ tert-Butyl-3-(2-(2-azidoethoxy)ethoxy)propanoate (0.18 g, 0.69 mmol) was dissolved in MeOH (3.0 ml, 60 µl concentrated HCl) and hydrogenated with Pd/C (10 wt%, 20 mg) under H2 for 30 min. The catalyst was filtered through a pad of celite, washing with MeOH. The filtrate was evaporated to give a colorless oil ( 0.15 g, yield 93%). MS ESI m/z calculated for C11H24NO4 [M+H]+ 234.16; found 234.14.

Example 34. Synthesis of 3-(2-(2-azidoethoxy)ethoxy)propanoic acid

tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2.51 g, 9.68 mmol), dissolved in 1,4dioxane (30 ml), was treated with 10 ml HCl (conc.) at room temperature . The mixture was stirred for 35 min, diluted with EtOH (30 ml) and toluene (30 ml), and evaporated in vacuo. The crude mixture was purified on silica gel using methanol (5 to 10%) and 1% formic acid in methylene chloride as eluent to give the title compound (1.63 g, 83% yield), ESI MS m/z. C ₇ H12N ₃ O4 [M-H] ^- , calc. 202.06, found 202.30.

Example 35 Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate

To 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (1.60 g, 7.87 mmol) in 30 ml dichloromethane was added NHS (1.08 g, 9.39 mmol) and EDC (3.60 g , 18.75 mmol) with stirring. After 8 hours, TLC analysis indicated completion of the reaction, the reaction mixture was evaporated and purified on silica gel using ethyl acetate (5% to 10%) in methylene chloride as eluant to give the title compound (1.93 g, 82% yield). . ESI MS m/z C11H17N4O6 [M+H] ⁺ , calc. 301.11, found 301.20.

Example 36 Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate

NHS (3.0 g, 26.08 mmol) was added to 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (4.50 g, 18.21 mmol) in 80 ml dichloromethane with stirring. and EDC (7.60 g, 39.58 mmol). After 8 hours, TLC analysis indicated completion of the reaction, the reaction mixture was evaporated and purified on silica gel using ethyl acetate (5% to 10%) in methylene chloride as eluent to give the title compound (5.38 g, 86% yield) . ESI MS m/z C ₁₃ H ₂₀ N ₄ O ₇ [M+H] ⁺ , calc. 345.13, found 345.30.

Example 37. Synthesis of (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15- dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oic acid

- 84 044827

To a solution of (S)-2-((S)-2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-4-(tert-butoxy)-4oxobutanoic acid (2.81 g, 6.73 mmol ) 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy) was added to a mixture of DMA (70 ml) and 0.1 M NaH ₂ PO ₄ (50 ml, pH 7.5). ethoxy)propanoate (3.50 g, 10.17). The mixture was stirred for 4 hours, evaporated in vacuo, purified on silica gel using a mixture of methanol (5% to 15%) in methylene chloride containing 0.5% acetic acid as eluent to give the title compound (3.35 g , 77% yield). ESI MS m/z C ₂₈ H ₅₁ N ₆ O _n [M+H]+, calc. 647.35, found 647.80.

Example 38. Synthesis of (14S,17S)-tert-butyl-1-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)-17((4-(hydroxymethyl)phenyl)carbamoyl)-12.15 -dioxo-3,6,9-trioxa-13,16-diazanonadecane-19-oate

(14S,17S)-1 -Azido-17-(2-(tert-butoxy)-2-oxoethyl)-14-(4-((tert-butoxycarbonyl)amino)butyl)12,15-dioxo-3,6 ,9-trioxa-13,16-diazaoctadecan-18-onic acid (3.30 g, 5.10 mmol) and (4aminophenyl)methanol (0.75 g, 6.09) in DMA (25 ml) was added EDC ( 2.30 g, 11.97 mmol). The mixture was stirred overnight, evaporated in vacuo, purified on silica gel using methanol (5% to 8%) in methylene chloride as eluent to give the title compound (3.18 g, 83% yield). MS ESI m/z C ₃₅ H ₅₈ N ₇ On [M+H] ⁺ , calc. 752.41, found 752.85.

Example 39. Synthesis of (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17((4-(hydroxymethyl)phenyl)carbamoyl)-12,15- dioxo-3,6,9-trioxa-13,16-diazanonadecane-19-oate

To a solution of (14S,17S)-tert-butyl 1-azido-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo- 3,6,9-trioxa-13,16-diazanonadecane-19-oate (1.50 g, 1.99 mmol) in THF (35 ml) was added Pd/C (200 mg, 10% Pd, 50% humidity) in a hydrogenation vessel. The mixture was shaken at a pressure of 1 atm. H ₂ overnight, filtered through Celite (filtration aid) and the filtrate was evaporated to give the title compound (1.43 g, 99% yield), which was immediately used for the next step without further purification. ESI MS m/z C ₃₅ H ₆₀ N ₅ O _n [M+H] ⁺ , calculated 726.42, found 726.70.

Example 40 Synthesis of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecane-1-oic acid

To a solution of (S)-2-(2-amino-3-methylbutanamido)acetic acid (Val-Gly) (1.01 g, 5.80 mmol) in a mixture of DMA (50 ml) and 0.1 M NaH ₂ PO ₄ (50 ml, pH 7.5) 2,5-dioxopyrrolidin-1-yl 3-(2-(2azidoethoxy)ethoxy)propanoate (1.90 g, 6.33) was added. The mixture was stirred for 4 hours, evaporated in vacuo, purified on silica gel using a mixture of methanol (5% to 15%) in methylene chloride containing 0.5% acetic acid as eluent to give the title compound (1.52 g, yield 73%). ESI MS m/z C14H26N5O6 [M+H] ⁺ , calc. 360.18, found 360.40.

Example 41 Synthesis of (S)-2,5-dioxopyrrolidin-1-yl 15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa3,6-diazapentadecane-1-oate

To a solution of (S)-15-azido-5-isopropyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecane-1-oic acid (1.50 g, 4.17 mmol) in 40 ml dichloromethane, NHS (0.88 g, 7.65 mmol) and EDC (2.60 g, 13.54 mmol) were added with stirring. After 8 hours, TCM analysis indicated completion of the reaction, the reaction mixture was evaporated and purified on silica gel using ethyl acetate (5% to 20%) in methylene chloride as eluent to give the title compound (1.48 g, 78% yield) . ESI MS m/z

- 85 044827

C ₁₈ H ₂₉ N ₆ O ₈ [M+H]+, calculated for 457.20, found 457.50.

Example 42. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid

CbzHN^ _x z\^CO ₂ H

A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H2O (40 ml) was cooled to 0°C and a solution of CbzCl (16.1 g, 95 mmol) was added dropwise in THF (32 ml). After 1 h, the reaction mixture was allowed to warm to room temperature and stirred for 3 h. THF was removed in vacuo and the pH of the aqueous solution was adjusted to 1.5 by adding 6 N HCl. Extracted with ethyl acetate and the organic layer was washed with saturated aqueous sodium chloride, dried and evaporated to give the title compound (16.4 g, 92% yield). ESI MS m/z calculated for C ₁₂ H ₁₆ NO ₅ [M+H]+ 238.10, found 238.08.

Example 43. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate

CbzHN^-s^CO^Bu

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to a solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 ml). After stirring at room temperature overnight, the reaction mixture was filtered and the filtrate was evaporated. The residue was dissolved in ethyl acetate, washed with 1 N HCl, saturated aqueous sodium chloride and dried over Na ₂ SO ₄ . Evaporation and purification by column chromatography (10-50% EtOAc/hexane mixture) gave the title compound (7.5 g, 37% yield). MS ESI m/z calc. for C ₁₆ H ₂₃ NO ₄ Na [M+Na]+316.16, found 316.13.

Example 44 Synthesis of tert-butyl 4-aminobutanoate

H ₂ N^^CO ₂ ^z Bu tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 ml), and Pd/C catalyst (10% wt., 100 mg), then hydrogenated (1 atm) at room temperature for 3 hours. The catalyst was filtered and all volatiles were removed in vacuo to give the title compound (272 mg, 90% yield). MS ESI m/z calc. for C ₈ H ₁₈ NO ₂ [M+H]+ 160.13, found 160.13.

Example 45. Synthesis of di-tert-butyl 3,3'-(benzylazanediyl)dipropanoate ° 2 ''aUVi

VP

A mixture of phenylmethanamine (2.0 ml, 18.29 mmol, 1.0 eq.) and tert-butyl acrylate (13.3 ml, 91.46 mmol, 5.0 eq.) was refluxed at 80°C for overnight and then evaporated. The crude product was purified by column chromatography on SiO ₂ (20:1 hexane/EtOAc) to give the title compound as a colorless oil (5.10 g, 77% yield). ESI MS m/z: calculated for C21H34NO4 [M+H]+ 364.2, found 364.2. ¹ H NMR (400 MHz, CDC^) δ 7.38 -7.21 (m, 5H), 3.58 (s, 2H), 2.76 (t, J=7.0 Hz, 4H), 2 .38 (t, J=7.0 Hz, 4H), 1.43 (s, 17H).

Example 46 Synthesis of di-tert-butyl 3,3'-azanediyldipropanoate

Oh Oh

N

Pd/C (0.20 g, 10 % Pd/C, 50% humidity) in a hydrogenation vessel. The mixture was shaken overnight under H ₂ and then filtered through a pad of celite. The filtrate was evaporated to give the title compound as a colorless oil (1.22 g, 89% yield). ESI MS m/z: calculated for C14H28NO4 [M+H]+ 274.19, found 274.20.

Example 47. Synthesis of tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)propanamido)-butanoate

To a solution of tert-butyl 4-aminobutanoate (1.00 g, 6.28 mmol, 1.0 eq.) and ZL-alain (2.10 g, 9.42 mmol, 1.5 eq.) in anhydrous DCM ( 50 ml) at 0°C, HATU (3.10 g, 8.164 mmol, 1.3 eq.) and TEA (2.6 ml, 18.8 mmol, 3.0 eq.) were added. The reaction mixture was stirred at 0°C for 10 min, then warmed to room temperature and stirred overnight. The mixture was diluted with DCM and washed with water and saturated aqueous sodium chloride, dried over anhydrous Na2SO4, evaporated and purified by _SiO2 column chromatography (10:3 petroleum ether/ethyl acetate) to give the title compound as a colorless oil (1, 39 g, 61% yield). MS ESI m/z: calculated for C ₁₉ H ₂₉ N ₂ O ₅ Na [M+H]+ 387.2, found 387.2.

Example 48 Synthesis of tert-butyl 4-(2-aminopropanamido)butanoate

- 86 044827

Pd/C (0. 20 g, 10 wt.%, 10% humidity) in a bottle for hydrogenation. The mixture was shaken for 2 hours and then filtered through Celite (filtration aid) and evaporated to give the title compound as a light yellow oil (0.838 g, 95% yield). MS ESI m/z: calc. for C11H23N2O3 [M+H]+ 231.16, found 231.15.

Example 49. Synthesis of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid

ABOUT

TPA ( 5 ml). After stirring for 90 minutes, the reaction mixture was diluted with anhydrous toluene and evaporated, this operation was repeated three times to obtain the title compound as a light yellow oil (2.0 g, theoretical yield), which was directly used in the next step. MS ESI m/z calc. for C ₂₁ H ₂₈ NO ₄ [M+H]+ 358.19, found 358.19.

Example 50 Synthesis of perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate

ΝΒη ₂

ABOUT

To a solution of 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoic acid (2.00 g, 5.59 mmol, 1.0 eq.) in anhydrous DCM (30 mL) at 0°C, add DIPEA until neutral pH, and then PFP (1.54 g, 8.38 mmol, 1.5 eq) and DIC (1.04 ml, 6.70 mmol, 1.2 eq) were added. After 10 min, the reaction mixture was warmed to room temperature and stirred overnight. The mixture was filtered, evaporated and purified by SiO ₂ column chromatography (15:1 petroleum ether/ethyl acetate) to give the title compound as a colorless oil (2.10 g, 72% yield). MS ESI m/z: calcd. for C27H27F5NO4 [M+H]+ 524.2, found 524.2.

Example 51 Synthesis of tert-butyl 2-benzyl-13-methyl-11,14-dioxo-1-phenyl-5,8-dioxa-2,12,15-triase nonadecane-19-oate

To a solution of tert-butyl 4-(2-aminopropanamido)butanoate (0.736 g, 3.2 mmol, 1.0 eq.) and perfluorophenyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (2.01 g, 3.84 mmol, 1.2 eq.) in anhydrous DMA (20 ml) was added DIPEA (1.7 ml, 9.6 mmol, 3.0 eq.) at 0°C. After stirring at 0°C for 10 min, the reaction mixture was warmed to room temperature and stirred overnight. Water (100 ml) was added and the mixture was extracted with EtOAc (3x100 ml). The combined organic layers were washed with water (3x200 ml) and saturated aqueous sodium chloride (200 ml), dried over Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography on SiO ₂ (25:2 DCM/MeOH) to give the above the title compound appears as a colorless oil (1.46 g, 80% yield). MS ESI m/z: calcd. for C32H48N3O6 [M+H]+ 570.34, found 570.33.

Example 52. Synthesis of 2-benzyl-13-methyl-11,14-dioxo-1-phenyl-5,8-dioxa-2,12,15-triazanonedecane19-oic acid

To a solution of tert-butyl 2-benzyl-13-methyl-11,14-dioxo-1-phenyl-5,8-dioxa-2,12,15-triazanonedecane-19-oate (0.057 g, 0.101 mmol, 1.0 eq) in DCM (3 ml) at room temperature, TFA (1 ml) was added and stirred for 40 min. The reaction mixture was diluted with anhydrous toluene and then evaporated. This operation was repeated three times to obtain the title compound as a colorless oil (0.052 g, theoretical yield), which was used directly in the next step. MS ESI m/z: calculated for C ₂₈ H ₄₀ N ₃ O ₆ [M+H]+ 514.28, found 514.28.

Example 53. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid

CbzHN^^^CC^H

A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H ₂ O (40 ml) was cooled to 0°C, and then a solution of CbzCl (16.1 g, 95 mmol) in THF (32 ml). After 1 h, the reaction mixture was allowed to warm to room temperature and stirred for 3 h. THF was removed in vacuo and the pH of the aqueous solution was adjusted to 1.5 by adding 6 N HCl. Extracted with ethyl acetate and the organic layer was washed with saturated aqueous sodium chloride, dried and evaporated to give the title compound (16.4 g, 92% yield). ESI MS m/z calculated for

C ₁₂ H ₁₆ NO ₅ [M+H]+ 238.10, found 238.08.

Example 54. Synthesis of tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate (b/HX^/x^CO/Bn

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to a solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 ml). After stirring at room temperature overnight, the reaction mixture was filtered and the filtrate was evaporated. The residue was dissolved in ethyl acetate, washed with 1 N HCl, saturated aqueous sodium chloride and dried over Na ₂ SO ₄ . Evaporation and purification by column chromatography (10-50% EtOAc/hexane mixture) gave the title compound (7.5 g, 37% yield). ESI MS m/z calculated for C ₁₆ H ₂₃ NO ₄ Na [M+Na] ⁺ 316.16, found 316.13.

Example 55 Synthesis of tert-butyl 4-aminobutanoate tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 ml) and Pd/C catalyst (10 wt.%, 100 mg), then hydrogenated (1 atm) at room temperature for 3 hours. The catalyst was filtered and all volatiles were removed in vacuo to give the title compound (272 mg, 90% yield). ESI MS m/z calculated for C ₈ H ₁₈ NO ₂ [M+H] ⁺ 160.13, found 160.13.

Example 56. Synthesis of tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetate

2-(((Benzyloxy)carbonyl)amino)propanoic acid (0.84 g, 5 mmol), tert-butyl 2-aminoacetate (0.66 g, 5 mmol), HOBt (0.68 g, 5 mmol), EDC (1.44 g, 7.5 mmol) was dissolved in DCM (20 mL) followed by DIPEA (1.7 mL, 10 mmol). The reaction mixture was stirred at room temperature overnight, washed with H2O (100 ml) and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over MgSO ₄ , filtered, evaporated under reduced pressure and the residue was purified on a SiO 2 column to give the title product 1 (0.87 g, 52%). ESI: m/z: calc. for C17H25N2O5 [M+H] ⁺ : 337.17, found 337.17.

Example 57. Synthesis of 2-(2-(((benzyloxy)carbonyl)amino)propanamido)acetic acid o TFA o o 11 I DCM θ n J tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)propanamido )acetate (0.25 g, 0.74 mmol) was dissolved in DCM (30 ml), followed by the addition of TFA (10 ml). The mixture was stirred at room temperature overnight and evaporated to give the title compound, which was used for the next step without further purification. ESI: m/z: calc. for C ₁₃ H ₁₇ N ₂ O ₅ [M+H] ⁺ : 281.11, found 281.60.

Example 58 Synthesis of 2,2-dipropiolamidoacetic acid

2,2-Diaminoacetic acid (2.0 g, 22.2 mmol) in a mixture of EtOH (15 ml) and 50 mm NaH ₂ PO ₄ pH 7.5 buffer (25 ml) was added 2,5-dioxopyrrolidin-1-ylpropiolate (9.0 g, 53.8 mmol). The mixture was stirred for 8 hours, evaporated, pH adjusted to 3.0 with 0.1 M HCl, extracted with EtOAc (3x30 ml). The organic layers were combined, dried over Na ₂ SO ₄ , filtered, evaporated and purified on a SiO ₂ column eluting with MeOH/CH ₂ Cl ₂ (1:10 to 1:6) to give the title compound (3, 27 g, 76% yield). 1H NMR (CDCl3) 11.8 (br., 1H), 8.12 (d, 2H), 6.66 (m, 1H), 2.66 (s, 2H). ESI MS m/z: calculated for C ₈ H6N2O4 [M+H] ⁺ 195.03, found 195.20.

Example 59 Synthesis of perfluorophenyl-2,2-dipropiolamidoacetate

2,2-Dipropiolamidoacetic acid (2.01 g, 10.31 mmol), pentafluorophenol (2.08 g, 11.30 mmol), DIPEA (1.00 ml, 5.73 mmol) and EDC (4.01 g , 20.88 mmol) in CH2Cl2 (100 ml), stirred at room temperature overnight, evaporated and purified on a SiO ₂ column, eluting with EtOAc/CH ₂ Cl ₂ (1:15 to 1:8) , giving the title compound (3.08 g, 83% yield). ¹ H NMR (CDCl ₃ ) 8.10 (d,

2H), 6.61 (m, 1H), 2.67 (s, 2H). ESI MS m/z: calculated for C14H6F5N2O4 [M+H]+ 361.02, found 361.20.

Example 60. Synthesis of (S)-2-((S)-2-(2,2-dipropiolamidoacetamido)propanamido)propanoic acid

(S)-2-((S)-2-Aminopropanamido)propanoic acid (422) (1.10 g, 6.87 mmol) in a mixture of DMA (18 ml) and 50 mM NaH ₂ PO ₄ , pH 7.5 buffer (30 mL), perfluorophenyl 2,2-dipropiolamidoacetate (3.00 g, 8.33 mmol) was added. The mixture was stirred for 14 h, evaporated, pH adjusted to 3.0 with 0.1 M HCl, extracted with EtOAc (3x40 ml). The organic layers were combined, dried over Na ₂ SO ₄ , filtered, evaporated and purified on a SiO ₂ column eluting with MeOH/CH ₂ Cl ₂ (1:10 to 1:5) to give the title compound (1, 80 g, 78% yield). MS ESI m/z: calculated for C ₁₄ H ₁₇ N ₄ O ₆ [M+H] ⁺ 337.11, found 337.30.

Example 61. Synthesis of (S)-2,5-dioxopyrrolidin-1-yl 2-((S)-2-(2,2-dipropiolamidoacetamido)propanamido)propanoate

(S)-2-((S)-2-(2,2-dipropiolamidoacetamido)propanamido)propanoic acid (1.01 g, 3.00 mmol), NHS (0.41 g, 3.56 mmol), DIPEA (0.40 ml, 2.29 mmol) and EDC (1.51 g, 7.86 mmol) in CH2Cl2 (50 ml) were stirred at room temperature overnight, evaporated and purified on a SiO2 column, eluting with EtOAc/CH ₂ Cl ₂ (1:15 to 1:7) to give the title compound (1.05 g, 81% yield). MS ESI m/z: calculated for C ₁₈ H ₂₀ N ₅ O ₈ [M+H] ⁺ 434.12, found 434.40.

Example 62. Synthesis of di-tert-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15in-1,30-dioate

Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved in NMP (10 ml) and cooled to 0°C, tert-butyl compound 3-(2-(2-(2 -aminoethoxy)ethoxy))-ethoxy)propanoate (2.06 g, 7.43 mmol, 2.4 eq.), followed by portions of DMTMM (2.39 g, 8.65 mmol, 2.8 eq.). The reaction mixture was stirred at 0°C for 6 hours and then diluted with ethyl acetate and washed with water and saturated aqueous sodium chloride. The organic solution was evaporated and triturated with a mixture of solvents: ethyl acetate and petroleum ether. The solid was filtered off and the filtrate was evaporated and purified by column chromatography (80-90% EA/PE) to give a light yellow oil (2.26 g, >100% yield) which was used without further purification. ESI MS m/z [M+H] ⁺ 633.30.

Example 63 Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioic acid

The compound di-tert-butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30dioate (2.26 g) was dissolved in dichloromethane (15 ml) and cooled to 0°C, then treated with TFA (15 ml). The reaction mixture was warmed to room temperature and stirred for 45 min, and then the solvent and residual TPA were removed by rotary evaporation. The crude product was purified by column chromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86% yield over two steps). ESI MS m/z [M+H] ⁺ 521.24.

Example 64. Synthesis of di-tert-butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33hexaoxa-3,6,19 ,24,37,40-hexaazadotetracont-21-yne-1,42-dioate

To a solution of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diaza triacont-15-yne-1,30-dioic acid (1.38 g, 2.65 mmol) , tert -butyl 2-(2-aminopropanamido)propanoate (0.75 g, 3.47 mmol) in a mixture of DMA (40 ml) was added EDC (2.05 g, 10.67 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:5 to 1:1) to give the title compound (2.01 g, yield 82%, purity ~95% according to HPLC). ESI MS m/z calculated for C ₄₂ H ₇₃ N6O ₁₆

[M+H]+ 917.50, found 917.90.

Example 65. Synthesis of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa3,6,19,24,37, 40-hexaazadotetracont-21-yne-1,42-dioic acid di-di-tert-Butyl 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16 ,27,30,33-hexaoxa-3,6,19 24,37,40-hexaazadotetracont-21-yne-1,42-dioate (1.50 g, 1.63 mmol) was dissolved in a mixture of CH ₂ Cl ₂ ( 10 ml) and TFA (10 ml). The mixture was stirred overnight, diluted with toluene (20 ml), evaporated to give the title compound (1.33 g, 101% yield, ~92% HPLC purity), which was used in the next step without further purification. MS ESI m/z calc. C ₃₄ H ₅₆ N ₆ O ₁₆ [M+H]+805.37, found 805.85.

Example 66. Synthesis of bis(2,5-dioxopyrrolidin-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo10,13,16,27,30,33 - hexaoxa-3,6,19,24,37,40-hexaazadotetracont-21-in-1,42-dioate

To a solution of 2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa3,6,19,24,37,40- hexaazadotetracont-21-yne-1,42-dioic acid (1.30 g, 1.61 mmol) in a mixture of DMA (10 ml) was added NHS (0.60 g, 5.21 mmol) and EDC (1.95 g , 10.15 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:4 to 2:1) to give the title compound (1.33 g, 83% yield, purity ~95% according to HPLC). ESI MS m/z calculated for C42H63N8O20 [M+H]+ 999.40, found 999.95.

Example 67 Synthesis of 2,3-bis(2-bromoacetamido)succinyl dichloride

To 2,3-diaminosuccinic acid (5.00 g, 33.77 mmol) in THF/H ₂ O/DIPEA (125 ml/125 ml/8 ml) was added 2-bromoacetyl bromide (25.0 g, 125.09 mmol). The mixture was stirred overnight, evaporated and purified by column chromatography on SiO ₂ (H2O/CH3CN 5:95) to give 2,3-bis(2bromoacetamido)succinic acid (9.95 g, 76% yield) as light yellow oil. ESI MS m/z calculated for C ₈ H _n Br ₂ N ₂ O ₆ [M+H]+ 388.89, found 388.68.

2,3-bis(2-Bromoacetamido)succinic acid (3.50 g, 9.02 mmol) in dichloromethane (80 ml) was added oxalyl dichloride (5.80 g, 46.05 mmol) and DMF (0.01 ml) . The mixture was stirred for 2.5 hours, diluted with toluene, evaporated and co-evaporated with dichloroethane (2x20 ml) and toluene (2x15 ml) to dryness to give 2,3-bis(2-bromoacetamido)succinyl dichloride as a crude product (which is unstable ), which was used for the next step without further purification (3.90 g, 102% yield). MS ESI m/z calc. for С8Н9Вг2СШО4 [M+H]+ 424.82, found 424.90.

Example 68. Synthesis of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid

CbzHN NHCbz

_{Benzylcarbonochloridate (} 15 .0 g, 88.23 mmol) for 2 hours. The mixture was stirred for an additional 6 hours, evaporated and purified on a SiO ₂ column, eluting with a mixture of H2O/CH3CN (1:9) containing 1% formic acid, obtaining of the title compound (8.65 g, 76% yield, ~95% purity). ESI MS m/z calculated for C ₂₀ H ₂₁ N ₂ O ₈ [M+H]+ 417.12, found 417.60.

Example 69. Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(((benzyloxy)carbonyl)amino)succinate

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To a solution of 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (4.25 g, 10.21 mmol) in DMA (70 ml) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column, eluting with EtOAc/CH ₂ Cl ₂ (1:6) to give the title compound (5.42 g, 87% yield, ~95% purity). MS ESI m/z calc. For C ₂₈ H ₂₇ N ₄ O ₁₂ [M+H]+ 611.15, found 611.60.

Example 70. Synthesis of 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid θ JI \ Q A_ ? and o./A.o

ΗοΑΛοΐΙ Λ—/ _b ° HOAc/Ac _g O o i/CH _{1I ΤΓΦ} / _Η2θ ζο ¹ ^ DMF ^{H0 T} ^P

2,3-Diaminosuccinic acid (5.00 g, 33.77 mmol) in TGO/H ₂ O/DIPEA (125 ml/125 ml/2 ml) added maleic anhydride (6.68 g, 68.21 mmol) . The mixture was stirred overnight and evaporated to give 2,3-bis((7)-3-carboxyacrylamido)succinic acid (11.05 g, 99% yield) as a white solid. ESI MS m/z calculated for C ₁₂ H ₁₃ N ₂ O ₁₀ [M+H]+ 345.05, found 345.35.

To 2,3-bis((2)-3-carboxyacrylamido)succinic acid (11.05 g, 33.43 mmol) in a mixture of a solution of HOAc (70 ml), DMF (10 ml) and toluene (50 ml) was added anhydride (30 ml). The mixture was stirred for 2 h, heated to boiling with a Dean-Stark packing at 100°C for 6 h, evaporated, co-evaporated with EtOH (2x40 ml) and toluene (2x40 ml), and purified on a SiO ₂ column. eluting with H2O/CH3CN (1:10) to give the title compound (7.90 g, 76% yield, ~95% purity). MS ESI m/z calc. for Cl ₂ H ₉ N ₂ O ₈ [M+H]+ 309.03, found 309.30.

Example 71. Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)succinate

To a solution of 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid (4.00 g, 12.98 mmol) in DMF (70 ml) was added NHS (3.60 g, 31.30 mmol) and EDC (7.05 g, 36.72 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:6) to give the title compound (5.73 g, 88% yield, ~96% purity according to HPLC). ESI MS m/z calculated for C ₂₀ H ₁₅ N ₄ O ₁₂ [M+H]+ 503.06, found 503.45.

Example 72. Synthesis of (3S,6S,39S,42S)-di-tert-butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)22,23-bis(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)-3,42-bis((4-(hydroxymethyl)phenyl)carbamoyl)5,8,21,24,37,40-hexaoxo-11,14,17, 28,31,34-hexaoxo-4,7,20,25,38,41-hexaazatetratetracontane-1,44dioate

K (14S,17S)-tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl)phenyl)carbamoyl)-12,15-dioxo -3,6,9-trioxa-13,16-diazanonadecane-19-oatu (1.43 g, 1.97 mmol) and 2,3-bis(2,5-dioxo-2,5-dihydro-1H- Pyrrol-1-yl)succinic acid (0.30 g, 0.97 mmol) in DMA (25 ml) was added with EDC (1.30 g, 6.77 mmol). The mixture was stirred overnight, evaporated in vacuo, purified on silica gel using methanol (5% to 8%) in methylene chloride as eluent to give the title compound (1.33 g, 80% yield). MS ESI m/z C ₈₂ H ₁₂₃ N ₁₂ O ₂₈ [M+H] ⁺ , calc. 1722.85, found 1722.98.

Example 73 Synthesis of tert-butyl-1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate

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To a solution of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)propanoic acid (1.55 g, 6.27 mmol), tert-butyl 2-(2-aminopropanamido)propanoate (1.35 g, 6.27 mmol) to the DMA mixture (60 ml) was added EDC (3.05 g, 15.88 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:3) to give the title compound (2.42 g, 86% yield, ~95% purity according to HPLC). MS ESI m/z calc. For C ₁₉ H ₃₆ N ₅ O ₇ [M+H]+ 446.25, found 446.60

Example 74. Synthesis of 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18oic acid

To tert-butyl-1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (2.20 g, 4.94 mmol) in HCl (12M, 10 mL) was added to 1,4-dioxane (40 mL). The mixture was stirred for 40 min, diluted with dioxane (20 ml) and toluene (40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dryness to obtain the crude title product, which was used in the next step without further purification (1.92 g, 100% yield, ~94% purity by HPLC). ESI MS m/z calculated for C ₁₅ H ₂₈ N ₅ O ₇ [M+H]+ 390.19, found 390.45.

Example 75 Synthesis of 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl4,7,20,23,36,39 -hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracontane-1,42-dioic acid

To 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oic acid (1.90 g, 4.88 mmol) in DMA (40 ml) Pd/C (0.20 g, 50% humidity) was added. The system was evacuated and placed under 2 atm of hydrogen gas through a hydrogenation reactor with vigorous stirring. The reaction mixture was then stirred for 6 hours at room temperature and TLC showed consumption of the starting materials. The crude reaction mixture was passed through a thin layer of celite, washing with ethanol. The solvent was evaporated under reduced pressure to give the crude product, 1-amino-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oic acid in DMA, which was used directly at the next stage. MS ESI m/z ⁺ C ₁₅ H ₃₀ N ₃ O ₇ (M+H), calc. 364.20, found 364.30.

To the amine in DMA (~30 ml) was added 0.1 M NaH ₂ PO ₄ , pH 7.5 (20 ml), followed by the addition of bis(2,5-dioxopyrrolidin-1-yl) 2,3-bis(2,5 - dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinate (1.30 g, 2.59 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column, eluting with 8% water/CH3CN to give the title compound (1.97 g, 81% yield). MS ESI m/z ⁺ C ₄₂ H ₆₃ N ₈ O ₂₀ (M+H), calc. 999.41, found 999.95.

Example 76. Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol1-yl)-2,5,38,41-tetramethyl -4,7,20,23,36,39-hexaoxo-10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40hexaazadotetracontane-1,42-dioate

- 92 044827

To a solution of 21,22-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39hexaoxo -10,13,16,27,30,33-hexaoxa-3,6,19,24,37,40-hexaazadotetracontane-1,42-dnoic acid (1.50 g, 1.50 mmol) in DMA mixture ( 10 ml) NHS (0.60 g, 5.21 mmol) and EDC (1.95 g, 10.15 mmol) were added. The mixture was stirred overnight, evaporated and purified on a SiO2 column eluting with EtOAc/CH ₂ Cl ₂ (1:4 to 2:1) to give the title compound (1.50 g, 83% yield, ~ purity 95% according to HPLC data). ESI MS m/z calculated for C ₅₀ H ₆₉ N ₁₀ O ₂₄ [M+H]+ 1193.44, found 1193.95.

Example 77 Synthesis of (S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate

Z^^oh

Vos

Boc-L-proline (10.0 g, 46.4 mmol) dissolved in 50 ml THF was cooled to 0°C, to which BH3 in THF (1.0 M, 46.4 ml) was carefully added. The mixture was stirred at 0°C for 1.5 hours, then poured into ice water and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride (50 ml), dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to obtain the title compound (8.50 g, 91% yield) as a white solid. ¹ NMR (500 MHz, CDCl3) δ 3.94 (dd, J=4.9, 2.7 Hz, 2H), 3.60 (dd, J=18.7, 11.9, 9.3 Hz, 2H), 3.49-3.37 (m, 1H), 3.34-3.23 (m, 1H), 2.06-1.91 (m, 1H), 1.89-1.69 ( m, 2H), 1.65-1.51 (m, 1H), 1.49-0.40 (m, 9H).

Example 78 Synthesis of (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate

To a solution of (S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate (13.0 g, 64.6 mmol) in dimethyl sulfoxide (90 ml) was added triethylamine (40 ml) and stirring was continued for 15 minutes. The mixture was cooled in an ice bath and sulfur trioxide pyridine complex (35.98 g, 226 mmol) was added in portions over 40 min. The reaction mixture was warmed to room temperature and stirred for 2.5 hours. After adding ice (250 g), the mixture was extracted with dichloromethane (150 mlx3). The organic phase was washed with 50% citric acid solution (150 ml), water (150 ml), saturated sodium hydrogen carbonate solution (150 ml) and saturated sodium chloride solution (150 ml), dried over anhydrous Na ₂ SO ₄ . Removal of the solvent in vacuo gave the title aldehyde (10.4 g, 81% yield) as a thick oil, which was used without further purification. 1H NMR (500 MHz, CDCl ₃ ) δ 9.45 (s, 1H), 4.04 (s, 1H), 3.53 (dd, J=14.4, 8.0 Hz, 2H), 2, 00-1.82 (m, 4H), 1.44 (d, J=22.6 Hz, 9H).

Example 79 Synthesis of (4R,5S)-4-methyl-5-phenyl-3-propionyloxazolidin-2-one

n-Butyllithium in hexane (21.6 ml, 2.2 M, 47.43 mmol) was added dropwise at -78°C to a stirred solution of 4-methyl-5-phenyloxazolidin-2-one (8.0 g, 45. 17 mmol) in THF (100 ml) under N2. The solution was kept at -78°C for 1 hour, then propionyl chloride (4.4 ml, 50.59 mmol) was added slowly. The reaction mixture was heated to -50°C, stirred for 2 hours, then saturated ammonium chloride solution (100 ml) was added. The organic solvent was removed in vacuo and the resulting solution was extracted with ethyl acetate (3x100 ml). The organic layer was washed with saturated sodium hydrogen carbonate (100 ml) and saturated aqueous sodium chloride (100 ml), dried over Na ₂ SO ₄ , filtered and evaporated in vacuo. The residue was purified by column chromatography (20% ethyl acetate/hexane) to give the title compound as a thick oil (10.5 g, 98% yield). ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.45-7.34 (m, 3H), 7.30 (d, J=7.0 Hz, 2H), 5.67 (d, J=7, 3 Hz, 1H), 4.82-4.70 (m, 1H), 2.97 (dd, J=19.0, 7.4 Hz, 2H), 1.19 (t, J=7.4 Hz, 3H), 0.90 (d, J=6.6 Hz, 3H).

Example 80. Synthesis of (S)-tert-butyl 2-((1R,2R)-1-hydroxy-2-methyl-3-((4R,5S)-4-methyl-2-oxo-5phenyloxazolidin-3-yl )-3-oxopropyl)pyrrolidine-1-carboxylate

Et ₃ N (6.45 mL, 46.64 mmol) at 0°C followed by the addition of 1 M dibutylborone triflate in dichloromethane (42 ml, 42 mmol). The mixture was stirred at 0°C for 45 min, cooled to -70°C, then (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate (4.58 g, 22.97 mmol) in dichloromethane (40 ml) for 30 min. The reaction mixture was stirred at -70°C for 2 hours, at 0°C for 1 hour and at room temperature for 15 minutes, and then phosphate buffer solution (pH 7.38 ml) was added. After adding MeOH-30% H ₂ O ₂ (2:1, 100 ml) at a temperature below 10°C and stirring for 20 minutes, water (100 ml) was added and the mixture was evaporated in vacuum. To the remainder

- 93 044827 additional water (200 ml) was added and the mixture was extracted with ethyl acetate (3x100 ml). The organic layer was washed with 1 N KHSO4 (100 ml), sodium hydrogen carbonate solution (100 ml) and saturated aqueous sodium chloride (100 ml), dried over anhydrous Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by flash column chromatography (10-50% ethyl acetate/hexane) to give the title compound as a white solid (7.10 g, 71% yield). 1H NMR (500 MHz, CDCI3) δ 7.39 (dt, J=23.4, 7.1 Hz, 3H), 7.30 (d, J=7.5 Hz, 2H), 5.67 (d , J=7.1 Hz, 1H), 4.84-4.67 (m, 1H), 4.08-3.93 (m, 3H), 3.92-3.84 (m, 1H), 3.50 (d, J=9.0 Hz, 1H), 3.24 (d, J=6.7 Hz, 1H), 2.15 (s, 1H), 1.89 (dd, J=22 ,4, 14.8 Hz, 3H), 1.48 (d, J=21.5 Hz, 9H), 1.33 (d, J=6.9 Hz, 3H), 0.88 (d, J =6.4 Hz, 3H).

Example 81. Synthesis of (S)-tert-butyl 2-((1R,2R)-1-methoxy-2-methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidine-3 -yl)-3-oxopropyl)pyrrolidine-1-carboxylate

To a mixture of (S)-tert-butyl 2-((1R,2R)-1-hydroxy-2-methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidin-3-yl )-3-oxopropyl)pyrrolidine-1-carboxylate (5.1 g, 11.9 mmol) and molecular sieves (4A, 5 g) were added anhydrous dichloroethane (30 ml) under N2. The mixture was stirred at room temperature for 20 minutes and cooled to 0°C. Proton sponge (6.62 g, 30.9 mmol) was added followed by trimethyloxonium tetrafluoroborate (4.40 g, 29.7 mmol). Stirring was continued for 2 hours at 0°C and 48 hours at room temperature. The reaction mixture was filtered and the filtrate was evaporated and purified by column chromatography (20-70% ethyl acetate/hexane) to give the title compound as a white solid (1.80 g, 35% yield). 1H NMR (500 MHz, CDCI3) δ 7.46-7.27 (m, 5H), 5.65 (s, 1H), 4.69 (s, 1H), 3.92 (s, 1H), 3 .83 (s, 1H), 3.48 (s, 3H), 3.17 (s, 2H), 2.02-1.68 (m, 5H), 1.48 (d, J=22.3 Hz, 9H), 1.32 (t, J=6.0 Hz, 3H), 0.91 - 0.84 (m. 3H).

Example 82. Synthesis of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid ^Boc >) o

To a solution of (3)-tert-butyl 2-((1R,2R)-1-methoxy-2-methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidin-3-yl )-3-oxopropyl)pyrrolidine-1-carboxylate (1.80 g, 4.03 mmol) in THF (30 ml) and H2O (7.5 ml) was added 30% H ₂ O ₂ (1.44 ml, 14 .4 mmol) for 5 min at 0°C, then a solution of LiOH (0.27 g, 6.45 mmol) in water (5 ml) was added. After stirring at 0°C for 3 hours, 1N sodium sulfite (15.7 ml) was added and the mixture was allowed to warm to room temperature and stirred overnight. THF was removed in vacuo and the aqueous phase was washed with dichloromethane (3x50 ml) to remove additional oxazolidinone. The aqueous phase was acidified to pH 3 with 1 N HCl and extracted with ethyl acetate (3x50 ml). The organic layer was washed with saturated aqueous sodium chloride (50 ml), dried over Na2SO4, filtered and evaporated in vacuo to give the title compound as a colorless oil (1.15 g, 98% yield). 1H NMR (500 MHz, CDC13) δ 3.99 - 3.74 (m, 2H), 3.44 (d, J=2.6 Hz, 3H), 3.23 (s, 1H), 2.60 - 2.45 (m, 1H), 1.92 (tt, J=56.0, 31.5 Hz, 3H), 1.79 - 1.69 (m, 1H), 1.58 - 1.39 (m, 9H), 1.30 - 1.24 (m, 3H).

Example 83 Synthesis of (2R,3R)-methyl 3-methoxy-2-methyl-3-((8)-pyrrolidin-2-yl)propanoate

ΛΊ I ^OC1 ₂ I

MeOH _g ^Boc/ O ^N /θ θ

To a solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (0.86 g, 2.99 mmol) in MeOH (10 ml) thionyl chloride (1.08 ml, 14.95 mmol) was added slowly at 0°C. The reaction mixture was then warmed to room temperature and stirred overnight. The mixture was evaporated in vacuo and co-evaporated with toluene to give the title compound (0.71 g, 100% yield) as a white solid, which was immediately used in the next step without further purification. HRMS (High Resolution Mass Spectrometry) (ESI) m/z calc. for Ci ₀ H ₂₀ NO ₃ [M+H]+: 202.14, found: 202.14.

Example 84. Synthesis of (4S,5S)-ethyl 4-((tert-butoxycarbonylamino)-5-methyl-3-oxoheptanoate ^H O O

To an ice-cooled solution of N-Boc-L-isoleucine (4.55 g, 19.67 mmol) in THF (20 mL) was added 1,1'-carbonyldiimidazole (3.51 g, 21.63 mmol). After gas evolution ceased, the resulting mixture

- 94 044827 was stirred at room temperature for 3.5 hours.

A solution of freshly prepared isopropylmagnesium bromide in THF (123 mmol, 30 ml) was added dropwise to a pre-cooled (0°C) solution of ethyl hydrogen malonate (6.50 g, 49.2 mmol) at a rate to maintain the internal temperature below 5° WITH. The mixture was stirred at room temperature for 1.5 hours. This magnesium enolate solution was then cooled in an ice bath, followed by gradual addition of the imidazolide solution over 1 hour through a double-ended needle adapter at 0°C. The resulting mixture was stirred at 0°C for 30 minutes, then at room temperature. for 64 hours. The reaction mixture was added with 10% aqueous citric acid (5 ml) and acidified to pH 3 with additional 10% aqueous citric acid (110 ml). The mixture was extracted with ethyl acetate (3x150 ml). The organic extracts were washed with water (50 ml), saturated aqueous sodium hydrogen carbonate (50 ml) and saturated aqueous sodium chloride (50 ml), dried over Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by silica gel column chromatography using ethyl acetate/hexane (1:4) as eluent to give the title compound (5.50 g, 93% yield). ¹ H NMR (500 MHz, CDCl ₃ ) δ 5.04 (d, J=7.8 Hz, 1H), 4.20 (p, J=7.0 Hz, 3H), 3.52 (t, J =10.7 Hz, 2H), 1.96 (d, J=3.7 Hz, 1H), 1.69 (s, 2H), 1.44 (s, 9H), 1.28 (dd, J =7.1, 2.9 Hz, 3H), 0.98 (t, J=6.9 Hz, 3H), 0.92 - 0.86 (m, 3H).

Example 85 Synthesis of (3R,4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methylheptanoate

BoCx^zC^x^OEt ^N OH O

To a solution of (4S,5S)-ethyl 4-((tert-butoxycarbonyl)amino)-5-methyl-3-oxoheptanoate (5.90 g, 19.83 mmol) in ethanol (6 ml) at -60°C was added one serving sodium borohydride (3.77 g, 99.2 mmol). The reaction mixture was stirred for 5.5 hours at a temperature below -55°C, then a 10% aqueous solution of citric acid (100 ml) was added. The resulting solution was acidified to pH 2 with additional 10% aqueous citric acid, followed by extraction with ethyl acetate (3x100 ml). The organic extracts were washed with saturated aqueous sodium chloride (100 ml), dried over Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by column chromatography (1050% ethyl acetate/hexane) to give the pure title compound as a diastereomer (2.20 g, 37% yield) and a mixture of the two diastereomers (2.0 g, 34% yield, ratio about 9 :1). 1H NMR (500 MHz, CDCl ₃ ) δ 4.41 (d, J=9.3 Hz, 1H), 4.17 (tm, J=7.1, 3.6 Hz, 2H), 4.00 ( t, J=6.9 Hz, 1H), 3.55 (dd, J=11.7, 9.3 Hz, 1H), 2.56 -2.51 (m,2H), 2.44 (dd , J=16.4, 9.0 Hz, 1H), 1.79 (d, J=3.8 Hz, 1H), 1.60-1.53 (m, 1H), 1.43 (s, 9H), 1.27 (dd, J=9.3, 5.0 Hz, 3H), 1.03 - 0.91 (m, 7H).

Example 86. Synthesis of (3R,4S,5S)-4-((tert-butoxycarbonylamino)-3-hydroxy-5-methylheptanoic acid ^H OH O

To a solution of (3R,4S,5S)-ethyl-4-((tert-butoxycarbonyl)amino)-3-hydroxy-5-methylheptanoate (2.20 g, 7.20 mmol) in ethanol (22 ml) was added 1 N aqueous sodium hydroxide solution (7.57 ml, 7.57 mmol). The mixture was stirred at 0°C for 30 minutes, then at room temperature. for 2 hours. The resulting solution was acidified to pH 4 by adding 1N aqueous hydrochloric acid, which was then extracted with ethyl acetate (3x50 ml). Organic extracts were washed with 1N. aqueous potassium hydrogen sulfate (50 ml) and saturated aqueous sodium chloride (50 ml), dried over Na ₂ SO ₄ and evaporated in vacuo to give the compound (1.90 g, 95% yield). 1H NMR (500 MHz, CDCl ₃ ) δ 4.50 (d, J=8.7 Hz, 1H), 4.07 (d, J=5.5 Hz, 1H), 3.59 (d, J= 8.3 Hz, 1H), 2.56 - 2.45 (m, 2H), 1.76 - 1.65 (m, 1H), 1.56 (d, J=7.1 Hz, 1H), 1.45 (s, 9H), 1.26 (t, J=7.1 Hz, 3H), 0.93 (dd, J=14.4, 7.1 Hz, 6H).

Example 87. Synthesis of (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-methoxy-5-methylheptanoic acid

Sodium hydride ( 60% oil suspension, 1.93 g, 48.3 mmol) at 0°C. After stirring for 1 hour, methyl iodide (6.6 mL, 103.5 mmol) was added. Stirring was continued at 0°C for 40 hours until saturated aqueous sodium hydrogen carbonate solution (50 ml) was added, then water (100 ml) was added. The mixture was washed with diethyl ether (2x50 ml) and the aqueous layer was acidified to pH 3 with 1 N aqueous potassium hydrogen sulfate solution, then extracted

- 95 044827 with ethyl acetate (3x50 ml). The combined organic extracts were washed with 5% aqueous sodium thiosulfate (50 ml) and saturated aqueous sodium chloride (50 ml), dried over Na ₂ SO ₄ and evaporated in vacuo to give the title compound (1.00 g, 48% yield) . 1H NMR (500 MHz, CDCl ₃ ) δ 3.95 (d, J=75.4 Hz, 2H), 3.42 (d, J=4.4 Hz, 3H), 2.71 (s, 3H) , 2.62 (s, 1H), 2.56 -2.47 (m, 2H), 1.79 (s, 1H), 1.47 (s, 1H), 1.45 (d, J=3 .3 Hz, 9H), 1.13 - 1.05 (m, 1H), 0.96 (d, J=6.7 Hz, 3H), 0.89 (td, J=7.2, 2, 5 Hz, 3H).

Example 88 Synthesis of Boc-N-Me-L-Val-OH

Sodium hydride (3.68 g, 92 mmol) was added to a solution of Boc-L-Val-OH (2.00 g, 9.2 mmol) and methyl iodide (5.74 mL, 92 mmol) in anhydrous THF (40 mL). at 0°C. The reaction mixture was stirred at 0°C for 1.5 hours, then heated to room temperature. and stirred for 24 hours. Ice water (50 ml) was added to the reaction mixture. After adding water (100 ml), the reaction mixture was washed with ethyl acetate (3x50 ml) and the aqueous solution was acidified to pH 3, then extracted with ethyl acetate (3x50 ml). The combined organic phase was dried over Na2SO4 and evaporated to give Boc-N-Me-Val-OH (2.00 g, 94% yield) as a white solid. 1H NMR (500 MHz, CDCl ₃ ) δ 4.10 (d, J=10.0 Hz, 1H), 2.87 (s, 3H), 2.37 - 2.13 (m, 1H), 1, 44 (d, J=26.7 Hz, 9H), 1.02 (d, J=6.5 Hz, 3H), 0.90 (t, J=8.6 Hz, 3H).

Example 89. Synthesis of (2R,3R)-methyl 3-((S)-1-((3R,4S,5S)-4-((tert-butoxycarbonyl)-(methyl)amino)-3methoxy-5-methylheptanoyl) pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate

To a solution of (2R,3R)-methyl-3-methoxy-2-methyl-3-((S)-nirrolidin-2-yl)nponoate (0.71 g, 2.99 mmol) and (3R,4S,5S )-4-((tert-butoxycarbonyl)(methyl)amino)-3-methoxy-5-methylhetanoic acid (1 g, 3.29 mmol) in DMF (10 ml) at 0°C was added diethyl cyanophosphonate (545 μl, 3 .59 mmol), then Et ₃ N (1.25 mL, 8.99 mmol) was added. The reaction mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 ml), washed with 1 N aqueous potassium hydrogen sulfate (20 ml), water (20 ml), saturated aqueous sodium hydrogen carbonate (20 ml) and saturated aqueous sodium chloride (20 ml), dried over sulfate sodium and evaporated in vacuum. The residue was purified by silica gel column chromatography, eluting with ethyl acetate/hexane (1:5 to 2:1), to give the title white solid (0.9 g, 62% yield). MSVR (ESI) m/z calc. for C25H46N2O7 [M+H]+: 487.33, found: 487.32.

Example 90. Synthesis of (S)-tert-butyl 2-((lR,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylpropan-2yl)amino)-2 -methyl-3-oxopropyl)pyrrolidine-1-carboxylate \ 7 I H

Vos [f Ph /O O CO ₂ Me

To a solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (100 mg, 0.347 mmol) and L-phenylalanine methyl ester hydrochloride (107.8 mg, 0.500 mmol) in DMF (5 ml) at 0°C was added diethyl cyanophosphonate (75.6 μl, 0.451 mmol), then Et3N (131 μl, 0.94 mmol). The reaction mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was then diluted with ethyl acetate (80 ml), washed with 1 N aqueous potassium hydrogen sulfate (40 ml), water (40 ml), saturated aqueous sodium hydrogen carbonate (40 ml) and saturated aqueous sodium chloride (40 ml), dried over Na ₂ SO ₄ H was evaporated in vacuo. The residue was purified by column chromatography (15-75% ethyl acetate/hexane) to give the title compound (130 mg, 83% yield) as a white solid. 1H NMR (500 MHz, CDCl ₃ ) δ 7.28 (dd, J=7.9, 6.5 Hz, 2H), 7.23 (t, J=7.3 Hz, 1H), 7.16 ( s, 2H), 4.81 (s, 1H), 3.98 - 3.56 (m, 5H), 3.50 (s, 1H), 3.37 (d, J=2.9 Hz, 3H ), 3.17 (dd, J=13.9, 5.4 Hz, 2H), 3.04 (dd, J=14.0, 7.7 Hz, 1H), 2.34 (s, 1H) , 1.81 - 1.69 (m, 2H), 1.65 (s, 3H), 1.51 1.40 (m, 9H), 1.16 (d, J=7.0 Hz, 3H) .

Example 91 General procedure for removing Boc functional groups using trifluoroacetic acid.

Trifluoroacetic acid (1.0 ml) was added to a solution of N-Boc amino acid (1.0 mmol) in methylene chloride (2.5 ml). After stirring at room temperature for 1-3 hours, the reaction

- 96 044827

the mixture was evaporated in vacuo. Co-evaporation with toluene gave an unprotected product, which was used without any further purification.

Example 92. Synthesis of (2R,3R)-methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3dimethylbutanamido )-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate

Boc-Val-OH

O BroP, DIPEA BocHN^/% CH ₂ C1 ₂ * j

To a solution of the product (2R,3R)-methyl 3-methoxy-3-((S)-1-((3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl)pyrrolidine-2 -yl)-2-methylpropanoate (715 mg, 1.85 mmol) without protecting groups and Boc-ValOH (1.2 g, 5.56 mmol) in DCM (20 ml) was added BroP (1.08 g, 2.78 mmol), then diisopropylethylamine (1.13 mL, 6.48 mmol) was added. The mixture was protected from light and stirred at 0°C for 30 min, then at room temperature for 48 h. The reaction mixture was diluted with ethyl acetate (50 ml), washed with 1 N aqueous potassium hydrogen sulfate (20 ml), water (20 ml) , saturated aqueous sodium hydrogen carbonate (20 ml) and saturated aqueous sodium chloride (20 ml), dried over Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by silica gel column chromatography, eluting with ethyl acetate/hexane (1:5 to 4:1) to give the title compound (0.92 g, 85% yield) as a white solid. MSVR (ESI) m/z calc. for C30H55N3O8 [M+H]+: 586.40, found: 586.37.

Example 93. Synthesis of (2R,3R)-methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)-2-methylpropanamido)- N,3-dimethylbutαnamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2methylpropanoate

To a solution of the unprotected product (2R,3R)-methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3 -dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2methylpropanoate (50 mg, 0.085 mmol) and perfluorophenyl 2-(dimethylamino)-2-methylpropanoate (74.5 mg, 0.085 mmol) 25 mmol) in DMF (2 ml) at 0°C was added DIPEA (44 μl, 0.255 mmol). The reaction mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was diluted with ethyl acetate (30 ml), washed with water (10 ml) and saturated aqueous sodium chloride (10 ml), dried over sodium sulfate and evaporated in vacuum. The residue was purified by silica gel column chromatography eluting with ethyl acetate/hexane (1:5 to 5:1) to give the title compound (50 mg, 100% yield). MSVR (ESI) m/z calc. for C ₃₁ H ₅₈ N ₄ O ₇ [M+H] ⁺ : 599, found: 599.

Example 94. Synthesis of (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)-2-methylpropαnamido)N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid

To a solution of (2R,3R)-methyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)-2-methylpropanαamido)N,3 -dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoate (50 mg, 0.0836 mmol) in 1,4-dioxane (3 ml) at 0-4° A solution of lithium hydroxide (14 mg, 0.334 mmol) in water (3 ml) was added dropwise over 5 min. The reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was acidified to pH 7 with 1 N HCl and evaporated in vacuo, and then used in the next step without further purification. MSVR (ESI) m/z calc. for C ₃₀ H ₅₇ N ₄ O ₇ [M+H] ⁺ : 585.41, found: 585.80.

Example 95. Synthesis of (2R,3R)-perfluorophenyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylαamino)-2methylpropanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methyl si-2-methylpropanoate

To a solution of (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylαamino)-2-methylpropanamido)-N,3 - 97 044827 dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoic acid (0.0836 mmol) and PFP (18.5 mg, 0.1 mmol) in DCM (2 ml) DIC (12.7 mg, 0.1 mmol) was added at

0°C. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was evaporated in vacuo and used in the next step without further purification. MSVR (ESI) m/z calc. for C ₃₆ H ₅₆ F ₅ N ₄ O ₇ [M+H]+: 751.40, found: 751.70.

Example 96 Synthesis of (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(4-hydroxy-3-nitrophenyl)propanoate

To a solution of Boc-L-tyrosine methyl ester (5 g, 16.9 mmol) in THF (50 ml) was added tert-butyl nitrite (10 ml, 84.6 mmol), then the reaction mixture was stirred for 5 hours at room temperature. The reaction mixture was evaporated and purified by silica gel column chromatography using ethyl acetate/hexane (1:10 to 1:5) to give the compound (4.5 g, 78% yield) as a yellow solid. MSVR (ESI) m/z calc. for C ₁₅ H ₂₁ N ₂ O ₇ [M+H] ⁺ : 341.13, found: 341.30.

Example 97. Synthesis of (B)-methyl 3-(3-amino-4-hydroxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoate

Pd/C (0.2 d) and stirred for 2 hours in a hydrogen atmosphere. The mixture was filtered and the filtrate was evaporated in vacuo to give the title compound (1.7 g, 95% yield) as a white solid. MSVR (ESI) m/z calc. for C ₁₅ H ₂₃ N ₂ O ₅ [M+H] ⁺ : 311.15, found: 311.30.

Example 98 Synthesis of compound A-1

To a solution of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioic acid (95 mg, 0.182 mmol) and (B)- methyl 3-(3-amino-4-hydroxyphenyl)-2-(tert-butoxycarbonylamino)propanoate (56.6 mg, 0.182 mmol) in DMF (5 ml) at 0°C and added EDC (128.5 mg, 0.338 mmol), followed by the addition of DIPEA (64 μl, 0.365 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The mixture was diluted with ethyl acetate (30 ml), washed with water (10 ml) and saturated aqueous sodium chloride (10 ml), dried over sodium sulfate and evaporated in vacuo. The residue was purified by silica gel column chromatography, eluting with DCM/MeOH (20:1 to 10:1), to give Compound A-1 (68 mg, 47% yield). MSVR (ESI) m/z calc. for C ₃₇ H ₅₅ N ₄ O ₁₅ [M+H] ⁺ : 795.36, found: 795.30.

Example 99. Synthesis of compound A-2

ΤΦΚ

DXM η ₂ ν

To a solution of compound A-1 (32 mg, 0.04 mmol) in DCM (3 ml) was added TFA (1 ml) at 0°C. The reaction mixture was warmed to room temperature and stirred for 30 min, diluted with toluene, concentrated, co-evaporated with toluene, and then used in the next step without further purification. MSVR (ESI) m/z calc. for C ₃₃ H ₄₇ N ₄ O ₁₅ [M+H] ⁺ : 795.36, found: 795.30.

Example 100. Synthesis of compound A-3

- 98 044827

DMA/DIPEA

To a solution of (2R,3R)-perfluorophenyl 3-((S)-1-((3R,4S,5S)-4-((S)-2-(2-(dimethylamino)-2methylpropanamido)-N,3- dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3methoxy-2-methylpropanoate (20 mg, 0.027 mmol) and compound A-2 (31.7 mg, 0.04 mmol) in DMA (2 ml) DIPEA (9 µl, 0.053 mmol) was added at 0°C. The reaction mixture was warmed to room temperature and stirred for 30 minutes. The mixture was evaporated in vacuo and purified by preparative HPLC (C-18, 250 mm x 10 mm, eluted with H2O/CH3CN (9 ml/min, from 90% water to 40% water in 40 min) to obtain compound A-3 (14 mg , 42% yield) HRMS (ESI) m/z calculated for C ₆₂ H ₁₀₁ N ₈ O ₁₉ [M+H]+: 1261.71 found: 1261.30.

Example 101. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)- 3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3phenylpropanoate

¹ O O CO ₂ Me

To a solution of the Boc-unprotected product (S)-tert-butyl 2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1oxo-3-phenylpropan-2-yl)amino) -2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.29 mmol) and (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-methoxy-5-methylheptane acid (96.6 mg, 0.318 mmol) in DMF (5 ml) at 0°C was added diethyl cyanophosphonate (58 μl, 0.347 mmol), then Et ₃ N (109 μl, 0.78 mmol) was added. The reaction mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 ml), washed with 1 N aqueous potassium hydrogen sulfate (40 ml), water (40 ml), saturated aqueous sodium hydrogen carbonate (40 ml) and saturated aqueous sodium chloride (40 ml), dried over Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by column chromatography (15-75% ethyl acetate/hexane) to give the title compound (150 mg, 81% yield) as a white solid. LC-MS (ESI) m/z calc. for C ₃₄ H ₅₅ N ₃ O ₈ [M+H] ⁺ : 634.40, found: 634.40.

Example 102. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl )amino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate.

To a solution of the Boc-unprotected product (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl)amino)- 3-methoxy-5-methylheptanoyl)-pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate (0.118 mmol) and Boc-Val-OH (51.8 mg, 0.236 mmol) in DCM ( 5 ml) at 0°C, BroP (70.1 mg, 0.184 mmol) was added, then diisopropylethylamine (70 μl, 0.425 mmol) was added. The mixture was protected from light and stirred at 0°C for 30 min, then at room temperature for 2 days. The reaction mixture was diluted with ethyl acetate (80 ml), washed with 1 N aqueous potassium hydrogen sulfate (40 ml), water (40 ml), saturated aqueous sodium hydrogen carbonate (40 ml) and saturated aqueous sodium chloride (40 ml), dried over Na ₂ SO ₄ and evaporated in vacuum. The residue was purified by column chromatography (20-100% ethyl acetate/hexane) to give the title compound (67 mg, 77% yield) as a white solid. LC-MS (ESI) m/z calc. for C ₃₉ H ₆₄ N ₄ O ₉ [M+H] ⁺ : 733.47, found: 733.46.

Example 103. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((6S,9S,12S, 13R)-12-((S)-sec-butyl)- 6, 9diisopropyl-13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecan-15-oil) pyrrolidin-2-yl)-3 methoxy-2-methylpropanamido)-3-phenylpropanoate

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To a solution of the Boc-unprotected product (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((tert-butoxycarbonyl )αmino)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3methoxy-2-methylpropanamido)-3-phenylpropanoate (0.091 mmol) and Boc-N-Me-Val-OH (127 mg, 0.548 mmol) in DMF (5 ml) at 0°C was added diethyl cyanophosphonate (18.2 μl, 0.114 mmol), then N-methylmorpholine (59 μl, 0.548 mmol) was added. The reaction mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 ml), washed with 1 N aqueous potassium hydrogen sulfate (40 ml), water (40 ml), saturated aqueous sodium hydrogen carbonate (40 ml) and saturated aqueous sodium chloride (40 ml), dried over sulfate sodium and evaporated in vacuum. The residue was purified by column chromatography (20-100% ethyl acetate/hexane) to give the title compound (30 mg, 39% yield) as a white solid. LC-MS (ESI) m/z calc. for C45H75N5O10 [M+H]+: 846.55, found: 846.56.

Example 104. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2 -((S)-3-methyl-2(methylamino)butanamido)butanamido)-3-methoxy-5-methyl-heptanoyl)pyrrolidin-2-yl)-3-methoxy-2methylpropanamido)-3-phenylpropanoate ¹ O ¹ O _h O _X O O CO ₂ Me

To a solution of (S)-methyl 2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6, 9-diisopropyl-13methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecan-15-oil) pyrrolidin-2-yl)-3methoxy-2 -methylpropanamido)-3-phenylpropanoate (75.0 mg, 0.0886 mmol) in methylene chloride (5 ml) was added trifluoroacetic acid (2 ml) at room temperature. After stirring at room temperature for 1 hour, the reaction mixture was evaporated in vacuum. Co-evaporation with toluene gave the unprotected title product, which was used without further purification.

Example 105. Synthesis of (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2- ((S)-3-methyl-2(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2methylpropanamido)-3-phenylpropanoic acid.

(S)-Methyl-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-(( S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methyl-heptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3phenylpropanoate (25 mg, 0.030 mmol ) in a mixture of conc. HCl (0.3 ml) and 1,4-dioxane (0.9 ml) were stirred at RT. within 35 min. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), evaporated and co-evaporated with EtOH/toluene (2:1) to obtain the title compound as a white solid (22 mg, ~100% yield ), which was used in the next step without further purification. LC-MS (ESI) m/z calc. for C ₃₉ H ₆₆ N ₅ O ₈ [M+H] ⁺ : 732.48, found: 732.60.

Example 106. Synthesis of (2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-azido-17-((R)-βtor-butyl) -11,14diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazacosan-20-oil) pyrrolidin-2-yl)-3- methoxy-2-methylpropanamido)-3-phenylpropanoic acid

To crude (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-(( S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)-pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (22 mg, 0.030 mmol) in a mixture of DMA (0.8 ml) and NaH ₂ PO ₄ buffer solution (pH 7.5, 1.0 M, 0.7 ml) 2,5-dioxopyrrolidin-1-yl 3-(2-( 2-azidoethoxy)ethoxy)propanoate (18.0 mg, 0.060 mmol) in four portions over 2 hours. The mixture was stirred overnight, evaporated and purified by column chromatography on SiO ₂ (CH ₃ OH/CH ₂ Cl ₂ /HOAc 1 :8:0.01) to give the title compound (22.5 mg, 82% yield). LC-MS (ESI) m/z calc. for C46H ₇₇ N8Oii [M+H] ⁺ : 917.56, found: 917.60.

Example 107. Synthesis of (2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-amino-17-((R)-βtor-butyl) -11,14diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazacosan-20-oil) pyrrolidin-2-yl)-3- methoxy-2-methylpropanamido)-3-phenylpropanoic acid

- 100 044827

K (2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-azido-17-((R)-sec-butyl)-11, 14-diisonpropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazacosan-20-oyl)pyrrolidin-2-yl)-3 methoxy- 2-Methylpropanamido)-3-phenylpropanoic acid (22.0 mg, 0.024 mmol) in methanol (5 ml) was added to the hydrogenation vessel with Pd/C (5 mg, 10% Pd, 50% humidity). After evacuating the air and passing H2 at 172 kPa (25 psi), the mixture was shaken for 4 hours, filtered through celite. The filtrate was evaporated to give the crude title product (~20 mg, 92% yield), which was used in the next step without further purification. ESI MS m/z ⁺ C46H79N6O11 (M+H), calc. 891.57, found 891.60.

Example 108. Synthesis of (S)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6 ,9-diisopropyl13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecan-15-oyl)pyrrolidin-2yl)-3-methoxy- 2-methylpropanamido)-3-phenylpropanoic acid

To a solution of (S)-methyl 2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6, 9-diisopropyl-13methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecan-15-oyl)pyrrolidin-2-yl)-3methoxy-2 -methylpropanamido)-3-phenylpropanoate (30 mg, 0.035 mmol) in THF (1.0 ml) was added LiOH in water (1.0 M, 0.8 ml). The mixture was stirred at room temperature for 35 minutes, neutralized with 0.5 MH ₃ PO ₄ to pH 6, evaporated and purified using SiO ₂ column chromatography (CH ₃ OH/CH ₂ Cl ₂ /HOAc 1:10:0 .01) to obtain the title compound (25.0 mg, 85% yield). LC-MS (ESI) m/z calculated for C44H74N5O10 [M+H] ⁺ : 832.54, found: 832.60.

Example 109. Synthesis of (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2- ((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid n «Υ ⁴ O I and ¹ O ¹ o _x o >) o CO ₂ H

To a solution of (S)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6.9 -diisopropyl-13-methoxy-2,2,5,11 -tetramethyl-4,7,10-trioxo-3 -oxa-5,8,11 -triazapentadecan-15-oil)pyrrolidin-2-yl)-3 - methoxy-2-methylpropanamido)-3-phenylpropanoic acid (25 mg, 0.030 mmol) in dioxane (2.0 ml) was added HCl (12.0 M, 0.6 ml). The mixture was stirred at room temperature for 30 min, diluted with dioxane (4 ml) and toluene (4 ml), evaporated and purified by column chromatography on a C-18 HPLC column, eluting with MeOH and water (L200 mmx F20 mm, v=9 ). ml/min, from 5% methanol to 40% methanol in 40 min) to obtain the title compound (20.0 mg, 90% yield). LC-MS (ESI) m/z calc. for C ₃₉ H ₆₆ N ₅ O ₈ [M+H] ⁺ : 732.48, found: 732.90.

Example 110. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((5S,8S,11S,14S,15R)-14-((S)-sec-butyl )-8,11diisopropyl-15-methoxy-5,7,13-trimethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazaheptadecane-17-oil) pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate

HATU ( HATU ( 0.135 g, 0.356 mmol, 2.0 eq.) and NMM (0.12 ml, 1.07 mmol, 6.0 eq.). The reaction mixture was stirred at 0°C for 10 min, then warmed to room temperature and stirred overnight. The mixture was diluted with DCM and washed with water and saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ , evaporated and purified by SiO ₂ column chromatography (20:1 DCM/MeOH) to give the title compound as a white foam solid (0.148 g, 88% yield). MS ESI m/z: calculated for C ₅₁ H ₇₉ N ₆ Oh [M+H] ⁺ 951.6, found 951.6.

Example 111. Synthesis of (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)- 2-((S)-2-amino-Nmethylpropanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)- 3-phenylpropanoate

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To the solution (8)-methyl 2-((2R,3R)-3-((S)-1-(5S,8S,11S,14S,l5R)-14-((S)-sec-butyl)-8 ,11-diisopropyl15-methoxy-5,7,13-trimethyl-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13-tetraazaheptadecan-17oyl)pyrrolidin-2-yl )-3-methoxy-2-methylpropanamido)-3-phenylpropanoate (0.148 g, 0.156 mmol, 1.0 equiv.) in MeOH (5 ml) was added Pd/C (0.100 g, 10% Pd/C, 50% humidity ) in a hydrogenation vessel. The mixture was shaken for 5 hours, then filtered through a pad of celite. The filtrate was evaporated to give the title compound as a white foamy solid (0.122 g, 96% yield). MS ESI m/z: calculated for C ₄₃ H ₇₃ N ₆ O ₉ [M+H]+ 817.5, found 817.5.

Example 112. Synthesis of (S)-2-((2R,3R)-3-((S)-1-((8S,11S,14S,17S,20S,21R)-20-((S)-second- bumΉl)-14,17diisopropyl-21-methoxy-8,11,13,19-tetramethyl-3,6,9,12,15,18-hexaoxo-5-propiolamido-4,7,10,13,16,19hexazatricos -1-in-23-oil)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (A-4)

To compound (8)-methyl 2-((2R,3R)-3-((S)-1-(3RAS,5S)-4-((S)-2-((S)-2-((S )-2-amino-N-methylpropanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate (20 mg, 0.027 mmol) in a mixture of DMA (2 ml) and 0.1 M Na ₂ HPO ₄ , pH 8.0 (1 ml), (8)-2,5-dioxopyrrolidin-1-yl 2-((8) -2-(2,2-dipropiolamidoacetamido)propanamido)propanoate (20.1 mg, 0.046 mmol) in three portions over 3 hours, and then the mixture was stirred for an additional 12 hours. The mixture was evaporated, and purified by reverse phase HPLC (200 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water in 40 min, v = 8 ml/min) to obtain the title compound (22.1 mg, 78% yield) . MS ESI m/z: calculated for C ₅₃ H ₈₀ N ₉ O ₁₃ [M+H] ⁺ 1050.58, found 1050.96.

Example 113 Synthesis of (Z)-4-hydrazinyl-4-oxobut-2-enoic acid, hydrochloride salt

Оу°уО NH ₂ NH ₂ *HCI о О \=Г DMA ' ΗΟ^^/νΗΝΗ,

To hydrazine hydrochloride (7.00 g, 102.1 mmol) in DMA (100 ml) was added maleic anhydride (10.01 g). The mixture was stirred overnight, evaporated and recrystallized in EtOH to give the title compound (12.22 g, 92% yield). MS ESI m/z: calculated for C ₄ H ₇ N ₂ O ₃ [M+H] ⁺ 131.04, found 131.20.

Example 114. Synthesis of (2S)-2-((2R,3R)-3-(2S)-1-((11S,14S,17S,18R)-17-((S)-sec-butyl)-11, 14 diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-1-((bis(2-(Z)-3-carboxyacrylhydrazinyl)phosphoryl)amino)-3,6-dioxa-10, 13,16-triazaicosan-20-oil) pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3phenylpropanoic acid (A-5)

To the compound (Z)-4-hydrazinyl-4-oxobut-2-enoic acid HCl salt (22.0 mg, 0.132 mmol) in a mixture of THF (5 ml) and DIPEA (10 μl, 0.057 mmol) was added at 0°C POCl ₃ (10.1 mg, 0.0665 mmol). After stirring at 0°C for 20 min, the mixture was warmed to room temperature and continued stirring for an additional 4 h.

Then (S)-2-((2R,3R)-3-((S)-1-((11S,14S,17S,18R)-1-amino-17-((S)-sec- butyl)11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oil) pyrrolidin-2 yl)- 3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (60 mg, 0.067 mmol) and DIPEA (20 μl, 0.114 mmol). The mixture was stirred at 50°C overnight, evaporated and purified by reverse phase HPLC (200 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water for 40 min, v=8 ml/min) to obtain the title compound (25.6 mg, 31% yield). MS ESI m/z: calculated for C ₅₄ H ₈₄ N ₈₈ O ₁₈ P [M+H] ⁺ 1195.59, found 1196.10.

Example 115. Synthesis of (S,E)-2-methyl-N-(3-methylbutan-2-ylidene)propan-2-sulfonamide δ Η

To a solution of (S)-2-methylpropane-2-sulfonamide (100 g, 0.825 mol, 1.0 eq.) in 1 L of THF was added

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Ti(OEt) ₄ (345 ml, 1.82 mol, 2.2 eq.) and 3-methyl-2-butanone (81 ml, 0.825 mol 1.0 eq.) under N ₂ at room temperature. The reaction mixture was heated to reflux for 16 hours, then cooled to room temperature and poured into ice water. The mixture was filtered and the filter layer was washed with EtOAc. The organic layer was separated, dried over anhydrous Na ₂ SO ₄ and evaporated to leave a residue which was purified by vacuum distillation (15-20 mmHg, 95°C) to give the title product (141 g, 90% yield) in the form of a yellow oil. 1H NMR (500 MHz, CDCl ₃ ) δ 2.54 - 2.44 (m, 1H), 2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, J=6 ,9, 5.1 Hz, 6H). ESI MS m/z calculated for C^NaNOS [M+Na]+ 212.12; found 212.11.

Example 116 Synthesis of (2S,3S)-2-azido-3-methylpentanoic acid

H,.Λ -/ ⁴ with ₂ n

To a solution of NaN ₃ (20.0 g, 308 mmol) in a mixture of water (50 ml) and dichloromethane (80 ml), cooled to 0°C, Tf ₂ O (10 ml, 59.2 mmol, 2.0 eq.). After addition, the reaction mixture was stirred at 0°C for 2 hours, then the organic phase was separated and the aqueous phase was extracted with dichloromethane (2x40 ml). The combined organic phases were washed with saturated NaHCO ₃ solution and used as is. A dichloromethane solution of trifluoroazide was added to a mixture of (L)-isoleucine (4.04 g, 30.8 mmol, 1.0 eq), K2CO3 (6.39 g, 46.2 mmol, 1.5 eq), CuSO ₄ -5H ₂ O (77.4 mg, 0.31 mmol, 0.01 eq.) in water (100 ml) and methanol (200 ml). The mixture was stirred at room temperature. for 16 hours. Organic solvents were removed under reduced pressure, and the aqueous phase was diluted with water (250 ml) and acidified to pH 6 with concentrated HCl, and diluted with phosphate buffer (0.25 M, pH 6.2, 250 ml). The aqueous layer was washed with EtOAc (5x100 ml) to remove the sulfonamide by-product and then acidified to pH 2 with concentrated HCl, extracted with EtOAc (3x150 ml). The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give the title product (4.90 g, 99% yield) as a colorless oil. ^1H NMR (500 MHz, CDC^) δ 12.01 (s, 1H), 3.82 (d, J=5.9 Hz, 1H), 2.00 (ddd, J=10.6, 8, 6, 5.5 Hz, 1H), 1.54 (d.sq.d., J=14.8, 7.5, 4.4 Hz, 1H), 1.36 - 1.24 (m, 1H ), 1.08 - 0.99 (m, 3H), 0.97 - 0.87 (m, 3H).

Example 117. Synthesis of DN-methylpipecolic acid '^CO ₂ H

To a solution of D-pipecolic acid (10.0 g, 77.4 mmol, 1.0 equiv.) in methanol (100 ml) was added formaldehyde (37% aqueous solution, 30.8 ml, 154.8 mmol, 2.0 eq.), then Pd/C (10 wt.%, 1.0 g). The reaction mixture was stirred under H2 (1 atm) overnight and then filtered through celite, washing the filter bed with methanol. The filtrate was evaporated under reduced pressure to give the title compound (10.0 g, 90% yield) as a white solid.

Example 118 Synthesis of (R)-perfluorophenyl 1-methylpiperidine-2-carboxylate

To a solution of DN-methylpipecolic acid (2.65 g, 18.5 mmol) in EtOAc (50 ml) was added pentafluorophenol (3.75 g, 20.4 mmol) and DCC (4.21 g, 20.4 mmol). The reaction mixture was stirred at room temperature for 16 hours and then filtered through celite. The layer on the filter was washed with 10 ml EtOAc. The filtrate was used for the next step without further purification or evaporation. MS ESI m/z calculated for C ₁₃ H ₁₃ F ₅ NO ₂ [M+H] ⁺ 309.08; found 309.60.

Example 119 Synthesis of perfluorophenyl 2-(dimethylamino)-2-methylpropanoate

To a solution of 2-(dimethylamino)-2-methylpropanoic acid (5.00 g, 38.10 mmol) in ethyl acetate (200 ml) at 0°C was added 2,3,4,5,6-pentafluorophenol (10.4 g , 57.0 mmol) followed by the addition of DIC (8.8 ml, 57.0 mmol). The reaction mixture was warmed to room temperature, stirred overnight and filtered. The filtrate was evaporated to give the title compound (12.0 g, >100% yield), which was used in the next step without further purification. ESI MS m/z calculated for C12H13F5NO2 [M+H] ⁺ 298.08; found 298.60.

Example 120 Synthesis of 2,2-diethoxyethanethanioamide

OEt s

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2,2-Diethoxyacetonitrile (100 g, 0.774 mol, 1.0 eq.) was mixed with an aqueous solution of (NH4)2S (48%, 143 ml, 1.05 mol, 1.36 eq.) in methanol (1.5 l) at room temperature. After stirring for 16 hours, the reaction mixture was evaporated and the residue was dissolved in dichloromethane, washed with saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ H and evaporated. The residue was triturated with a mixture of petroleum ether and dichloromethane solvents. After filtration, the desired title product was obtained as a white solid (100 g, 79% yield). 1H NMR (500 MHz, CDCl ₃ ) δ 7.81 (d, J=71.1 Hz, 2H), 5.03 (s, 1H), 3.73 (dq, J=9.4, 7.1 Hz, 2H), 3.64 (dq. J=9.4, 7.0 Hz, 2H), 1.25 (t, J=7.1 Hz, 6H).

Example 121 Synthesis of ethyl 2-(diethoxymethyl)thiazole-4-carboxylate

OEt

JL N ^CO ₂ Et A // * g of molecular sieves (3A) were added to a mixture of 2,2-diethoxyethanethanioamide (100 g, 0.61 mol, 1.0 eq.) and ethyl bromopyruvate (142 ml, 1.1 mol, 1.8 eq.) in 1 L of EtOH. The mixture was heated to reflux (internal temperature about 60°C) for 1 hour, then the ethanol was removed by rotary evaporation and the residue was dissolved in dichloromethane. The solid was filtered and the filtrate was evaporated and purified by column chromatography (PE/EtOAc 5:1-3:1) to give the title compound (thiazole carboxylate) (130 g, 82% yield) as a yellow oil.

Example 122 Synthesis of ethyl 2-formylthiazole-4-carboxylate

To a solution of 2-(diethoxymethyl)thiazole-4-carboxylate (130 g, 0.50 mol in acetone (1.3 L) was added 2 N HCl (85 ml, 0.165 mol 0.33 eq.) The reaction mixture was heated to boiling reflux (internal temperature about 60°C), monitored by TLC analysis until complete consumption of the starting material (about 1-2 hours).The acetone was removed under reduced pressure and the residue was dissolved in dichloromethane (1.3 L), washed with saturated solution NaHCO ₃ , water and saturated aqueous sodium chloride solution, and then dried over anhydrous Na ₂ SO _4. The solution was filtered and evaporated under reduced pressure. The crude product was purified by recrystallization from petroleum ether and diethyl ether to give the title compound as white solid (40 g, 43% yield) ¹ H NMR (500 MHz, CDCl ₃ ) δ 10.08 - 10.06 (m, 1H), 8.53 - 8.50 (m, 1H), 4, 49 (kv, J=7.1 Hz, 2H), 1.44 (t, J=7.1 Hz, 3H) ESI MS m/z calculated for C ₇ H ₈ NO ₃ S [M+H] ⁺ 186.01; found 186.01.

Example 123. Synthesis of ethyl 2-((R,E)-3-(((S)-tert-butylsulfinyl)imino)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate

To a solution of diisopropylamine (121 ml, 0.86 mol, 4.0 eq.) in dry THF (300 ml) was added nbutyllithium (2.5 M, 302 ml, 0.76 mol, 3.5 eq.) at -78°C in an N2 atmosphere. The reaction mixture was heated to 0°C for 30 minutes and then cooled again to -78°C. Add (S,E)-2-methyl-N-(3-methylbutan-2ylidene)propane-2-sulfonamide (57 g, 0.3 mol, 1.4 eq.) in THF (200 ml). The reaction mixture was stirred for 1 hour before OtrOisoPrE (168.5 g, 0.645 mol, 3.0 eq.) in THF (350 ml) was added dropwise. After stirring for 1 hour, ethyl 2-formylthiazole-4-carboxylate (40 g, 0.215 mol, 1.0 eq.) dissolved in THF (175 ml) was added dropwise and the resulting reaction mixture was stirred for 2 hours. Completion reactions were shown by TLC analysis. A mixture of acetic acid and THF (v/v 1:4, 200 ml) was added to the reaction mixture, then poured into ice water, extracted with EtOAc (4x500 ml). The organic phase was washed with water and saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by column chromatography (DCM/EtOAc/PE 2:1:2) to give the title compound (60 g, 74% yield) as a colorless oil. 1H NMR (500 MHz, CDCl ₃ ) δ 8.13 (s, 1H), 6.63 (d, J=8.2 Hz, 1H), 5.20 - 5.11 (m, 1H), 4, 43 (kv, J=7.0 Hz, 2H), 3.42 - 3.28 (m, 2H), 2.89 (dt, J=13.1, 6.5 Hz, 1H), 1.42 (t, J=7.1 Hz, 3H), 1.33 (s, 9H), 1.25 - 1.22 (m, 6H). ESI MS m/z calculated for C ₁₆ H ₂₆ NaN ₂ O ₄ S ₂ [M+Na]+ 397.13, found 397.11.

Example 124. Synthesis of ethyl 2-((1R,3R)-3-((S)-1,1-dimethylethylsulfinamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate.

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A solution of ethyl 2-((R,E)-3-(((S)-tert-butylsulfinyl)imino)-1-hydroxy-4-methylpentyl)thiazole-4carboxylate (23.5 g, 62.7 mmol), dissolved in THF (200 ml), cooled to -45°C. Ti(0Et)4 (42.9 mL, 188 mmol, 3.0 eq) was added slowly. After addition was complete, the mixture was stirred for 1 hour before NaBH ₄ (4.75 g, 126 mmol, 2.0 eq.) was added in portions. The reaction mixture was stirred at 45°C for 3 hours. TLC analysis showed incomplete consumption of some starting materials. HOAc/THF (1:4 v/v, 25 ml) was poured into the reaction mixture, followed by EtOH (25 ml). The reaction mixture was poured onto ice (100 g) and warmed to room temperature. After filtration through celite, the organic phase was separated and washed with water and saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by column chromatography (EtOAc/PE 1:1) to give the title product (16.7 g, 71% yield) as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.10 (s, 1H), 5.51 (d, J=5.8 Hz, 1H), 5.23 - 5.15 (m, 1H), 4 .41 (kv, J=7.0 Hz, 2H), 3.48 - 3.40 (m, 1H), 3.37 (d, J=8.3 Hz, 1H), 2.29 (t, J=13.0 Hz, 1H), 1.95 - 1.87 (m, 1H), 1.73 1.67 (m, 1H), 1.40 (t, J=7.1 Hz, 3H) , 1.29 (s, 9H), 0.93 (d, J=7.3 Hz, 3H), 0.90 (d, J=7.2 Hz, 3H). ESI MS m/z calculated for C ₁₆ H ₂₈ NaN ₂ O ₄ S2 [M+Na]+ 399.15, found 399.14.

Example 125. Synthesis of ethyl 2-((1R,3R)-3-amino-1-hydroxy-4-methylpentyl)thiazole-4carboxylate hydrochloride

Ύ' he HCIH^AxSpN _ooe(

S-U

To a solution of ethyl 2-((1R,3R)-3-((S)-1,1-dimethylethylsulfinamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate (6.00 g, 16.0 mmol, 1.0 eq.) in ethanol (40 ml), 4N was slowly added. HCl in dioxane (40 ml) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 2.5 hours, then evaporated and triturated with petroleum ether. The white title solid (4.54 g, 92% yield) was collected and used in the next step.

Example 126. Synthesis of ethyl 2-((1R,3R)-3-((2S,3S)-2-azido-3-methylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate

(2S,3S)-2-Azido-3-methylpentanoic acid (5.03 g, 28.8 mmol, 2.0 eq.) was dissolved in THF (120 ml) and cooled to 0°C, to which NMM ( 6.2 mL, 56.0 mmol, 4.0 eq.) and isobutyl chloroformate (3.7 mL, 28.8 mmol, 2.0 eq.). The reaction mixture was stirred at 0°C for 30 min and at room temperature. 1.0 hours, and then cooled again to 0°C. Ethyl 2((1R,3R)-3-amino-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate hydrochloride (4.54 g, 14.7 mmol, 1.0 eq.) was added portionwise. After stirring at 0°C for 30 min, the reaction mixture was heated to room temperature. and stirred for 2 hours. Water was added to the reaction mixture at 0°C, and the reaction mixture was extracted with ethyl acetate three times. The combined organic layers were washed with 1 N HCl, saturated NaHCO ₃ and saturated aqueous sodium chloride, dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by column chromatography (0-30% EtOAc/PE) to give a white solid of the title compound (4.55 g, 74% yield).

Example 127. Synthesis of ethyl 2-((1R,3R)-3-((2S,3S)-2-azido-3-methylpentanamido)-4-methyl-1-((triethylsilyl)oxy)pentyl)thiazol-4- carboxylate

To a solution of ethyl 2-((1R,3R)-3-((2S,3S)-2-azido-3-methylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate (5.30 g, 12 .8 mmol, 1.0 eq.) in CH ₂ Cl ₂ (50 ml) was added imidazole (1.75 g, 25.6 mmol, 2.0 eq.), then chlorotriethylsilane (4.3 ml, 25.6 mmol, 2.0 eq.) at 0°C. The reaction mixture was left to warm to room temperature. for 1 hour and stirred for one additional hour. A saturated sodium chloride solution was added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic phases were dried, filtered, evaporated under reduced pressure and purified by column chromatography with a gradient of 15-35% EtOAc in petroleum ether to give the title product (6.70 g, 99% yield) as a white solid. 1H NMR (500 MHz, CDCl ₃ ) δ 8.12 (s, 1H), 6.75 (d, J=8.0 Hz, 1H), 5.20 - 5.12 (m,

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1H), 4.44 (kv, J=7.0 Hz, 2H), 4.06 -3.97 (m, 1H), 3.87 (d, J=3.8 Hz, 1H), 2, 14 (d, J=3.8 Hz, 1H), 2.01 - 1.91 (m,

3H), 1.42 (t, J=7.1 Hz, 3H), 1.34 - 1.25 (m, 2H), 1.06 (d, J=6.8 Hz, 3H), 1, 00 - 0.93 (m, 18Н), 0.88 (dd, J=19.1,

6.8 Hz, 6H). ESI MS m/z calculated for C24H44N ₃ O4SSi [M+H]+ 526.28, found 526.28.

Example 128. Synthesis of ethyl 2-((1R,3R)-3-((2S,3S)-2-azudo-N,3-dimethylpentanamido)-4-methyl-1-((triethylsilyl)oxy)pentyl)thiazol- 4 carboxylates

A solution of ethyl 2-((1R,3R)-3-((2S,3S)-2-azido-3-methylpentanamido)-4-methyl-1-((triethylsilyl)oxy)pentyl)thiazole-4-carboxylate ( 5.20 g, 9.9 mmol, 1.0 eq) in THF (50 ml) was cooled to -45°C and KHMDS (1M in toluene, 23.8 ml, 23.8 mmol, 2.4 eq) was added .). The resulting mixture was stirred at -45°C for 20 minutes followed by the addition of methyl iodide (1.85 ml, 29.7 mmol, 3.0 eq.). The reaction mixture was warmed to room temperature for 4.5 hours, then EtOH (10 ml) was added to the reaction. The crude product was diluted with EtOAc (250 ml) and washed with saturated aqueous sodium chloride (100 ml). The aqueous layer was extracted with EtOAc (3x50 ml). The organic layers were dried, filtered, evaporated and purified by column chromatography with a gradient of 15-35% EtOAc in petroleum ether to give the title product (3.33 g, 63% yield) as a light yellow oil. ^1H NMR (500 MHz, CDC^) δ 8.09 (s, 1H), 4.95 (d, J=6.6 Hz, 1H), 4.41 (q, J=7.1 Hz, 2H ), 3.56 (d, J=9.5 Hz, 1H), 2.98 (s, 3H), 2.27 - 2.06 (m, 4H), 1.83 - 1.70 (m, 2H), 1.41 (t, J=7.2 Hz, 3H), 1.29 (ddd, J=8.9, 6.8, 1.6 Hz, 3H), 1.01 (d, J =6.6 Hz, 3H), 0.96 (dt, J=8.0, 2.9 Hz, 15H), 0.92 (d, J=6.6 Hz, 3H), 0.90 (d , J=6.7 Hz, ZN). ESI MS m/z calculated for C25H46N5O4SS1 [M+H] ⁺ 540.30, found 540.30.

Example 129. Synthesis of ethyl 2-((3S,6R,8R)-3-((S)-sec-butyl)-1O,10-diethyl-6-isopronyl-5-methyl-1((R)-1- methylpiperidin-2-yl)-1,4-dioxo-9-oxa-2,5-diaza-10-siladodecan-8-yl)thiazol-4-carboxylate

Dry Pd/C (10 wt.%, 300 mg) and ethyl 2-((1R,3R)-3-((2S,3S)-2-azido-N,3-dimethylpentanamido)-4methyl-1-(( triethylsilyl)oxy)pentyl)thiazole-4-carboxylate (3.33 g, 6.61 mmol) was added to (R)perfluorophenyl 1-methylpiperidine-2-carboxylate in EtOAc. The reaction mixture was stirred under hydrogen atmosphere for 27 hours and then filtered through a pad of celite, washing the filter cake with EtOAc. The combined organic portions were evaporated and purified by column chromatography with a gradient of 0-5% methanol in EtOAc to give the title product (3.90 g, 86% yield). ESI MS m/z calculated for C ₃₂ H ₅₉ N ₄ O ₅ SSi [M+H] ⁺ 639.39, found 639.39.

Example 130. Synthesis of ethyl 2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methyl piperidine-2-carboxamido)pentanamido)-1- hydroxy-4-methylpentyl)thiazole-4-carboxylate.

Ethyl 2-((3S,6R,8R)-3 -((S)-sec-butyl)-10,10-diethyl-6-isopropyl-5 -methyl-1 -((R)-1 -methylpiperidin2-yl )-1,4-dioxo-9-oxa-2,5-diaza-10-sidadodecan-8-yl)thiazol-4-carboxylate (3.90 g, 6.1 mmol) was dissolved in a deoxygenated AcOH/water/ THF (v/v/v 3:1:1, 100 ml), and stirred at room temperature. for 48 hours. The reaction mixture was then evaporated and purified by column chromatography (2:98 to 15:85 MeOH/EtOAc) to give the title compound (2.50 g, 72% yield over 2 steps). ESI MS m/z calculated for C ₂₆ H ₄₅ N ₄ O ₅ S [M+H] ⁺ 525.30, found 525.33.

Example 131. Synthesis of 2-((1R,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperudin-2-carboxamido)pentanamido)-1-hydroxy- 4-methylpentyl)thiazole-4-carboxylic acid

An aqueous solution of LiOH (0.4 N, 47.7 mL, 19.1 mmol, 4.0 eq.) was added to the solution of ethyl 2-((1R,3R)-3((2S,3S)-N,3- dimethyl 2-((R)-1-methylpiperudin-2-carboxamido)-pentanamudo)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate (2.50 g, 4.76 mmol, 1.0 eq. ) in dioxane (47.7 ml) at 0°C. The reaction mixture was stirred at room temperature. for 2 hours and then evaporated. Column chromatography on SiO ₂ (100% CH ₂ Cl ₂ then CH ₂ Cl ₂ /MeOH/NH ₄ OH 80:20:1) gave the title compound (2.36 g, 99% yield) as an amorphous solid matter. ESI MS m/z calculated for C ₂₄ H ₄₁ N ₄ O ₅ S [M+H] ⁺ 497.27, found 497.28.

Example 132. Synthesis of 2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpineridin-2-car-106 044827 boxamido )pentanamido)-4-methylpentyl)thiazole-4-carboxylic acid

To a solution of 2-(( 1 R,3R)-3-((2S,3 S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-1-hydroxy- 4-methylpentyl)thiazole-4-carboxylic acid (2.36 g, 4.75 mmol) in pyridine (50 ml) at 0°C, acetic anhydride (2.25 ml, 24 mmol) was added slowly. The reaction mixture was warmed to room temperature for 2 hours and stirred at room temperature. for 24 hours. The reaction mixture was evaporated and the residue was purified using reverse phase HPLC (C^ column, 50 mm (d) x 250 (mm), 50 ml/min, 10-90% acetonitrile/water for 45 min) with obtaining the title compound (2.25 g, 88% yield) as an amorphous white solid. ESI MS m/z calculated for C ₂ 6H43N ₄ O ₆ S [M+H] ⁺ 539.28, found 539.28.

Example 133. Synthesis of (1 E,3E)-3-((28,38)-X,3-dimethyl-2-((I)-1-methyl1[neridin-2-carboxamido)pentanamido)-4-methyl- 1-(4-(perfluorobenzoyl)thiazol-2-yl)pentyl acetate

To a solution of 2-(( 1 R,3R)-1 -acetoxy-3-((28,3 8)-M,3-dimethyl-2-((E)-1 -methylpiperidine-2-carboxamido)pentanamido)- 4-methylpentyl)thiazole-4-carboxylic acid (860 mg, 1.60 mmol, 1.0 eq.) in dichloromethane (20 ml) was added pentafluorophenol (440 mg, 2.40 mmol, 1.5 eq.) and N ,N'-diisopropylcarbodiimide (220 mg, 1.75 mmol, 1.1 eq.) at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. After removing the solvent under reduced pressure, the reaction mixture was diluted with EtOAc (20 ml), then filtered through celite. The filtrate was evaporated and purified by SiO ₂ column chromatography (1:10 to 1:3 EtOAc/DCM) to give the title compound (935.30 mg, 82% yield), which was directly used for the next step. MS ESI m/z calculated for C ₃ 2H ₄ 2F ₅ N ₄ O ₆ S [M+H] ⁺ 704.28, found 704.60.

Example 134. Synthesis of ethyl 2-((6S,9R,l^)-6-((8)-sec-butyl)-13,13-diethyl-9-isopropyl-2,3,3,8tetramethyl-4,7 - dioxo-12-oxa-2,5,8-triaza-13-silapentadecan-11-yl)thiazol-4-carboxylate

Dry Pd/C (10 wt.%, 300 mg) and ethyl 2-((1E,3E)-3-((28.38)-2-azido-N,3-dimethylpentanamido)-4methyl-1-(( triethylsilyl)oxy)pentyl)thiazole-4-carboxylate (3.33 g, 6.16 mmol) was added to perfluorophenyl 2-(dimethylamino)-2-methylpropanoate (--2.75 g, 1.5 equiv. crude) in EtOAc. The reaction mixture was stirred under hydrogen atmosphere for 27 hours and then filtered through a pad of celite, washing the filter cake with EtOAc. The combined organic portions were evaporated and purified by column chromatography with a gradient of 0-5% methanol in EtOAc to give the title product (3.24 g, 84% yield). ESI MS m/z calculated for C3iH ₅ 9N ₄ O ₅ SSi [M+H] ⁺ 626.39, found 626.95.

Example 135. Synthesis of ethyl 2-((1 E,3E)-3-((28.38)-2-(2-(dimethylamino)-2-methylpropanamido)-M,3dimethylpentanamido)-1-hydroxy-4-methylpentyl )thiazole-4-carboxylate

Ethyl 2-((6S,9R,l 1E)-6-((8)-sec-butyl)-13,13-diethyl-9-isopropyl-2,3,3,8-tetramethyl-4,7-dioxo12 -oxa-2,5,8-triaza-13-silapentadecan-11-yl)thiazol-4-carboxylate (3.20 g, 5.11 mmol) was dissolved in deoxygenated AcOH/water/THF (v/v). /vol. 3:1:1, 100 ml) and stirred at room temperature for 48 hours. The reaction mixture was then evaporated and purified using SiO ₂ column chromatography (2:98-15:85 MeOH/EtOAc) to obtain the indicated in the title compound (2.33 g, 89% yield). ESI MS m/z calculated for C25H ₄₅ N ₄ O ₅ S [M+H] ⁺ 512.30, found 512.45.

Example 136. Synthesis of 2-((1 E,3E)-3-((28.38)-2-(2-(dimethylamino)-2-methylpropanamido)-K,3-dimethylpentanamido)-1-hydroxy-4- methylpentyl)thiazole-4-carboxylic acid

An aqueous solution of LiOH (0.4 N, 47.7 mL, 19.1 mmol, 4.0 eq.) was added to the solution of ethyl 2-((lR,3R)-3

- 107044827 ((2S,3S)-2-(2-(dimethylamino)-2-methylpropanamido)-N,3-dimethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate (2.30 g, 4 .50 mmol, 1.0 eq.) in dioxane (50 ml) at 0°C. The reaction mixture was stirred at room temperature for 2 hours and then evaporated. Purification on a SiO ₂ column (100% CH ₂ Cl ₂ /MeOH/NH ₄ OH 80:20:1) gave the title compound (2.13 g, 98% yield) as an amorphous solid. ESI MS m/z calculated for C ₂₃ H ₄₁ N ₄ O ₅ S [M+H]+ 485.27, found 485.55.

Example 137. Synthesis of 2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12 -oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxylic acid

To a solution of 2-((1R,3R)-3-((2S,3S)-2-(2-(dimethylamino)-2-methylpronanamido)-N,3-dimethylpentanamido)-1-hydroxy-4-methylpentyl)triazole -4-carboxylic acid (2.10 g, 4.33 mmol) in pyridine (50 ml) at 0° C., acetic anhydride (2.25 ml, 24 mmol) was slowly added. The reaction mixture was warmed to room temperature for 2 hours and stirred at room temperature. for 24 hours. The reaction mixture was evaporated and the residue was purified using reverse phase HPLC (C ₁₈ column, 50 mm (d) x 50 (mm), 50 ml/min, 10-90% acetonitrile/water for 45 min) with obtaining the title compound (1.95 g, 86% yield) as an amorphous white solid. ESI MS m/z calculated for C25H43N4O6S [M+H]+ 526.28, found 526.80.

Example 138. Synthesis of perfluorophenyl 2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo- 12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxylate

To a solution of 2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopronyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12oxa-2 ,5,8-triazatetradecane-11-yl)thiazol-4-carboxylic acid (1.90 g, 3.61 mmol, 1.0 eq.) in dichloromethane (70 ml) was added pentafluorophenol (1.00 g, 5. 43 mmol, 1.5 eq.) and N,N'-diisonropylcarbodiimide (512 mg, 3.96 mmol, 1.1 eq.) at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. After removing the solvent under reduced pressure, the reaction mixture was diluted with EtOAc (80 ml), then filtered through celite. The filtrate was evaporated and purified by SiO ₂ column chromatography (1:10 to 1:3 EtOAc/DCM) to give the title compound (2.09 g, 84% yield), which was used directly for the next step. MS ESI m/z calculated for C ₃₁ H ₄₂ F ₅ N ₄ O ₆ S [M+H]+693.27, found 693.60.

Example 139. Synthesis of tert-butyl-2-(triphenylphosphoranylidene)propanoate

Ph ₃ P=(

CO ₂ *Vi

A mixture of tert-butyl 2-bromopropanoate (15.5 g, 74.1 mmol, 1.0 eq) and triphenylphosphine (19.4 g, 74.1 mmol, 1.0 eq) in dry acetonitrile (45 ml ) was stirred at room temperature for 18 hours. Acetonitrile was removed under reduced pressure and toluene was added to dissolve the white precipitate. The toluene was then decanted and the white solid was dissolved in dichloromethane (100 ml) and transferred to a separatory funnel. 10% NaOH (100 ml) was added to the funnel and after shaking the organic layer immediately turned yellow. The organic layer was separated and the aqueous layer was extracted once with dichloromethane (30 ml). The dichloromethane layers were combined and washed once with saturated aqueous sodium chloride (50 ml), then dried over Na ₂ SO ₄ , filtered and evaporated to give the ylide as a yellow solid (16.8 g, 58%).

Example 140. Synthesis of (S)-methyl 3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate.

To a mixture of Boc-L-Tyr-OMe (20.0 g, 67.7 mmol, 1.0 eq.), K2CO3 (14.0 g, 101.6 mmol, 1.5 eq.) and KI (1. 12 g, 6.77 mmol, 0.1 eq.) in acetone (100 mL), BnBr (10.5 mL, 81.3 mmol, 1.2 eq.) was slowly added. The mixture was then heated to reflux overnight. Water (250 ml) was added and the reaction mixture was extracted with EtOAc (3x100 ml). The combined organic layers were washed with saturated aqueous sodium chloride (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and purified by SiO ₂ column chromatography (4:1 hexane/EtOAc isomer mixture) to give a white solid as shown in title compound (26.12 g, 99% yield). 1H NMR (500 MHz,

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CDC13) δ 7.44-7.41 (m, 2H), 7.41-7.36 (m, 2H), 7.35-7.30 (m, 1H), 7.04 (d, J= 8.5 Hz, 2H), 6.93 - 6.89 (m, 2H),

5.04 (s, 2H), 4.97 (d, J=7.7 Hz, 1H), 4.55 (d, J=6.9 Hz, 1H), 3.71 (s, 3H), 3.03 (dd, J=14.4, 5.7 Hz, 2H), 1.44 (d,

J=18.6 Hz, 10H). ESI MS m/z calculated for C ₂₂ H ₂₇ NO ₅ Na [M+Na]+408.18, found 408.11.

Example 141 Synthesis of (S)-tert-butyl(1-(4-(benzyloxy)phenyl)-3-oxopropan-2-yl)carbamate

BocHN.

To a solution of (S)-methyl 3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate (26.1 g, 67.8 mmol, 1.0 eq.) in anhydrous dichloromethane ( 450 ml) at -78°C, DIBAL (1.0M in hexane isomer mixture, 163 ml, 2.2 eq.) was added over 1 hour. The mixture was stirred at -78°C for 3 hours and then 50 ml was added ethanol 1N was added drop by drop. HCl until pH 4 was reached. The resulting mixture was heated to 0°C. The layers were separated and the aqueous layer was further extracted with EtOAc (3x100 ml). The combined organic solutions were washed with saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ and evaporated. Trituration with PE/EtOAc and filtration gave the title compound as a white solid (18.3 g, 76% yield). MS ESI m/z calculated for C ₂₂ H ₂₇ NO ₅ Na [M+Na] ⁺ 378.11, found 378.11.

Example 142 Synthesis of (S,Z)-tert-butyl 5-(4-(benzyloxy)phenyl)-4-((tert-oxycarbonyl)amino)-2methylpent-2-enoate

BocHN.

*Q&O ₂ C^

ΌΒη (S)-tert-Butyl (1-(4-(benzyloxy)phenyl)-3-oxopropan-2-yl)carbamate (0.84 g, 2 mmol, 1.0 eq.) was dissolved in dry dichloromethane (50 ml), tert-butyl 2-(triphenylphosphoranylidene)propanoate (1.6 g, 4 mmol, 2.0 eq.) was added and the solution was stirred at room temperature for 1.5 h, the completion of the reaction was determined by TLC. Purification by column chromatography (10-50% EtOAc/hexane) gave the title compound (1.16 g, 98% yield).

Example 143 Synthesis of (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2methylpentanoate

BocHN.

^l BuO ₂ C^ (S,Z)-tert-Butyl 5-(4-(benzyloxy)phenyl)-4-((tert-oxycarbonyl)amino)-2-methylpent-2-enoate (467 mg x 1 mmol) dissolved in methanol (30 ml) and hydrogenated (1 atm) with Pd/C catalyst (10 wt.%, 250 mg) at room temperature overnight. The catalyst was filtered off and the filtrate was evaporated under reduced pressure to give the title compound (yield 379 mg, 99%).

Example 144 Synthesis of (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)-5-(4-hydroxy-3-nitrophenyl)-2-methylpentanoate

BocHN ^l BuO ₂ C

NO ₂ (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2-methylpentanoate (379 mg, 1 mmol, 1.0 eq.) was dissolved in THF (20 ml ), a solution of tert-butyl nitrite (315 mg, 3 mmol, 3.0 eq.) in THF (2 ml) was added. The reaction mixture was stirred at room temperature for 3 hours and then poured into water, extracted with EtOAc (2x50 ml) and the combined organic phases were washed with saturated aqueous sodium chloride (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. Purification by column chromatography (10-50% EtOAc/hexane) gave the title compound (300 mg, 71% yield).

Example 145 Synthesis of (4R)-tert-butyl 5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

BocHN. 'BuOjC sl ^{he 4} nh ₂ (4R)-tert-Butyl 4-((tert-butoxycarbonyl)amino)-5-(4-hydroxy-3-nitrophenyl)-2-methylpentanoate (200 mg, 0.47 mmol) was dissolved in EtOAc (30 ml) and mixed with palladium catalyst (10% on carbon, 100 mg), then hydrogenated (1 atm) at room temperature. for 2 hours. The catalyst was filtered and all volatiles were removed in vacuo to give the title compound (185 mg, 99%).

Alternatively, (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)-5-(4-hydroxy-3-nitrophenyl)-2methylpentanoate (56 mg x 0.132 mmol) was dissolved in EtOAc (20 ml) and mixed with Pd/C catalyst (10 wt.%, 50 mg) and hydrogenated (1 atm) at room temperature for 3 hours. The catalyst was filtered and all volatiles were removed in vacuo to give the title compound (52 mg, 99% yield). . MS ESI m/z calculated for C ₂₁ H ₃₅ N ₂ O ₅ [M+H] ⁺ 395.25, found 395.26.

- 109 044827

Example 146 Synthesis of (4R)-tert-butyl 4-((tert-butoxycarbonyl)amino)-5-(4-((tert-butyldimethylsilyl)oxy)-3-nitrophenyl)-2-methylpentanoate

In about cHN^^ ^_θ TV S 'ViOgO^ ΝΟ ₂

Imidazole (408 mg, 6 mmol) and tert-butylchlorodimethylsilane (602 mg, 4 mmol). The resulting solution was stirred at room temperature. for 3 hours. The reaction mixture was then washed with saturated aqueous sodium chloride (50 ml), dried over anhydrous Na ₂ SO ₄ , evaporated and purified by column chromatography (10% to 30% EtOAc/hexane mixture) to give the above title compound (344 mg, 64% yield).

Example 147 Synthesis of (4R)-tert-butyl 5-(3-amino-4-((tert-butyldimethylsilyl)oxy)phenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoatene

OTBS *ΒιιΟ ₂ Γ^^ NH ₂ (4R)-tert-Butyl 4-((tert-butoxycarbonyl)amino)-5-(4-((tert-butyldimethylsilyl)oxy)-3-nitrophenyl)-2-methylpentanoate ( 200 mg, 0.37 mmol) dissolved in EtOAc (30 ml), mixed with palladium catalyst (10 wt.%, on carbon, 100 mg) hydrogenated (1 atm) at room temperature. for 2 hours. The catalyst was filtered and all volatiles were removed in vacuo to give the title compound (187 mg, 99% yield).

Example 148. Synthesis of 2-(1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)-4-((2R)-5-(tert- butoxy)-2-((tert-butoxycarbonyl)amino)-4-methyl-5-oxopentyl)phenyl 1azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane -18-oata

EDC/DMA/DIPEA

To a solution of 1-azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oic acid (1.50 g, 3.85 mmol) and (4R )-tert-butyl 5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.75 g, 1.90 mmol) in DMA (40 ml) added EDC (2.05 g, 10.67 mmol) and DIPEA (0.70 ml, 4.0 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:5 to 1:1) to give the title compound (2.01 g, 82% yield, purity ~95% according to HPLC). ESI MS m/z calculated for C ₅₁ H ₈₅ N ₁₂ O ₁₇ [M+H]+ 1137.61, found 1137.90.

Example 149. Synthesis of (4R)-tert-butyl 5-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)3,6,39,42-tetramethyl- 2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18,19,20,21, 22,23,24,25,26, 27,29,30,32,33,35,36,37,38,39,40,41,42,43,44-hexatracontahydro-2H-benzo[b][1 ,14,17,20,31,34,37,4,7,10, 23,28,41,44]heptaoxa-heptazazacyclohexatetracontin-46-yl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

2-( 1 -Azido-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)-4-((2R)-5-(tert-butoxy)-2-( (tert-butoxycarbonyl)amino)-4-methyl-5-oxopentyl)phenyl 1-azido-14,17-dimethyl-12,15- 110 044827 dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18 -oate (900 mg, 0.79 mmol) was dissolved in EtOAc (30 ml), stirred with palladium catalyst (10 wt.%, on carbon, 100 mg) and hydrogenated (1 atm) at room temperature. for 4 hours. The catalyst was filtered and all volatiles were removed in vacuo to give 2(1-amino-14,17-dimethyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecanamido)-4 -((2R)-5-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-methyl-5-oxopentyl)phenyl 1-amino-14,17-dimethyl-12,15-dioxo- 3,6,9-trioxa-13,16-diazaoctadecane-18-oate (815 mg, 96% yield), which was used immediately without further purification. ESI MS m/z calculated for C ₅₁ H ₈₈ N ₈ O ₁₇ [M+H]+ 1085.62, found 1085.95.

To diamine (810 mg, 0.75 mmol) and 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinic acid (231 mg, 0.75 mmol) in DMA (10 ml), EDC (1.25 g, 6.51 mmol) and DIPEA (0.35 ml, 2.0 mmol) were added. The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:5 to 1:1) to give the title compound (844 mg, 83% yield, ~95% purity according to HPLC data). MS ESI m/z calculated for C ₆₃ H ₉₂ N ₁₀ O ₂₃ [M+H]+ 1357.63, found 1357.95.

Example 150. Synthesis of (2R)-1-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,39,42-tetramethyl-2, 5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18,19,20,21,22, 23,24,25,26,27,29,30,32,33, 35,36,37,38,39,40,41,42,43,44-hexatriacontahydro-2H-benzo[b][1,14 ,17,20,31,34,37,4,7,10,23,28,41,44]heptaoxaheptaazacyclohexatetracontin-46-yl)-4-carboxypentane-2-aminium

(4R)-tert-Butyl 5-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,39,42-tetramethyl2,5,8 ,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18,19,20,21,22,23,24 ,25,26,27,29,30,32,33,35, 36,37,38,39,40,41,42,43,44-hexatriacontahydro-2H-benzo[b][1,14,17, 20,31,34,37,4,7,10,23,28,41,44]gentaoxaheptaazacyclohexatetracontin-46-yl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (840 mg, 0.62 mmol) was dissolved in a mixture of CH ₂ Cl ₂ (6 ml) and TFA (4 ml). The mixture was stirred overnight, diluted with toluene (10 ml), evaporated to give the title compound (7.43 g, 100% yield, ~91% HPLC purity), which was used in the next step without further purification. MS ESI m/z calc. C ₅₄ H ₇₆ N ₁₀ O ₂₁ [M+H]+ 1200.51, found 1200.95.

Example 151. Synthesis of (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid

To a solution of (4R)-4-(2-(( 1 R,3R)-1 -acetoxy-3-((2S,3 S)-N,3-dimethyl-2-((R)-1 -methylpiperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-amino-4-hydroxyphenyl)-2-methylpentanoic acid (Huang Y. et al., Med Chem. #44, 249 ^th ACS National Meeting, Denver, CO, Mar. 2226, 2015; WO 2014009774) (100 mg, 0.131 mmol) in a mixture of DMA (10 ml) and NaH ₂ PO ₄ buffer solution (pH 7.5, 1.0 M, 0. 7 ml) 2,5-dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)ethoxy)propanoate (80.0 mg, 0.266 mmol) was added in four portions over 2 hours. The mixture was stirred overnight, evaporated and purified by preparative C ₁₈ HPLC (3.0 x 25 cm, 25 mL/min), eluting from 80% water/methanol to 10% water/methanol over 45 min to give the title compound (101.5 mg, 82% exit). LC-MS (ESI) m/z calc. for C45H ₇ oN ₉ OhS [M+H]+: 944.48, found: 944.70.

Example 152. Synthesis of (4R)-4-(2-((1R,3R)-1-acetoxu-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylπuπperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3-(2-(2-(2-aminoethoxy))ethoxy)propanamido)-4-hydroxyphenyl)-2- methylpentanoic acid

To a solution of (4R)-4-(2-(( 1 R,3R)-1 -acetoxy-3-((2S,3 S)-N,3-dimethyl-2-((R)-1 -methylpiperidin- 2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-(3-(2-(2-azidoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid (100 .0 mg, 0.106 mmol) in methanol (25 ml) containing 0.1% HCl, Pd/C (25 mg, 10% Pd, 50% humidity) was added to the hydrogenation vessel. After evacuating the air in the vessel and passing H ₂ at 35 psi, the mixture was shaken for 4 hours, filtered through celite. The filtrate was evaporated and purified by C ₁₈ preparative HPLC (3.0 x 25 cm, 25 ml/min), eluting from 85% water/methanol to 15% water/methanol over 45 min to give the title compound (77.5 mg, 79% yield). LC-MS (ESI) m/z calc. For

C ₄₅ H ₇₂ N ₇ O11S [M+H]+: 918.49, found: 918.60.

Example 153 Synthesis of (4R)-tert-butyl 5-(4-acetoxy-3-nitrophenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

BocHN 'BuO ₂ C

E-OAc ^no ₂

To a solution of compound 190 (107.1 mg, 0.252 mmol) in dichloromethane (4.0 ml) at 0° C., acetic anhydride (0.11 ml, 1.17 mmol) and triethylamine (0.16 ml) were successively added. The reaction mixture was then warmed to room temperature and stirred for 1 hour, diluted with dichloromethane and washed with water and saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The residue was purified by column chromatography (0-15% EA/PE) to give a colorless oil (120.3 mg, theoretical yield). ESI MS m/z calculated for C ₂₃ H ₃₅ N ₂ O ₈ [M+H] ⁺ 467.23, found 467.23.

Example 154 Synthesis of (4R)-tert-butyl 5-(4-acetoxy-3-aminophenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

ΝΗ ₂ (4R)-tert-Butyl 5-(4-acetoxy-3-nitrophenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (120.3 mg, 0.258 mmol) was dissolved in ethyl acetate (5 ml ) and acetic acid (0.5 ml). Pd/C (10 wt%, 10 mg) was added to the mixture, and the mixture was stirred under H2 at room temperature for 30 minutes and then filtered through a pad of celite, washing with ethyl acetate. The filtrate was evaporated and purified by column chromatography (0-25% EA/PE) to give a yellow oil (120.9 mg, theoretical yield). ESI MS m/z calculated for C ₂₃ H ₃₇ N ₂ O ₆ [M+H] ⁺ 437.26, found 437.28.

Example 155. Synthesis of (4R)-ethyl 5-(3-(4-(((benzyloxy)carbonyl)amino)butanamido)-4-((tert-butyldimethylsilyl)oxy)phenyl)-4-((tert-butoxycarbonyl) amino)-2-methylpentanoate /—rVoTBS

BocHN-< ^X =< o

HN-^NHCbz

_EtO2C—

2,5-Dioxopyrrolidin-1-yl 4-(((benzyloxy)carbonyl)amino)butanoate (0.396 g, 1.2 mmol) and (4R)ethyl 5-(3-amino-4-hydroxyphenyl)-4-( (tert-butoxycarbonyl)amino)-2-methylpentanoate (0.44 g, 1.2 mmol) was dissolved in EtOH (10 ml) and phosphate buffer solution (pH=7.5, 0.1 M, 2 ml) was added. The reaction mixture was stirred at room temperature overnight and then the solvent was removed under reduced pressure and the residue was purified by SiO ₂ column chromatography to obtain the title product (0.485 g, 70%). ESI: m/z: calculated for C ₃₁ H ₄₄ N ₃ O ₈ [M+H] ⁺ : 586.31, found 586.31.

Example 156. Synthesis of (4R)-ethyl 5-(3-(4-aminobutanamido)-4-((tert-butyldimethylsilyl)oxy)phenyl)4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

(4R)-Ethyl 5-(3-(4-(((benzyloxy)carbonyl)amino)butanamido)-4-((tert-butyldimethylsilyl)oxy)phenyl)-4-((tert-butoxycarbonyl)amino)-2 -Methyl pentanoate (0.35 g, 0.5 mmol) was dissolved in MeOH (5 ml) and then Pd/C (10 wt%, 35 mg) was added. The reaction mixture was stirred at room temperature under H ₂ overnight, then filtered through celite and the filtrate was evaporated under reduced pressure to give the title product (0.22 g, 79% yield). ESI MS m/z: calculated for C ₂₉ H ₅₂ N ₃ O ₆ Si [M+H] ⁺ : 566.35, found 566.35.

Example 157. Synthesis of 2-((6S,9S,12R,14R)-9-((S)-sec-butyl)-14-hydroxy-6,12-diisopropyl-2,2,5,11tetramethyl-4,7 ,10-trioxo-3-oxa-5,8,11-triazatetradecane-14-yl)thiazol-4-carboxylic acid

Pentafluorophenol (39 mg, 0.21 mmol) and DCC (32 mg, 0.154 mmol) were added to a solution of Boc-N-Me-L-Val-OH (33 mg, 0.14 mmol) in EtOAc. The reaction mixture was stirred at room temperature for 16 hours and then filtered through a pad of celite, washing with EtOAc. The filtrate was evaporated and redissolved in DMA (2 ml), and then 2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentane-112 044827 do)-1- hydroxy-4-methylpentyl)thiazole-4-carboxylic acid (52 mg, 0.14 mmol) and DIPEA (48.5 μl, 0.28 mmol). The reaction mixture was stirred at room temperature for 24 hours, then evaporated and purified by reverse phase HPLC ( _C18 column, 10-100% acetonitrile/water) to give the title compound (40.2 mg, 49% yield) . ESI MS m/z: calculated for C ₂₈ H ₄₉ N ₄ O ₇ S

[M+H]+: 585.32, found 585.32.

Example 158. Synthesis of 2-((6S,9S,12R,14R)-9-((S)-βtor-butyl)-6,12-di-isonropyl-2,2,5,11-tetramethyl4,7,10 ,16-tetraoxo-3,15-dioxa-5,8,11-triazaheptadecan-14-yl)thiazol-4-carboxylic acid γ no oac vs 0^iz ^c02H

2-((6S,9S,12R, 14R)-9-((S)-sec-Butyl)-14-hydroxy-6,12-diisopropyl-2,2,5,11-tetramethyl-4,7,10trioxo -3-oxa-5,8,11-triazatetradecane-14-yl)thiazol-4-carboxylic acid (40 mg, 0.069 mmol) was dissolved in pyridine (8 ml), acetic anhydride (20.4 mg, 0.2 ) mmol) at 0°C and the reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was evaporated and the residue was purified by SiO ₂ column chromatography with a DCM/MeOH gradient to give the title product (48.1 mg, ~100% yield). MS ESI m/z: calculated for C ₃₀ H ₅₁ N ₄ O ₈ S [M+H]+ 627.33, found 627.33.

Example 159. Synthesis of (4R)-4-(2-((6S,9S,12R,14R)-9-((S)-sec-butyl)-6,12-diisopropyl-2,2,5,11tetramethyl- 4,7,10,16-tetraoxo-3,15-dioxa-5,8,11-triazaheptadecan-14-yl)thiazol-4-carboxamido)-2methyl-5-phenylpentanoic acid

To a solution of 2-((6S,9S, 12R, 14R)-9-((S)-sec-butyl)-6,12-diisopropyl-2,2,5,11-tetramethyl-4,7,10,16tetraoxo Pentafluorophenol (21.2 mg, 0.115 mmol) and DCC ( 17.4 mg, 0.085 mmol). The reaction mixture was stirred at room temperature for 16 hours and then filtered through a pad of celite, washing with EtOAc. The filtrate was evaporated and redissolved in DMA (4 ml), and then (4R)-4-amino-2-methyl-5-phenylpentanoic acid (20.7 mg, 0.1 mmol) and DIPEA (26.8 µl, 0.154 mmol). The reaction mixture was stirred at room temperature for 24 hours, then evaporated and purified by reverse phase HPLC (C ₁₈ column, 10-100% acetonitrile/water) to give the title compound (63 mg, ~100% yield). MS ESI m/z: calculated for C ₄₂ H ₆₆ N ₅ O ₉ S [M+H]+ 816.45, found 816.45.

Example 160. Synthesis of hydrochloride (4R)-4-(2-((3S,6S,9R,11R)-6-((S)-βtor-butyl)-3,9-diisonropyl-8methyl-4,7,13 -trioxo-12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-2-methyl-5phenylpentanoic acid

(4R)-4-(2-((6S,9S, 12R,14R)-9-((S)-sec-Butyl)-6,12-diisopropyl-2,2,5,11-tetramethyl-4, 7,10,16tetraoxo-3,15-dioxa-5,8,11-triazaheptadecan-14-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (60 mg, 0.073 mmol) in ethyl acetate (3 ml) and hydrochloric acid (0.8 ml, 12 M). The mixture was stirred for 30 min and diluted with toluene (5 ml) and dioxane (5 ml). The mixture was evaporated and co-evaporated to dryness with dioxane (5 ml) and toluene (5 ml). The resulting crude title product (57.1 mg, 103% yield) was used in the next step without further purification. MS ESI m/z: calculated for C ₃₇ H ₅₈ N ₅ O ₇ S [M+H]+ 716.40, found 716.60.

Example 161. Synthesis of (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy)carbonyl)amino)propanamido)αcetamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino) -2-methylpentanoate

2-(2-(((Benzyloxy)carbonyl)amino)propanamido)acetic acid (0.2 g, 0.7 mmol), (4R)-tert-butyl-5-(3-amino-4-hydroxyphenyl)-4- ((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.19 g, 0.48 mmol) and HATU (0.18 g, 0.48 mmol) were dissolved in DCM (20 ml) followed by the addition of TEA ((134

- 113 044827 µl, 0.96 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and the residue was purified on a SiO ₂ column to give the title product (0.3 g, 95%). ESI: m/z: calculated for C ₃₄ H ₄₉ N ₄ O ₉ [M+H]+: 657.34, found 657.34.

Example 162 Synthesis of (4R)-tert-butyl-5-(3-(2-(2-aminopropanamido)acetamido)-4-hydroxyphenyl)-4((tert-butoxycarbonyl)amino)-2-methylpentanoate

In the hydrogenation vessel, Pd/C (0.1 g, 33 wt%, 50% moisture) was added to the (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy)))carbonyl solution )amino)propanamido)acetamido)-4-hydroxyphenyl)-4-((tertbutoxycarbonyl)amino)-2-methylpentanoate (0.3 g, 0.46 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H2, then filtered through celite (filtration aid) and the filtrate was evaporated to give the title compound (0.21 g, 87%) used for the next step without further purification. ESI: m/z: calculated for C ₂₆ H ₄₃ N ₄ O ₇ [M+H]+: 523.31, found 523.31.

Example 163. Synthesis of B-1 (tubulysin fragment having a bis-linker)

o θ %Ai'V ^Β -ι II

5-(3-(2-(2-Aminopropanamido)acetamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2methylpentanoate (0.11 g, 0.2 mmol), 4,17-dioxo -4 7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne1,30-dioic acid (0.104 g, 0.2 mmol), HATU (0.07 g, 0.2 mmol) dissolved in DCM (10 ml) followed by the addition of TEA (55 µl, 0.4 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give product B-1 (0.046 g, 23%). ESI: m/z: calculated for C ₄₈ H ₇₅ N ₆ O ₁₇ [M+H]+: 1007.51, found 1007.52. Example 164. Synthesis of B-2 (tubulysin fragment having a bis-linker)

Compound B-1 (0.046 g, 0.045 mmol) dissolved in DCM (1 ml), TFA (1 ml) was added and the reaction mixture was stirred at room temperature for 2 hours, evaporated and co-evaporated with DCM/toluene to obtain the crude compound B-2 (38.6 mg, 100% yield), used in the next step without additional purification. ESI: m/z: calculated for C ₃₉ H ₅₉ N ₆ O ₁₅ [M+H]+: 851.40, found 851.95.

Example 165. Synthesis of B-3 (analog of tubulisin having a bis-linker)

To a solution of compound B-2 (38.6 mg, 0.045 mmol) in DMA (4 ml) was added perfluorophenyl 2-114 044827 ((6S,9R,11R)-6-((S)-sec-butyl)-9- isopropyl 2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxylate (31.14 mg, 0.045 mmol) , then DIPEA (28 μl, 0.159 mmol) was added and the reaction mixture was stirred overnight. The solution was then evaporated and purified using HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN in 45 min, C-18 column, 10 mm (l) x 250 mm (l), 9 ml/min) with obtaining the title product (7.9 mg, 13%). ESI: m/z: calculated for C ₆₄ H ₉₉ N ₁₀ O ₂₀ S [M+H]+: 1359.67, found 1359.62.

Example 166. Synthesis of (4R)-tert-butyl-5-(3-(2-(((benzyloxy)carbonyl)amino)-3-methylbutanamido)-4hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2 -methyl pentanoate

HATU, TEA, DXM

(4R)-tert-Butyl-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.2 g, 0.51 mmol), 2-(( (benzyloxy)carbonyl)amino)-3-methylbutanoic acid (0.13 g, 0.51 mmol), HATU (0.2 g, 0.51 mmol) was dissolved in DCM (20 ml) followed by the addition of TEATEA (110 µl , 0.8 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was then removed under reduced pressure and purified by a SiO ₂ column to give the title product 12 (0.29 g, 90%). ESI: m/z: calculated for C ₃₄ H ₅₀ N ₃ O ₈ [M+H] ⁺ : 628.35, found 628.35.

Example 167 Synthesis of (4R)-tert-butyl-5-(3-(2-amino-3-methylbutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

In a hydrogenation vessel, add Pd/C (0.1 g, 33 wt%, 50% humidity) to a solution of (4R)-tert-butyl-5-(3-(2-(((benzyloxy)carbonyl)amino)-3 -methylbutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.29 g, 0.46 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H ₂ then filtered through Celite (auxiliary filter). The filtrate was evaporated to give the title compound (0.23 g, 100%) and used in the next step without further purification. ESI: m/z: calculated for C ₂₆ H ₄₄ N ₃ O ₆ [M+H] ⁺ : 494.64, found 494.64.

Example 168. Synthesis of (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy)carbonyl)amino)propanamido)-3methylbutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl) )amino)-2-methylpentanoate

(4R)-tert-Butyl-5-(3-(2-amino-3-methylbutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.23 g, 0. 46 mmol), 2-(((benzyloxy)carbonyl)aminopropanoic acid (0.10 g, 0.46 mmol) and HATU (0.18 g, 0.46 mmol) were dissolved in DCM (20 ml) followed by the addition of TEATEA (110 µl, 0.8 mmol) The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product (0.3 g, 95%).ESI: m /z: calculated for C ₃₇ H ₅₅ N ₄ O ₉ [M+H] ⁺ : 699.39, found 699.35.

Example 169 Synthesis of (4R)-tert-butyl-5-(3-(2-(2-aminopropanamido)-3-methylbutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

In a hydrogenation vessel, Pd/C (0.1 g, 33 wt%, 50% humidity) was added to the (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy)))carbonyl) solution. amino)propanamido)-3-methylbutanamido)-4-hydroxyphenyl)4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.3 g, 0.43 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H ₂ then filtered through Celite (filtration aid) and the filtrate was evaporated to give the title compound (0.22 g, 93%) which was used in the next step without further purification. ESI: m/z: calculated for C ₂₉ H ₄₉ N ₄ O ₇ [M+H] ⁺ : 565.35, found 565.31.

Example 170. Synthesis of B-4 (tubulysin fragment having a bis-linker)

- 115 044827

(4R)-tert-Butyl-5-(3-(2-(2-aminopropanαamido)-3-methylbutαnamido)-4-hydroxyphenyl)-4-((tertbutoxycarbonyl)amino)-2-methylpentanoate (0.05 g, 0.09 mmol), 11,14-dioxo-4,7,18,21-tetraoxa-10,15diazatetracos-12-yne-1,24-dioic acid (0.038 g, 0.09 mmol), HATU (0.067 g , 0.18 mmol) was dissolved in DCM (10 ml) followed by the addition of TEA (55 μl, 0.4 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give product B-4 (0.01 g, 12%). ESI: m/z: calculated for C ₄₇ H ₇₃ N ₆ O ₁₅ [M+H]+: 961.51, found 961.52.

Example 171. Synthesis of B-5 (tubulysin fragment having a bis-linker)

About η

TPA/DXM

ΒμΗΝ

and s

Compound B-4 (0.01 g, 0.01 mmol) was dissolved in DCM (1 ml) followed by TFA (0.8 ml). The reaction mixture was stirred at room temperature for 2 hours and evaporated, obtaining compound B-5 (10 mg) for the next step without further purification. ESI: m/z: calculated for C38H56N6O13 [M+H]+: 804.39, found 804.65.

Example 172. Synthesis of B-6 (analog of tubulisin having a bis-linker)

To a solution of compound B-5 (~10 mg) in DMA (4 ml) was added pentafluoric acid compound (6.92 mg, 0.01 mmol) and DIPEA (3.4 μl, 0.02 mmol). The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN after 45 min, C-18 column, 10 mm (d) x 250 mm (l), 9 ml/min) to obtain product B-6 (8.1 mg, 62%). ESI: m/z: calculated for C ₆₃ H ₉₇ N ₁₀ O ₁₈ S [M+H]+: 1313.66, found 1313.66.

Example 173. Synthesis of B-7 (tubulysin fragment having a bis-linker)

(4R)-tert-Butyl-5-(3-(2-(2-aminopropanαamido)acetamido)-4-hydroxyphenyl)-4-((tert-butoxyαr- 116 044827 bonyl)amino)-2-methylpentanoate (0. 21 g, 0.4 mmol) 11,14-dioxo-4,7,18,21-tetraoxa-10,15-diazatetracos-12-yne-1,24-dioic acid (0.17 g, 0.4 mmol ), HATU (0.15 g, 0.4 mmol) was dissolved in DCM (10 mL), followed by the addition of TEA (110 μL, 0.8 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give product B-7 (0.126 g, 34%). ESI: m/z: calculated for C ₄₄ H ₆₇ N ₆ O ₁₅ [M+H]+: 919.46, found 919.46.

Example 174. Synthesis of B-8 (tubulysin fragment having a bis-linker)

Compound B-7 (0.041 g, 0.045 mmol) was dissolved in DCM (1 ml), followed by the addition of TFA (1 ml). The reaction mixture was stirred at room temperature for 2 hours and evaporated, yielding compound B-8, which was used in the next step without further purification. ESI: m/z: calculated for C ₃₅ H ₅₁ N ₆ O ₁₃ [M+H] ⁺ : 763.35, found 763.80.

Example 175. Synthesis of B-9 (analog of tubulisin having a bis-linker)

To a solution of compound B-8 (9.1 mg, 0.012 mmol) in DMA (1 ml) was added pentafluoric acid compound (8.3 mg, 0.012 mmol) and DIPEA (1.4 μl, 0.008 mmol). The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN after 45 min, C-18 column, 10 mm (d) x 250 mm (l), 9 ml/min) to obtain the title compound B-9 (4.7 mg, 31%). ESI: m/z: calculated for C ₆₀ H ₉₁ N ₁₀ O ₁₈ S [M+H] ⁺ : 1271.62, found 1271.62.

Example 176. Synthesis of (4R)-tert-butyl-5-(3-(2-(((benzyloxy)carbonyl)amino)propanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

(4R)-tert-Butyl-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.3 g, 0.76 mmol), 2-(( (benzyloxy)carbonyl)aminopropanoic acid (0.17 g, 0.76 mmol), HATU (0.29 g, 0.76 mmol) were dissolved in DCM (20 ml) followed by the addition of TEA (110 μl, 0.8 mmol The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product (0.43 g, 95%).ESI: m/z: calculated for C ₃₂ H ₄₆ N ₃ O ₈ [M+H] ⁺ : 600.32, found 600.32.

Example 177 Synthesis of (4R)-tert-butyl-5-(3-(2-aminopropanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

In the hydrogenation vessel, add Pd/C (0.1 g, 33 wt.%, 50% humidity) to a solution of (4R)-tert-butyl-5-(3-(2-(((benzyloxy)carbonyl)amino)propanamido )-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.3 g, 0.5 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H ₂ and then filtered through celite (filtration aid). The filtrate was evaporated to give the title compound (0.24 g, 100%), which was used in the next step without further purification. ESI: m/z: calculated for C ₂₄ H ₄₀ N ₃ O ₆ [M+H] ⁺ : 466.28, found

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466.28.

Example 178. Synthesis of (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy)carbonyl)amino)propanamido)propanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino) -2-methylpentanoate

(4R)-tert-Butyl-5-(3-(2-aminopropanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2methylpentanoate (0.24 g, 0.5 mmol), 2- (((benzyloxy)carbonyl)amino)propanoic acid (0.11 g, 0.5 mmol) and HATU (0.2 g, 0.5 mmol) were dissolved in DCM (20 ml) followed by the addition of TEA (110 μl, 0.8 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product (0.28 g, 85%). ESI: m/z: calculated for C ₃₅ H ₅₁ N ₄ O9 [M+H]+: 671.36, found 671.35.

Example 179 Synthesis of (4R)-tert-butyl-5-(3-(2-(2-aminopropanamido)propanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

Pd/C (0.028 g, 10 wt%, 50% humidity) was added to the (4R)-tert-butyl-5-(3-(2-(2-(((benzyloxy))carbonyl)amino) solution to the hydrogenation flask. propanamido)propanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.28 g, 0.42 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H ₂ and then filtered through celite (filtration aid). The filtrate was evaporated to give the title compound (0.18 g, 100%), which was used in the next step without further purification. ESI: m/z: calculated for C ₂₇ H ₄₅ N ₄ O ₇ [M+H] ⁺ : 437.32, found 437.31.

Example 180. Synthesis of B-10 (tubulisin fragment having a bis-linker)

(4R)-tert-Butyl-5-(3-(2-(2-aminopropanamido)propanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.064 g, 0.12 mmol) 11,14-dioxo-4,7,18,21-tetraoxa-10,15diazatetracos-12-yne-1,24-dioic acid (0.042 g, 0.097 mmol) and HATU (0.073 g, 0.194 mmol) were dissolved in DCM (10 ml) followed by TEA (27.5 µl, 0.2 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product B-10 (0.074 g, 82%). ESI: m/z: calculated for C ₄₅ H ₆₉ N ₆ O ₁₅ [M+H] ⁺ : 933.47, found 933.46.

Example 181. Synthesis of B-11 (tubulisin fragment having a bis-linker)

Compound B-10 (0.074 g, 0.08 mmol) was dissolved in DCM (1 ml), followed by the addition of TFA (1 ml). The reaction mixture was stirred at room temperature for 2 hours and evaporated to obtain compound B-11, which was used in the next step without further purification.

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Example 182. Synthesis of B-12 (analog of tubulisin having a bis-linker)

To a solution of compound B-11 (62.08 mg, 0.08 mmol) in DMA (1 ml) was added pentafluoride compound (55.36 mg, 0.08 mmol), then DIPEA (27 μl, 0.16 mmol) was added ), the reaction mixture was stirred overnight. The solution was then evaporated and purified using HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN in 45 min, C-18 column, 10 mm (l) x 250 mm (l), 9 ml/min) with obtaining the title product B-12 (20 mg, 20%). ESI: m/z: calculated for C ₆₀ H ₉₁ N ₁₀ O ₁₈ S [M+H]+: 1285.63, found 1285.63.

Example 183. Synthesis of B-13 (tubulisin fragment having a bis-linker). (4R)-tert-butyl-5-(3amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.19 g, 0.48 mmol), 11,14dioxo-4, 7,18,21-tetraoxa-10,15-diazatetracos-12-yne-1,24-dioic acid (0.173 g, 0.4 mmol) and HATU (0.3 g, 0.8 mmol) were dissolved in DCM ( 50 ml), then TEA (110 µl, 0.8 mmol) was added

The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product B-13 (0.25 g, 80%). ESI: m/z: calculated for C ₃₉ H ₅₉ N ₄ O ₁₃ [M+H] ⁺ : 791.40, found 791.40.

Example 184. Synthesis of B-14 (tubulisin fragment having a bis-linker)

Compound B-13 (0.1 g, 0.14 mmol) was dissolved in DCM (1 ml) and then TFA (0.8 ml) was added. The reaction mixture was stirred at room temperature for 2 hours and then evaporated, yielding compound B-14, which was used in the next step without further purification.

Example 185. Synthesis of B-15 (analog of tubulisin having a bis-linker)

To a solution of compound B-14 (88.76 mg, 0.14 mmol) in DMA (1 ml) was added pentafluoride compound (96.88 mg, 0.14 mmol), then DIPEA (47.5 μl, 0.14 mmol) was added. 28 mmol), the reaction mixture was stirred overnight. The solution was then evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN in 45 min, C-18 column, 10 mm (l) x 250 mm (l), 9 ml/min) for obtaining the title product B-15 (40 mg, 25%). ESI: m/z: calculated for

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C55H83N8O16S [M+H]+: 1143.56, found 1143.56.

Example 186. Synthesis of (4R)-tert-butyl-5-(3-(4-(((benzyloxycarbonyl)amino)butanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2- methyl pentanoate o

BoHH .OH and l XHCbz . ί Г| T HO -- X _{HA4Uj TEA)} D _CM (4R)-tert-Butyl-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.2 g, 0.5 mmol), 4-(((benzyloxy)carbonyl)amino)butanoic acid (0.12 g, 0.5 mmol) and HATU (0.2 g, 0.5 mmol) were dissolved in DCM (50 ml) , and then TEA (110 μL, 0.8 mmol) was added. The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product (0.26 g, 85%). ESI: m/z: calculated for C ₃₃ H ₄₈ N ₃ O ₈ [M+H]+: 614.34, found 614.34.

Example 187 Synthesis of (4R)-tert-butyl-5-(3-(4-aminobutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate

In the hydrogenation flask, add Pd/C (0.028 g, 10 wt.%, 50% humidity) to a solution of (4R)tert-butyl-5-(3-(4-(((benzyloxy)carbonyl)amino)butanamido) -4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (0.09 g, 0.15 mmol) in MeOH (10 ml). The mixture was shaken overnight at 1 atm H2 and then filtered through celite (filtration aid). The filtrate was evaporated to give the title compound (0.07 g, 100%), which was used in the next step without further purification. ESI: m/z: calculated for C ₂₅ H ₄₂ N ₃ O ₆ [M+H]+: 480.30, found 480.31.

Example 188. Synthesis of B-16 (tubulisin fragment having a bis-linker)

HATU, TEA, DXM I

(4R)-tert-Butyl-5-(3-(4-aminobutanamido)-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2methylpentanoate (39 mg, 0.08 mmol), 11,14- dioxo-4,7,18,21-tetraoxa-10,15-diazatetracos-12-yne-1,24dioic acid (43 mg, 0.1 mmol) and HATU (30.4 mg, 0.08 mmol) were dissolved in DCM (20 ml) followed by TEA (22 µl, 0.16 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column to give the title product B-16 (42 mg, 60%). ESI: m/z: calculated for C ₄₃ H ₆₆ N ₅ O ₁₄ [M+H]+: 876.45, found 876.40.

Example 189. Synthesis of B-17 (tubulisin fragment having a bis-linker)

Compound B-16 (17 mg, 0.019 mmol) was dissolved in DCM (0.8 mL) followed by TEA (0.5 mL). The reaction mixture was stirred at room temperature for 2 hours and then evaporated to give compound B-17 (17 mg, >100%), which was used in the next step without further purification. ESI: m/z: calculated for C ₃₄ H ₅₀ N ₅ O ₁₂ [M+H]+: 720.34, found 720.70.

Example 190. Synthesis of B-18 (analog of tubulisin having a bis-linker)

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To a solution of compound B-17 (13.6 mg, 0.019 mmol) in DMA (1 ml) was added pentafluoride active acid compound (13 mg, 0.019 mmol) and DIPEA (6.4 μl, 0.038 mmol). The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H2O gradient (10% MeCN to 70% MeCN in 45 min, C-18 column, 10 mm (l) x 250 mm (l), 9 ml/ min) to obtain the title product B-18 (9.9 mg, 42%). ESI: m/z: calculated for C ₅₉ H ₉₀ N ₉ O ₁₇ S [M+H]+: 1228.61, found 1228.60.

Example 191. Synthesis of (4R)-tert-butyl-4-((tert-butoxycarbonyl)amino)-5-(3-(4-(2,5-dioxo-2,5dihydro-1H-pyrrol-1-yl) butanamido)-4-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)phenyl)-2-methylpentanoate

(4R)-tert-Butyl-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (68 mg, 0.17 mmol), 4-(2.5 -Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (94.5 mg, 0.52 mmol) and HATU (161.5 mg, 0.425 mmol) were dissolved in DCM (50 ml) followed by by adding TEA (73 µl, 0.52 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column eluting with EtOAc/DCM (1:10) to give the title product (98 mg, 80%). ESI: m/z: calculated for C ₃₇ H ₄₉ N ₄ O11 [M+H]+: 725.33, found 725.34.

Example 192. Synthesis of (2R)-4-carboxy-1-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)4-((4-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)phenyl)pentan-2-amine, TPA salt

(4R)-tert-Butyl-4-((tert-butoxycarbonyl)amino)-5 -(3 -(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)butanamido) -4-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)phenyl)-2-methylisopropylpentanoate (98 mg, 0.135 mmol) was dissolved in DCM (5 ml) followed by the addition of TFA (3 ml). The reaction mixture was stirred at room temperature for 2 hours and then evaporated to give the title compound (95 mg, >100% yield), which was used in the next step without further purification. ESI: m/z: calculated for C ₂₈ H ₃₃ N ₄ O ₉ [M+H]+: 569.22, found 569.60.

Example 193. Synthesis of (4R)-4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-2,3,3,8-tetramethyl-4,7, 13-trioxo12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-5-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)butanamido)-4-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)phenyl)-2-methylpentanoic acid (B-19)

To a solution of (2R)-4-carboxy-1-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)-4-((4- 121 044827 (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)phenyl)pentan-2-amine, TPA salt (76.9 mg, 0.135 mmol) in DMA (1 ml) pentafluoric acid compound (44 mg, 0.06 mmol) and DIPEA (45.8 μl, 0.27 mmol) were added. The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN after 45 min, C-18 column, 10 mm (d) x 250 mm (l), 9 ml/min) to obtain the title product B-19 (37 mg, 55%). ESI: m/z: calculated for C53H73N8O14S [M+H]+: 1077.49, found 1077.50.

Example 194. Synthesis of (4R)-tert-butyl-4-((tert-butoxycarbonyl)amino)-5-(3-(3-(2-(2-(2-(2,5-dioxo2,5-dihydro -1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-4-((3-(2-(2-(2-(2,5-dioxo-2,5-dihydro1 H-pyrrol-1 yl)ethoxy)ethoxy)ethoxy)propanoyl)oxy)phenyl)-2-methylpentanoate

(4R)-tert-butyl-5-(3-amino-4-hydroxyphenyl)-4-((tert-butoxycarbonyl)amino)-2-methylpentanoate (100 mg, 0.25 mmol), 3-(2-( 2-)(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanoic acid (75 mg, 0.25 mmol) and HATU (190 mg, 0.5 mmol) was dissolved in DCM (50 ml) followed by the addition of TEA (73 µl, 0.5 mmol). The reaction mixture was stirred at room temperature overnight, evaporated under reduced pressure and purified on a SiO ₂ column eluting with EtOAc/DCM (1:3) to give the title product (180.05 mg, 75%). ESI: m/z: calculated for C ₄₇ H ₆₉ N ₄ O ₁₇ [M+H]+: 961.45, found 961.81.

Example 195. Synthesis of (2R)-4-carboxy-1 -(3 -(3 -(2-(2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl )ethoxy))ethoxy)ethoxy)propanamido)-4-((3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy )ethoxy)propanoyl)oxy)phenyl)pentan-2-amine, TPA salt

(4R)-tert-Butyl-4-((tert-butoxycarbonyl)αmino)-5-(3-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H ))pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-4-((3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl )ethoxy)ethoxy)ethoxy)propanoyl)oxy)phenyl)-2-methylpentanoate (180.0 mg, 0.187 mmol) was dissolved in DCM (12 ml) followed by the addition of TFA (6 ml). The reaction mixture was stirred at room temperature for 2 hours, then concentrated and evaporated to dryness with DCM/toluene to give the title compound (155 mg, >100% yield), which was used in the next step without further purification. ESI: m/z: calculated for C ₃₈ H ₅₄ N ₄ O ₁₅ [M+H]+: 805.35, found 805.60.

Example 196. Synthesis of (4R)-4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,3,3,8-tetramethyl-4, 7, 13-trioxo-12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-5-(3-(3-(2-(2-(2-(2,5 -dioxo2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-4-((3-(2-(2-(2-(2,5-dioxo-2,5- dihydro1H-pyrrol-1-yl)ethoxy) ethoxy)ethoxy)ethoxy)propanoyl)oxy)phenyl)-2-methylpentanoic acid (B20)

To a solution of (2R)-4-carboxy-1-(3-(3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl))ethoxy )ethoxy)ethoxy)propanamido)-4-((3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxy) propanoyl)oxy)phenyl)pentan-2-amine, TFA salt (43 mg, 0.06 mmol) in DMA (1 ml), pentafluoride active acid compound (48.5 mg, 0.06 mmol) and DIPEA ( 34 µl, 0.2 mmol). The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN after 45 min, C-18 column, 10 mm (d) x 250 mm (l), 9 ml/min) to obtain the title product B-20 (35 mg, 45%). ESI: m/z: calculated for C ₅₉ H ₈₅ N ₈ O ₁₈ S [M+H]+: 1313.61, found 1313.85.

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Example 197. Synthesis of (4R)-5-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,39,42tetramethyl-2,5, 8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18,19,20,21,22,23, 24,25,26,27,29,30, 32,33,35,36,37,38,39,40,41,42,43,44-hexatriacontahydro-2H-benzo[b][1,14,17 ,20,31,34,37,4,7,10,23,28,41, 44]heptaoxaheptaazacyclohexatetracontin-46-ul)-4-(2-((6S,9R,11R)-6-((S) -sec-butyl)-9-isopropyl2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-2methylpentane acids (B-21)

DMA/pH 7.5

AT 21

To a solution of (2R)-1-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,39,42-tetramethyl2,5,8, 21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18,19,20,21,22,23,24, 25,26,27,29,30,32,33,35, 36,37,38,39,4O,41,42,43,44-hexatriacontahydro-2H-benzo[b][1,14,17,20 ,31,34,37,4,7,1O,23,28,41,44]heptaoxaheptaazacyclohexatetracontin-46-yl)-4-carboxypentan-2-amine TFA salt (60 mg, 0.050 mmol) in DMA (1.5 ml) pentafluoride active acid compound (44 mg, 0.06 mmol) and 0.1 M NaH ₂ PO ₄ , pH 7.5, 0.8 ml were added. The reaction mixture was stirred overnight, evaporated and purified by HPLC with a MeCN/H ₂ O gradient (10% MeCN to 70% MeCN after 45 min, C-18 column, 10 mm (d) x 250 mm (l), 8 ml/min) to obtain the title product B-21 (44 mg, 52% yield). ESI: m/z: calculated for C ₇₉ H ₁₁₇ N ₁₄ O ₂₆ S [M+H]+: 1709.79, found 1709.55.

Example 198. Synthesis of (4R)-4-(2-((4R,6R,9S,12S,15S,18S)-9-((S)-sec-butyl)-6,12-diisopropyl7,13,15, 18-tetramethyl-2,8,11,14,17,20,23-heptaoxo-21-propiolamido-3-oxa-7,10,13,16,19,22-hexaazapentacos-24-in-4-yl) thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (B-22)

K O soon

K hydrochloride (4R)-4-(2-((3S,6S,9R,11R)-6-((S)-sec-butyl)-3,9-diisopropyl-8-methyl-4,7,13trioxo- 12-oxa-2,5,8-triazatetradecane-11-yl)thiazol-4-carboxamido)-2-methyl-5-phenylpentanoic acid (25 mg, 0.034 mmol) in a mixture of DMA (2 ml) and 0.1 M Na2HPO4 , pH 8.0 (1 ml), (S)-2,5dioxopyrrolidin-1-yl 2-((S)-2-(2,2-dipropiolamidoacetamido)propanamido)propanoate (23.1 mg, 0.053 mmol) was added in three portions over 3 hours, and then the mixture was stirred for a further 12 hours. The mixture was evaporated and purified using reverse phase HPLC (200 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water in 40 min, v=8 ml/min) to obtain the title compound (30.0 mg, 85% yield). MS ESI m/z: calculated for C ₅₁ H ₇₁ N ₉ O ₁₂ S [M+H] ⁺ 1034.49, found 1034.90.

Example 199. Synthesis of (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidin- 2-carboxamido) pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(4-hydroxy-3-(3-(2-(2((bis((Z)-3-carboxyacrylhydrazinyl)phosphoryl)amino )ethoxy)ethoxy)propanamido)phenyl)-2-methylpentanoic acid (B-23). (B-23) o

[I hu-on

POCl ₃ (10.1 mg, 0 .0665 mmol). After stirring at 0°C for 20 minutes, the mixture was warmed to room temperature and continued stirring for an additional 4 hours. Compound (4R)-4-(2-((1R,3R)-1-acetoxy-3) was then added to the mixture ((2S,3S)-N,3-dimethyl-2-((R)-l-methylpiperidin-2-carboxamido)pentanamido)-4-methylpentyl)thiazol-4carboxamido)-5-(3-(3-(2 -(2-aminoethoxy)ethoxy)propanamido)-4-hydroxyphenyl)-2-methylpentanoic acid (60 mg, 0.065 mmol) and DIPEA (20 μl, 0.114 mmol). The mixture was stirred at 50°C overnight, evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water for 40 min, v=8 ml/min) to obtain the title compound (23.1 mg, 32% yield). ESI MS m/z: calculated for C ₅₃ H ₈₁ NnO ₁₈ PS [M+H] ⁺ 1222.51, found

- 123 044827

1222.80.

Example 200. Synthesis of (1R,3R)-1-(4-(((2R)-5-((2-aminoethyl)amino)-1-(22,23-bis(2,5-dioxo-2,5dihydro )-1H-pyrrol-1-yl)-3,6,39,42-tetramethyl-2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8 ,9,10,12,13,15,16, 18,19,20,21,22,23,24,25,26,27,29,30,32,33,35,36,37,38,39 ,40,41,42,43,44-hexatriacontahydro-2H-benzo[b][1,14,17,20,31,34,37,4,7,10,23,28,41,44]heptaoxageptaase- cyclohexatetracontin-46-yl)-4-methyl-5oxopentan-2-yl)carbamoyl)thiazol-2-yl)-3-((2S,3S)-2-(2-(dimethylamino)-2-methylpropanamido)-N ,3dimethylpentanamido)-4-methylpentyl acetate (B-24)

Compound B-21 (22.0 mg, 0.0129 mmol) in DMA (1 ml) was added EDC (15.0 mg, 0.078 mmol), ethane-1,2-diamine hydrochloride salt (8.0 mg, 0.060 mmol ) and DIPEA (0.010 ml, 0.060 mmol). The mixture was stirred overnight, evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water for 40 min, v = 8 ml/min) with obtaining the title compound (14.0 mg, 62% yield). ESI MS m/z: calculated for C ₈₁ H ₁₂₃ N ₁₆ O ₂₅ S [M+H]+1751.85, found 1751.20.

Example 201. Synthesis of (1R,3R)-1-(4-(((28R)-1-amino-29-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrole1- )yl)-3,6,39,42-tetramethyl-2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12, 13,15,16,18,19,20,21,22,23, 24,25,26,27,29,30,32,33,35,36,37,38,39,40,41,42, 43,44-hexatriacontahydro-2H-benzo[b][1,14,17,20,31,34, 37,4,7,10,23,28,41,44]heptaoxaheptaase-cyclohexatetracontin-46-yl)- 26 methyl-25-oxo-3,6,9,12,15,18, 21-heptaoxa-24-azanonacosan-28-yl carbamoyl)thiazol-2-yl)-3-((2S,3S)-2- (2-(dimethylamino)-2-methylpropanamido)-N,3-dimethylpentanamido)-4-methylpentyl acetate (B-25)

Compound B-21 (22.0 mg, 0.0129 mmol) in DMA (1 ml) was added with EDC (15.0 mg, 0.078 mmol), 3,6,9,12,15,18,21-heptaoxatricosan-hydrochloride 1,23-diamine (26.0 mg, 0.059 mmol) and DIPEA (0.010 ml, 0.060 mmol). The mixture was stirred overnight, evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C18 column, 10-100% acetonitrile/water for 40 min, v = 8 ml/min) to obtain the above in the title compound (14.5 mg, 55% yield). ESI MS m/z: calculated for C95H151N16O32S [M+H] ⁺ 2060.03, found 2060.80.

Example 202 Synthesis of (1R, 3R)-1-(4-(((28R)-29-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) 3,6,39,42-tetramethyl-2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15, 16,18,19,20,21,22,23,24,25, 26,27,29,30,32,33,35,36,37,38,39,40,41,42,43,44- hexatriacontahydro-2H-benzo[b][1,14,17,20,31,34,37, 4,7,10,23,28,41,44]heptaoxaheptaase-cyclohexatetracontin-46-yl)-1-hydroxy- 26-methyl-25-oxo3,6,9,12,15,18,21 -heptaoxa-24-azanonacosan-28-yl)carbamoyl)thiazol-2-yl)-3 -((2S,3S)-2 -(2(dimethylamino)-2-methylpropanamido)-N,3-dimethylpentanamido)-4-methylpentyl acetate (B-26)

Compound B-21 (22.0 mg, 0.0129 mmol) in DMA (1 ml) was added with EDC (15.0 mg, 0.078 mmol) and 23amino-3,6,9,12,15,18,21-heptaoxatricosane -1-ol (22.0 mg, 0.059 mmol). The mixture was stirred overnight, evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C18 column, 10-100% acetonitrile/water for 40 min, v = 8 ml/min) with obtaining the title compound (14.1 mg, 53% yield). MS ESI m/z: calculated for C ₉₅ H ₁₅₀ N ₁₅ O ₃₃ S [M+H] ⁺ 2061.02, found 2061.74.

Example 203. Synthesis of (2S)-tert-butyl 2-((4R)-5-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)3,6 ,39,42-tetramethyl-2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16,18 ,19,20,21,22,23,24,25,26, 27,29,30,32,33,35,36,37,38,39,40,41,42,43,44-hexatriconthydro-2H -benzo[b][1,14,17,20,31,34,37,4,7,10, 23,28,41,44]heptaoxahepta-azacyclohexatetracontin-46-yl)-4-(2-(( 6S,9R,11R)-6-((S)-sec-butyl)-9isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8-triazatetradecane -11-yl)thiazol-4-carboxy- 124 044827 samido)-2-methylpentanamido)-6-((tert-butoxycarbonyl)amino)hexanoate (B-27)

Compound B-21 (25.0 mg, 0.0146 mmol) in DMA (1 ml) was added with EDC (15.0 mg, 0.078 mmol) and (S)tert-butyl 2-amino-6-((tert-butoxycarbonyl )amino)hexanoate (9.0 mg, 0.030 mmol). The mixture was stirred overnight, evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water for 40 min, v = 8 ml/min) to give the title compound (20.5 mg, 71% yield). MS ESI m/z: calculated for C ₉₄ H ₁₄₄ N ₁₆ O ₂₉ S [M+H]+ 1994.00, found 1994.85.

Example 204. Synthesis of (2S)-6-amino-2-((4R)-5-(22,23-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)3, 6,39,42-tetramethyl-2,5,8,21,24,37,40,43-octaoxo-3,4,5,6,7,8,9,10,12,13,15,16, 18,19,20,21,22,23,24,25,26, 27,29,30,32,33,35,36,37,38,39,40,41,42,43,44-hexatriaconthydro- 2H-benzo[b][1,14,17,20,31,34,37,4,7,10, 23,28,41,44]heptaoxaheptaase-cyclohexatetracontin-46-yl)-4-(2-( (6S,9R,11R)-6-((S)-sec-butyl)-9isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8- triazatetradecane-11-yl)thiazol-4-carboxamido)-2-methylpentanamido)hexanoic acid (B-28)

Compound B-27 (20.0 mg, 0.010 mmol) was dissolved in DCM (1 ml), followed by the addition of TFA (1 ml). The reaction mixture was stirred at room temperature for 2 h, then evaporated and purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C ₁₈ column, 10-100% acetonitrile/water for 40 min, v = 8 ml /min) to obtain the title compound (13.5 mg, 73% yield). ESI: m/z: calculated for C ₈₅ H ₁₂₉ N ₁₆ O ₂₇ S [M+H]+: 1837.89, found 1838.20.

Example 205 Synthesis of (2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride

Y^CO ₂ Me no·»···/ y

Vnh

To a solution of trans-4-hydroxy-L-proline (15.0 g, 114.3 mmol) in dry methanol (250 ml), thionyl chloride (17 ml, 231 mmol) was added dropwise at 0-4°C. The resulting mixture was stirred at room temperature overnight, evaporated, crystallized from EtOH/hexane to give the title compound (18.0 g, 87% yield). ESI MS m/z 168.2 ([M+Na]+).

Example 206. Synthesis of (2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate ^XO ₂ Me ^.. .....<x

Vos

Boc ₂ O (30, 0 g, (137.6 mmol) in three portions over 4 hours. After stirring for an additional 4 hours, the reaction mixture was evaporated to ~350 ml and extracted with EtOAc (4x80 ml).The combined organic layers were washed with saturated aqueous sodium chloride (100 ml ), dried (MgSO ₄ ), filtered, evaporated and purified by column chromatography on SiO ₂ (1:1 hexane/EtOAc isomer mixture) to give the title compound (22.54 g, 86% yield). ESI MS m /z 268.2 ([M+Na]+).

Example 207. Synthesis of (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate /-^CO ₂ Me

The title compound obtained by Dess-Martin oxidation is described in: Franco Manfre et al., J. Org. Chem. 1992, 57, 2060-2065). An alternative Swern oxidation procedure is as follows: to a solution of (COCl) ₂ (13.0 ml, 74.38 mmol) in CH ₂ Cl ₂ (350 ml), cooled to -78°C, add dry DMSO (26.0 ml ). The solution was stirred at -78°C for 15 min and then (2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate (8.0 g, 32.63 mmol) was added to CH2Cl2 (100 ml). After stirring at -78°C for 2 hours, triethylamine (50 mL, 180.3 mmol) was added dropwise and the reaction solution was warmed to room temperature. The mixture was diluted with aqueous NaH ₂ PO ₄ (1.0 M, 400 ml) and the phases were separated. The aqueous layer was extracted with CH ₂ Cl ₂ (2x60 ml).

- 125 044827

The organic layers were combined, dried over MgSO4, filtered, evaporated and purified by _SiO2 column chromatography (7:3 hexane/EtOAc) to give the title compound (6.73 g, 85% yield). ESI MS m/z 266.2([M+Na]+).

Example 208. Synthesis of (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1,2-dicarboxylate __.CO ₂ Me

To a suspension of methyl triphenylphosphonium bromide (19.62 g, 55.11 mmol) in THF (150 ml) at 0° C. was added potassium tert-butoxide (6.20 g, 55.30 mmol) in anhydrous THF (80 ml). After stirring at 0°C for 2 hours, the resulting yellow ylide was added to a solution of (S)-1-tert-butyl 2-methyl 4oxopyrrolidine-1,2-dicarboxylate (6.70 g, 27.55 mmol) in THF (40 ml). After stirring at room temperature for 1 hour, the reaction mixture was evaporated, diluted with EtOAc (200 ml), washed with H2O (150 ml), saturated aqueous sodium chloride (150 ml), dried over MgSO ₄ , evaporated and purified by column chromatography with SiO ₂ (9:1 hexane/EtOAc isomer mixture) to give the title compound (5.77 g, 87% yield). MS EI m/z 264 ([M+Na]+).

Example 209. Synthesis of (S)-methyl 4-methylenepyrrolidine-2-carboxylate hydrochloride ^XO ₂ Me ⁼ \^NH HC1

HCl (12 M, 10 ml). The mixture was stirred for 1 hour, diluted with toluene (50 ml), evaporated and crystallized from EtOH/hexane to give the title compound as the HCl salt (3.85 g, 92% yield). MS EI m/z 142.2 ([M+H]+).

Example 210. Synthesis of (S)-tert-butyl-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carboxylate ^CO ₂ Me ^Boc

_LiAlH4THF

He

To a solution of (S)-1-tert-butyl-2-methyl-4-methylenepyrrolidine-1,2-dicarboxylate (5.20 g, 21.56 mmol) in anhydrous THF (100 ml) at 0°C was added LiAlH ₄ (15 ml, 2M in THF). After stirring at 0°C for 4 hours, methanol (5 ml) and water (20 ml) were added to the reaction mixture. The reaction mixture was neutralized with 1 M HCl to pH 7, diluted with EtOAc (80 ml), filtered through celite, separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, dried over Na ₂ SO ₄ , evaporated and purified by SiO ₂ column chromatography (EtOAc/DCM 1:5) to give the title compound (3.77 g, 82% yield). MS EI m/z 236.40 ([M+Na]+).

Example 211 Synthesis of (S)-(4-methylenepyrrolidin-2-yl)methanolα, hydrochloride salt

Vnh on

HCl (12 M, 10 ml). The mixture was stirred for 1 hour, diluted with toluene (50 ml), evaporated and crystallized from EtOH/hexane to give the title compound as the HCl salt (2.43 g, 94% yield). MS EI m/z 115.1 ([M+H]+).

Example 212 Synthesis of 4-(benzyloxy)-3-methoxybenzoic acid

VpO^^^.

MeO'

CO ₂ H

To a mixture of 4-hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) in ethanol (350 ml) and aq. NaOH solution (2.0 M, 350 ml) was added with BnBr (140.0 g, 823.5 mmol). The mixture was stirred at 65°C for 8 hours, concentrated, co-evaporated with water (2x400 ml) and evaporated to ~ 400 ml, acidified to pH 3.0 with 6N. HCl. The solid was filtered, crystallized with EtOH, dried at 45°C in vacuo to give the title compound (63.6 g, 83% yield). ESI MS m/z 281.2 ([M+Na]+).

Example 213 Synthesis of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid

VPO

MeO

CO ₂ H

To a solution of 4-(benzyloxy)-3-methoxybenzoic acid (63.5 g, 246.0 mmol) in CH ₂ Cl ₂ (400 ml) and HOAc (100 ml) was added HNO3 (fuming, 25.0 ml, 528, 5 mmol). The mixture was stirred for 6 hours, evaporated, crystallized in EtOH, dried at 40°C in vacuum to give the title compound (63.3 g, 85% yield). ESI MS m/z 326.1 ([M+Na]+).

Example 214. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylene-126 044827 pyrrolidin-1-yl)methanone

A catalytic amount of DMF (30 µl) was added to a solution of 4-(benzyloxy)-5-methoxy-2nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 ml, 22.50 mmol) in anhydrous CH ₂ Cl ₂ (70 ml) and the resulting mixture was stirred at room temperature for 2 hours. Excess CH ₂ Cl ₂ and oxalyl chloride were removed on a rotor. Acetyl chloride was resuspended in fresh CH ₂ Cl ₂ (70 ml) and slowly added to a premixed solution of (S)-(4-methylenepyrrolidin-2-yl)methanol, hydrochloride salt (1.32 g, 8.91 mmol) and Et ₃ N (6 ml) in CH ₂ Cl ₂ at 0°C in an N ₂ atmosphere. The reaction mixture was left to warm to room temperature. and continued stirring for 8 hours. After removal of CH ₂ Cl ₂ and Et ₃ N, the residue was partitioned between H ₂ O and EtOAc (70/70 ml). The aqueous layer was further extracted

EtOAc (2x60 ml). The combined organic layers were washed with saturated aqueous sodium chloride (40 ml), dried (MgSO ₄ ) and evaporated. Purification of the residue by flash chromatography (silica gel, 2:8 hexane/EtOAc) gave the title compound (2.80 g, 79% yield). MS EI m/z 421.2 ([M+Na]+).

Example 215. Synthesis of (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)methanone

To (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (2.78 g, 8.52 mmol) in DCM (10 ml) and pyridine (10 ml) were added tert-butylchlorodimethylsilane (2.50 g, 16.66 mmol). The mixture was stirred overnight, evaporated and purified on a SiO ₂ column eluting with EtOAc/CH ₂ Cl ₂ (1:6) to give the title compound (3.62 g, 83% yield, ~95% purity). MS ESI m/z calculated for C ₂₇ H ₃₇ N ₂ O ₆ Si [M+H] ⁺ 513.23, found 513.65.

Example 216 Synthesis of (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone

To (S)-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone (2.80 g, 7.03 mmol) in DCM (30 ml)) and CH3SO3H (8 ml) PhSCH ₃ (2.00 g, 14.06 mmol) was added. The mixture was stirred for 0.5 h, diluted with DCM (40 ml), neutralized by carefully adding 0.1 M Na ₂ CO ₃ solution. The mixture was separated and the aqueous solution was extracted with DCM (2x10 ml). The organic layers were combined, dried over Na ₂ SO ₄ , evaporated and purified on a SiO ₂ column eluting with MeOH/CH ₂ Cl ₂ (1:15 to 1:6) to give the title compound (1.84 g , yield 85%, ~95% purity). ESI MS m/z calculated for C ₁₄ H ₁₇ N ₂ O ₆ [M+H] ⁺ 309.10, found 309.30.

Example 217. Synthesis of (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)2-(hydroxymethyl)- 4-methylenepyrrolidin-1-yl)methanone)

K (S)-(4-hydroxy-5-methoxy-2-nitrophenyl)(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)meth none (0.801 g, 2.60 mmol) in butanone (10 ml) Cs ₂ CO ₃ (2.50 g, 7.67 mmol) was added followed by 1,5-diiodopentane (415 mmol, 1.28 mmol). The mixture was stirred for 26 hours, evaporated and purified on a SiO ₂ column, eluting with MeOH/CH ₂ Cl ₂ (1:15 to 1:5), to give the title compound (0.675 g, 77% yield, purity ~95%). ESI MS m/z calculated for C ₃₃ H ₄₁ N ₄ O ₁₂ [M+H] ⁺ 685.26, found 685.60.

Example 218. Synthesis of (S)-((pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)

2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)methanone)

K (S)-((pentane-1,5-diylbis(oxy))bis(5-methoxy-2-nitro-4,1-phenylene))bis(((S)-2-(hydroxymethyl)4-methylenepyrrolidine -1-yl)methanone) (0.670 g, 0.98 mmol) in CH ₃ OH (10 ml) was added Na ₂ S ₂ O ₄ (1.01 g, 5.80 mmol) in H ₂ O (8 ml) . The mixture was stirred at room temperature for 30 hours. The reaction

- 127 044827 the mixture was concentrated and co-evaporated with DMA (2x10 ml) and EtOH (2x10 ml) under high vacuum to dryness to give the title compound (total weight 1.63 g) containing inorganic salts, which was used directly next reaction steps (without further separation). ESMS m/z 647.32 ([M+Na]+).

Example 219 Synthesis of C-1 (PBD dimer analogue having a bis-linker)

(3S,6S,39S,42S)-di-tert-Butyl 6,39-bis(4-((tert-butoxycarbonyl)amino)butyl)-22,23-bis(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3,42-bis((4-(hydroxymethyl)phenyl)carbamoyl)-5,8,21,24,37,40-hexaoxo-11,14,17,28, 31,34-hexaoxy-4,7,20,25,38,41-hexaazatetratetracontane-1,44-dioate (0.840 g, 0.488 mmol) in THF (8 ml) containing pyridine (0.100 ml, 1.24 mmol) At 0°C, a solution of triphosgene (0.290 mg, 0.977 mmol) in THF (3.0 ml) was added dropwise. The reaction mixture was stirred at 0°C for 15 min, then used directly in the next step.

(B)-((Pentan-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(hydroxymethyl)-4methylenepyrrolidine -1 -yl)methanone) containing inorganic salts (0.842 mg, ~0.49 mmol) was suspended in EtOH (10 ml) at 0°C and the trichloride in THF prepared above was added. The mixture was stirred at 0°C for 4 h, then warmed to room temperature for 1 h, evaporated and purified using reverse phase HPLC (250 (L) mm x 10 (d) mm, C18 column, 10-80% acetonitrile /water for 40 min, v=8 ml/min) to obtain the title compound (561.1 mg, 48% yield over three steps). MS ESI m/z: calculated for C ₁₁₇ H ₁₆₃ N ₁₆ O ₃₈ [M+H]+ 2400.12, found 2400.90.

Example 220 Synthesis of C-2 (PBD dimer analogue having a bis-linker)

Dess-Martin periodinane (138.0 mg, 0.329 mmol) was added to a solution of compound C-1 (132.0 mg, 0.055 mmol) in DCM (5.0 ml) at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 hours. A saturated solution of NaHCO ₃ /Na ₂ SO ₃ (5.0 ml/5.0 ml) was then added and the mixture was extracted with DCM (3x25 ml). The combined organic layers were washed with NaHCO ₃ /Na ₂ SO ₃ (5.0 ml/5.0 ml), saturated aqueous sodium chloride (10 ml), dried over Na ₂ SO ₄ , filtered, evaporated and purified using reverse phase HPLC (250 (L) mm x 10 (d) mm, C ₁₈ column, 10-80% acetonitrile/water for 40 min, v = 8 ml/min) to obtain the title compound (103.1 mg, 78% output) in the form of foam. MS ESI m/z: calculated for C ₁₁₇ H ₁₅₈ N ₁₆ O ₃₈ [M+H]+ 2396.09, found 2396.65.

Example 221 Synthesis of C-3 (PBD dimer analogue having a bis-linker)

- 128 044827

Compound C-2 (55.0 mg, 0.023 mmol) was dissolved in DCM (3 ml) followed by the addition of TFA (3 ml). The reaction mixture was stirred at room temperature for 2 hours, then evaporated and co-evaporated with DCM/toluene to dryness, yielding crude product C-3 (48.0 mg, 100% yield, 92% purity by HPLC), which was further purified by reverse phase HPLC (250 (L) mm x 10 (d) mm, C18 column, 5-60% acetonitrile/water for 40 min, v = 8 ml/min), yielding purified product C-3 (42, 1 mg, yield 88%, purity 96%) in the form of foam. ESI MS m/z: calculated for C ₉₉ H ₁₂₆ N ₁₆ O ₃₄ [M+H]+ 2083.86, found 2084.35.

Example 222 Synthesis of (S)-methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine 2-carboxylate.

BnO

MeO

A catalytic amount of DMF (30 µl) was added to a solution of 4-(benzyloxy)-5-methoxy-2nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride (2.0 ml, 22.50 mmol) in anhydrous CH ₂ Cl ₂ (70 ml) and the resulting mixture was stirred at room temperature for 2 hours. Excess CH ₂ Cl ₂ and oxalyl chloride were removed on a rotor. Acetyl chloride was resuspended in fresh CH ₂ Cl ₂ (70 ml) and slowly added to a premixed solution of (8)-methyl 4methylenepyrrolidine-2-carboxylate hydrochloride (1.58 g, 8.91 mmol) and Et ₃ N (6 ml) in CH ₂ Cl ₂ at 0°C in an N2 atmosphere. The reaction mixture was heated to room temperature. and stirring was continued for 8 hours. After removal of CH ₂ Cl ₂ and Et3N, the residue was partitioned between H2O and EtOAc (70/70 ml). The aqueous layer was further extracted with EtOAc (2x60 ml). The combined organic layers were washed with saturated aqueous sodium chloride (40 ml), dried (MgSO ₄ ) and evaporated. Purification of the residue by flash chromatography (silica gel, 2:8 hexane/EtOAc) gave the title compound (2.88 g, 76% yield). MS EI m/z 449.1 ([M+Na] ⁺ ).

Example 223. Synthesis of (8)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carbaldehyde.

To a vigorously stirred solution of (8)-methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4methylenepyrrolidine-2-carboxylate (2.80 g, 6.57 mmol) in anhydrous CH ₂ Cl ₂ ( 60 ml) DIBAL-H (1N in CH ₂ Cl ₂ , 10 ml) was added dropwise at -78°C in an N2 atmosphere. After stirring the mixture for an additional 90 min, the excess reagent was decomposed by adding 2 ml of methanol followed by 5% HCl (10 ml). The resulting mixture was left to warm to 0°C. The layers were separated and the aqueous layer was further extracted with CH ₂ Cl ₂ (3x50 ml). The combined organic layers were washed with saturated aqueous sodium chloride, dried (Mg8O ₄ ) and evaporated. Purification of the residue by flash chromatography (silica gel, 95:5 CHO ₃ /Me0H) gave the title compound (2.19 g, 84% yield). MSEI m/z 419.1 ([M+Na] ⁺ ).

Example 224. Synthesis of (8)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2a]azepin-5(11aH)-one

ΒηΟ^^,Ν^

ABOUT

A mixture of (8)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carbaldehyde (2.18 g, 5.50 mmol) and Na ₂ 8 ₂ O ₄ (8 .0 g, 45.97 mmol) in THF (60 ml) and H ₂ O (40 ml) were stirred at room temperature for 20 hours. Solvents were removed under high vacuum. The residue was resuspended in MeOH (60 ml) and HCl (6 M) was added dropwise until pH ~ 2 was reached. The resulting mixture was stirred at room temperature. for 1 hour. Most of the MeOH was removed from the reaction mixture, then

- 129 044827 EtOAc (100 ml) was added. The EtOAc solution was washed with saturated NaHCO3 solution, saturated aqueous sodium chloride solution, dried (MgSO ₄ ) and evaporated. Purification of the residue by flash chromatography (silica gel, 97:3 CHCl ₃ /MeOH) gave the title compound (1.52 g, 80%). EIMS m/z 372.1 ([M+Na]+).

Example 225. Synthesis of (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2a]azepin-5(11aH)-one

To a solution of (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a]azepin5(11aH)-one (1.50 g, 4.32 mmol) 25 ml CH3SO3H was added to 70 ml CH ₂ Cl ₂ at 0°C. The mixture was stirred at 0°C for 10 min, then at room temperature for 2 h, diluted with CH ₂ Cl ₂ , the pH was adjusted to 4 with a cold 1.0 N NaHCO ₃ solution and filtered. The aqueous layer was extracted with CH ₂ Cl ₂ (3x60 ml). The organic layers were combined, dried over Na ₂ SO ₄ , filtered, evaporated and purified by SiO ₂ column chromatography (CH ₃ OH/CH ₂ Cl ₂ 1:15) to give 811 mg (73% yield) of the title compound. ESMS m/z 281.1 ([M+Na]+).

Example 226. Synthesis of (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e ]pyrrolo[1,2-a][1,4]diazepin-5 (11 aH)-one).

To a stirred suspended solution of Cs ₂ CO ₃ (0.761 g, 2.33 mmol) in butanone (8 ml) was added (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-1H-benzo [e]pyrrolo[1,2-a]azepin-5 (11 aH)-one (401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.740 mmol). The mixture was stirred at room temperature overnight, evaporated and purified by SiO ₂ chromatography (EtOAc/CH ₂ Cl ₂

1:10) to obtain 337 mg (yield 78%) of the title product. ESMS m/z 607.2 ([M+Na]+).

Example 227. Synthesis of (S)-7-methoxy-8-((5-((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro- 1 H)benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1Hbenzo[e]pyrrolo[1 ,2-а][1,4]diazepin-5(11 аН)-one

To a solution of (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1Hbenzo[e]pyrrolo[1,2 -a][1,4]diazepin-5(11aH)-one) (150 mg, 0.256 mmol) in anhydrous dichloromethane (1 ml) and absolute ethanol (1.5 ml) was added sodium borohydride in methoxyethyl ether (85 μl, 0.5M, 0.042 mmol) at 0°C. After 5 minutes, the ice bath was removed and the mixture was stirred at room temperature for 3 hours, then cooled to 0°C, saturated ammonium chloride was added, diluted with dichloromethane and the phases were separated. The organic layer was washed with saturated aqueous sodium chloride, dried over anhydrous Na ₂ SO ₄ , filtered through celite and evaporated. The residue was purified by reverse phase HPLC (C ₁₈ column, acetonitrile/water). The appropriate fractions were extracted with dichloromethane and evaporated to give the title compound (64.7 mg, 43%), MS m/z 609.2 ([M+Na]+), 625.3 ([M+K] ⁺ ) and 627.2 ([M+Na+H ₂ O]+); a fully reduced compound was obtained (16.5 mg, 11%), MS m/z 611.2 ([M+Na]+), 627.2 ([M+K] ⁺ ), 629.2 ([M+ Na+H2O]+); and unreacted starting material was also removed (10.2 mg, 7%), MS m/z 607.2 ([M+Na]+), 625.2 ([M+Na+H2O]+).

Example 228. Synthesis of (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy)ethoxy)propanoyl)-7-methoxy-2methylene-5-oxo-2, 3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2- methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one

To a mixture of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro)-1Hbenzo [e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2 -a][1,4]diazepin-5(11aH)-one (60.0 mg, 0.102 mmol) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2azidoethoxy)ethoxy)propanoate (40.5 mg, 0.134 mmol) in dichloromethane (5 ml) was added EDC (100.5 mg, 0.520 mmol). The mixture was stirred at room temperature overnight, evaporated and purified by SiO ₂ column chromatography (EtOAc/CH ₂ Cl ₂ , 1:6) to give 63.1 mg (81% yield) of the title product. . MS ESI m/z C ₄₀ H5oN ₇ O9 [M+H] ⁺ , calc. 772.36, found 772.30.

Example 229. Synthesis of (8)-8-((5-(((8)-10-(3-(2-(2-aminoethoxy)ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,11 a-hexahydro-1 H-benzo[e]pyrrolo[1,2-a] [1,4]diazepin-8-yl)oxy)pentyl)oxy)-7methoxy- 2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one

To a solution of (8)-8-((5-(((8)-10-(3-(2-(2-azidoethoxy)ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo2,3, 5,10,11,11 a-hexahydro-1H-benzo[e]pyrrolo[1,2-a] [1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2methylene-2 ,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (60 mg, 0.078 mmol) in a mixture of THF (5 ml) and NaH ₂ PO ₄ buffer solution (pH 7.5, 1.0 M, 0.7 ml) PPh ₃ (70 mg, 0.267 mmol) was added. The mixture was stirred at room temperature overnight, evaporated and purified on C ₁₈ preparative HPLC, eluted with water/CH ₃ CN (90% water to 35% water over 35 min) to give 45.1 mg (79% yield) of the title in the product header after drying under high vacuum. MS ESI m/z C ₄₀ H ₅₂ N ₅ O ₉ [M+H] ⁺ , calc. 746.37, found 746.50.

Example 230. Synthesis of (S)-N-(2-((S)-8-((5-(((11S,11aS)-10-((S)-15-azido-5-isopropyl-4,7 -dioxo)10,13-dioxa-3,6-diazapentadecane-1-oil)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11ahexahydro-1H- benzo[e]pyrrolo [1,2-a][1,4]diazepin-8-yl) oxy) pentyl)-oxy)-7-methoxy-2-methylene-5oxo-2,3,11,11a-tetrahydro -1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2azidoethoxy)ethoxy)propanamido) -3-methylbutanamide

To a mixture of (8)-7-methoxy-8-((5-(((8)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro)-1Hbenzo [e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2 -a][1,4]diazepin-5(11aH)-one (60.0 mg, 0.102 mmol) and (8)-15-azido-5-isopropyl-4,7-dioxo-10,13dioxa-3, 6-Diazapentadecan-1-onic acid (90.2 mg, 0.25 mmol) in DMA (8 ml) was added with BrOP (240.2 mg, 0.618 mmol). The mixture was stirred at room temperature overnight, evaporated and _purified by _8iO2 column chromatography ( _CH3OH / _CH2Cl2 , 1:10 to 1:5) to give 97.1 mg (74% yield) of the title in the product title. ESI MS m/z C ₆₁ H ₈₇ N ₁₄ O ₁₇ [M+H] ⁺ , calc. 1287.63, found 1287.95.

Example 231. Synthesis of (8)-N-(2-((8)-8-((5-(((118.11a8)-10-((8)-15-amino-5-isopropyl-4,7 -dioxo)10,13-dioxa-3,6-diazapentadecane-1-oil)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11ahexahydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene-5oxo-2,3,11,11a-tetrahydro- 1H-benzo[e]pyrrole[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2aminoethoxy)ethoxy)propanamido)- 3-methylbutanamide (C-4)

To a solution of (8)-N-(2-((8)-8-((5-(((118.11a8))-10-((8)-15-azido-5-isopropyl-4,7- dioxo-10,13-dioxa3,6-diazapentadecane-1-oil)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11 a-hexahydro-1Hbenzo[ e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11atetrahydro-1H -benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)-2-oxoethyl)-2-(3-(2-(2-azidoethoxy)ethoxy)propanamido) -3-methylbutanamide (85 mg, 0.066 mmol) in a mixture of THF (5 ml) and NaH ₂ PO ₄ buffer solution (pH 7.5, 1.0 M, 0.7 ml) was added PPh ₃ (100 mg, 0.381 mmol) . The mixture was stirred at room temperature overnight. After confirmation by LC-MS, the formation of (8)-N-(2-((8)-8-((5-(((118.11a8)

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10-((8)-15-amino-5-isopropyl)-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1-oil)-11-hydroxy-7 methoxy-2-methylene- 5 -oxo-2,3,5,10,11,11 a-hexahydro-1 H-benzo[e]pyrrolo [1,2-a][1,4]diazepin-8yl)oxy)pentyl)oxy)- 7-methoxy-2-methylene-5-oxo-2,3,11,11 a-tetrahydro-1H-benzo[e]pyrrolo [1,2a][1,4]diazepin-10(5H)-yl) -2-oxoethyl)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3-methylbutanamide (ESI MS m/z C ₆₁ H ₉₀ N ₁₀ O ₁₇ [M+Na]+, calc. 1257.66, found 1257.90), added bis(2,5dioxopyrrolidin-1-yl) 2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)succinate (33 mg, 0.066 mmol). The mixture was stirred for 4 hours, evaporated and purified by C18 preparative HPLC, eluted with water/CH ₃ CN (90% water to 30% water in 35 minutes) to give 40.1 mg (40% yield) of the title. product title C-4 after drying under high vacuum. ESI MS m/z C ₇₃ H ₉₅ N ₁₂ O ₂₃ [M+H] ⁺ , calc. 1507.66, found 1507.90.

Example 232 Synthesis of nitro-a-amanitine

To a solution of a-amanitin (15.0 mg, 0.0163 mmol) in acetic acid (0.5 ml) and CH ₂ Cl ₂ (1 ml) was added 70% HNO ₃ (0.3 ml) at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then at room temperature for 2 hours. Water (5 ml) and DMA (4 ml) were then added, the reaction mixture was evaporated and purified by preparative HPLC (H ₂ O/MeCN) to obtain light -yellow solid (9.8 mg, 62% yield). ESI MS m/z: calculated for C39H54N11O16S [M+H]+ 963.34, found 964.95.

Example 233 Synthesis of nitro-b-amanitine

To a solution of β-amanitin (15.0 mg, 0.0163 mmol) in acetic acid (0.5 ml) and CH ₂ Cl ₂ (1 ml) was added 70% HNO3 (0.3 ml) at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then at room temperature for 2 hours. Water (5 ml) and DMA (4 ml) were then added, the reaction mixture was evaporated and purified by preparative HPLC (H ₂ O/MeCN) to obtain light -yellow solid (9.8 mg, 62% yield). ESI MS m/z: calculated for C ₃₉ H ₅₃ N ₁₀ O ₁₇ S [M+H]+ 965.32, found 965.86.

Example 234 Synthesis of amanitine conjugated analogue (D-1) having a bis-linker

ϋ n

To a solution of nitro-a-amanitin (9.0 mg, 0.0093 mmol) in DMA (1 ml) was added Pd/C (3 mg, 50% humidity), then hydrogenated (1 atm) at room temperature for 6 h. The catalyst was filtered, followed by the addition of 0.5 ml, 0.1 M NaH ₂ PO ₄ , pH 7.5 and bis(2,5-dioxopyrrolidin-1-yl) 21,22-bis(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo10,13,16,27,30,33-hexaoxa -3,6 19,24,37,40-hexaazadotetracontane-1,42-dioate (11.0 mg, 0.0092 mmol). The mixture was stirred at room temperature overnight, evaporated and purified on preparative HPLC with C18, eluted with water/ _CH3CN (90% water to 30% water over 35 min) to give 6.1 mg (yield 35%) of the product D-1 indicated in the title after drying in a high vacuum. MS ESI m/z C ₈₁ H ₁₁₆ N ₁₉ O ₃₁ S [M+H]+, calc. 1882.77, found 1882.20.

Example 235 Synthesis of amanitine conjugated analogue (D-1) having a bis-linker

Pd/C (3 mg, 50% humidity) was added to a solution of nitro-β-amanitin (9.0 mg, 0.0093 mmol) in DMA (1 ml), then hydrogenated (1 atm) at room temperature for 6 h The catalyst was filtered off followed by the addition of 0.5 ml, 0.1 M NaH ₂ PO ₄ , pH 7.5 and bis(2,5-dioxopyrrolidin-1-yl) 21,22-bis(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl)-2,5,38,41-tetramethyl-4,7,20,23,36,39-hexaoxo10,13,16,27,30,33-hexaoxa-3 ,6 19,24,37,40-hexaazadotetracontane-1,42-dioate (11.0 mg, 0.0092 mmol). The mixture was stirred at room temperature overnight, evaporated and purified by _C18 preparative HPLC, eluted with water/ _CH3CN (90% water to 30% water over 35 min) to give (7.0 mg, 40% yield) of title product D-2 after drying under high vacuum. MS ESI m/z C ₈₁ H ₁₁₅ N ₁₈ O ₃₂ S [M+H]+, calc. 1883.76, found 1884.10.

Example 236 General method for preparing a conjugate.

To a mixture of 2.0 ml of 10 mg/ml her2 antibody at pH 6.0 ~ 8.0, 0.70 ~ 2.0 ml of PBS buffer from 100 mM NaH ₂ PO ₄ , pH 6.5 ~ 8.5 buffers was independently added , TSER (16-20 μl, 20 mM in water) and compound A-3, A-4, A-5, B-3, B-6, B-9, B-12, B-15, B-18 , В-19, В-20, В-21, В-22, В-23, В-24, В-25, В-26, В-28, С-3, С-4, D-1 or D -2 (28-32 µl, 20 mM in DMA). The mixture was incubated at room temperature for 4-18 hours, then DHAA (135 μl, 50 mM) was added. After continuous incubation at room temperature overnight, the mixture was purified on a G-25 column eluted with 100 mM NaH ₂ PO ₄ 50 mM NaCl, pH 6.0-7.5, to obtain 12.8-18.1 mg of conjugate A -3a, А-4а, А-5а, В-3a, В-6а, В-9а, В-12а, В-15а, В-18а, В-19а, В-20а, В-21а, В-22а , B-23a, B-24a, B-25a, B-26a, B-28a, C-3a, C-4a, D-1a or D-2a (yield 75%-90%), respectively at 14.4 ~ 15.5 ml buffer. The drug/antibody ratio (DAR) of the conjugate was 3.1~4.2 as determined by UHPLC-QTOF mass spectrum. It is 94~99% monomer based on size exclusion HPLC analysis (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm IDx30 cm, 0.5 ml/min, 100 min), and one band in SDS-PAGE. . The structures of the conjugates are presented below:

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- 134 044827

- 135 044827

- 136 044827

where n=2.0 ~ 4.5.

Example 237. in vitro assessment of the cytotoxicity of the conjugate A-3a, A-4a, A-5a, B-3a, B-6a, B-9a, B-12a, B-15a, B-18a, B-19a, B- 20a, V-21a, V-22a, V-23a, V-24, V-25, V-26, V-28, S-3a, S-4a, D-1a or D-2a compared to T- DM1.

The cell line used in the cytotoxicity assays was NCI-N87, a human gastric carcinoma cell line; cells were grown in RPMI-1640 with 10% FBS. For the assay, cells (180 μl, 6000 cells) were added to each well in a 96-well plate and incubated for 24 h at 37°C with 5% CO2. Cells were then treated with test compounds (20 μl) at various concentrations in a suitable cell culture medium (total volume, 0.2 ml). Control wells contained cells and media but no test compounds. The plates were incubated for 120 hours at 37°C with 5% CO ₂ . MTT (5 mg/ml) (20 μl) was then added to the wells and the plates were incubated for 1.5 h at 37°C. The medium was carefully removed and then DMSO (180 μl) was added. After shaking for 15 min, absorbance was measured at 490 nm and 570 nm with a 620 nm reference filter. % inhibition was calculated using the following equation:

% inhibition=[1-(assay sample)/(control sample)] x 100.

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Cytotoxicity results IC ₅₀

DAR (Drug Ratio) Cells N87 (Ag+) 1C ₅₀ (nM) N87 cells (Ag+) IC90 (nM) A-Za conjugate 3.5 0.32 nM 0.91 nM Conjugate A-4a 3.8 0.17 nM 0.87 nM Conjugate A-5a 4.1 0.094 nM 0.31 nM Conjugate B-Za 3.8 0.14 nM 0.28 Conjugate V-ba 3.8 0.21 nM 0.62 Conjugate B-9a 3.6 0.17 nM 0.67 Conjugate B-12a 3.8 0.13 nM 0.06 Conjugate B-15a 3.6 0.29 nM 0.92 Conjugate B-18a 3.6 0.46 nM 1.20 Conjugate B-19a 3.5 0.12 nM 0.63 Conjugate B-20a 3.8 0.33 nM 0.96 Conjugate B-21a 3.8 0.42 nM 1.10 Conjugate B-22a 3.6 0.13 nM 0.33 Conjugate B-23a 3.6 0.18 nM 0.38 Conjugate B-24a 3.8 0.83 nM 1.46 Conjugate B-25a 3.8 0.72 nM 1.82 Conjugate B-26a 3.7 0.93 nM 1.93 Conjugate B-28a 3.6 0.45 nM 0.78 Conjugate C-Za 3.6 0.09 nM 0.17 Conjugate C-4a 3.7 0.26 nM 0.48 Conjugate D -1a 3.8 0.041 nM 0.087 Conjugate D-2a 3.9 0.033 nM 0.072 Conjugate T-1a 3.8 0.25 nM 0.51 T-DM1 3.5 0.12 nM 0.26

Example 238. Antitumor activity in vivo (BALB/c Nude mice with NCI-N87 xenograft tumor).

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In vivo effectiveness of conjugates A-3a, B-6a, B-12a, B-15a, B-18a, B-20a, B-21a, B-24a, B-28a, C-3a and D-2a along with T -DM1 was evaluated in xenograft tumor models of the human gastric carcinoma cell line N-87. Five-week-old female BALB/c Nude mice (104 animals) were subcutaneously inoculated into the area under the right shoulder with N-87 carcinoma cells (5x 106 cells/mouse) in 0.1 ml of serum-free medium. The tumor was allowed to grow for 8 days to an average size of 110 mm ³ . The animals were then randomly divided into 13 groups (8 animals per group). The first group of mice served as a control group and received phosphate-buffered saline (PBS) vehicle. 10 groups were treated with conjugates A-3a, B-6a, B-12a, B-15a, B-18a, B-20a, B-21a, B-24a, B-28a and T-DM1, respectively, at a dose of 3 mg/kg , administered intravenously. The remaining 2 groups were treated with conjugate C-3a and D1a, respectively, at a dose of 1 mg/kg administered intravenously. Three tumor measurements were taken every 4 days, and tumor volumes were calculated using the formula tumor volume=1/2 (length x width x height). At the same time, the weight of the animals was measured. Mice were sacrificed upon achieving one of the following criteria: (1) body weight loss was greater than 20% of pre-compound body weight, (2) tumor volume greater than 2000 mm ³ , (3) too sick to independently reach food source and water, or (4) skin necrosis. A mouse was considered tumor-free if there was no tumor on palpation.

The results are presented in Fig. 47. All 13 conjugates did not cause loss of body weight in animals. And the animals in the control group were sacrificed on day 50 because the tumor volume was more than 1800 mm ³ and the animals were very sick. Here, all 12 conjugates tested demonstrated antitumor activity. Animals in the groups of conjugates B-24a, C-3a, B-20a, B-21a and D-20a demonstrated better antitumor activity of the conjugates compared to T-DM1. But in animals in the groups of conjugates B-18a, B-15a, A-3a, B-6a, B-28a and B-12a, the conjugates showed worse antitumor activity compared to T-DM1. T-DM1 at a dose of 3 mg/kg inhibited tumor growth for 28 days, but it failed to eliminate tumors during the test. In contrast, conjugates B-20a, B21a and D-20a completely eradicated the tumors of some animals from day 15 to day 43. Tumor growth inhibition at these doses is listed below:

At the end of the experiment (day 50), animals in the PBS, A-3a, B-21a, T-DM1 and B-15a groups were sacrificed and tumors were removed, as shown in FIG. 48.

Example 239 Stability study of bis-linked conjugate compared to conventional mono-linked conjugates in mouse serum.

Forty-five female ICR mice, 6–7 weeks old, were divided into 3 groups. Each group included 15 mice to study the PK of one of the three ADCs. These 15 mice were further randomly divided into three groups (n=5). Each mouse was injected with T-DM1, B-21a and T-1a conjugates

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Claims (11)

(Huang Y. et al., Med Chem. #44, 249 ^th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO 2014009774) respectively at a dose of 10 mg/kg/mouse, i.v. bolus. Blood collection was performed in accordance with the NCI Rodent Blood Collection Guide. In general, mice in each group were bled one at a time to avoid more than two blood draws within 24 hours. Blood was collected from the retro-orbital blood sinus using a 70 μL capillary at time points 0 (pre-dose), 0.083, 0 .25, 0.5, 1, 4, 8, 24, 48, 96, 168, 312 and 504 hours after dosing. Plasma samples were analyzed for total antibodies and drug-conjugated antibodies using specific ELISA methods. Briefly, the concentration of conjugated antibody or total antibody concentration in mouse serum was measured as follows: 96-well ELISA plates were respectively coated overnight at 4°C with anti-DM1 antibody, anti-tubulisin antibody or anti-Her-2 Fab antibody (1 μg/ ml in 10 mM PBS, pH 7.2). The plates were then washed three times with PBS-T wash buffer (PBS/0.02% Tween20) and then blocked with 1% (wt/vol) BSA/PBS-T dilution buffer for 1 h at 37°C. After blocking buffer was removed, mouse serum standards or samples, each in triplicate, were diluted in 1% BSA/PBS-T buffer, incubated at 37°C for 1 h, then AP-conjugated donkey anti-human antibody was added for 30 min at 37°C after the plates were washed. The plates were washed again followed by the addition of pNPP substrate to develop color and then read on a microplate reader at 405 nm once the color reaction was quenched with 1 mol/L sodium hydroxide. The conjugated antibody concentration or total antibody concentration was obtained from fitting a four-parameter curve to a standard curve. As a result, as shown in FIG. 49, the PK behavior of total antibodies and drug-conjugated antibodies after administration of three ADCs is presented as typical biphasic clearance curves. Equivalence between plasma and peripheral tissues was achieved 8 hours after dosing. The elimination phase began 24 hours after administration and continued until the last sampling point. Thus, the conjugate exposure values (Auc _last ) for these three ADCs are 14981, 14713 and 16981 h/μg/kg for T-DM1, T-1a and B-21a, respectively. The volumes of distribution for all three conjugates are twice those of total blood volumes. The clearances (CL) of the conjugates are 0.59, 0.57 and 0.47 ml/h/kg, which is almost half that for general antibodies. The clearance of B-21a, both conjugate and total antibodies, is less than the other two ADCs, indicating that the bis-linked conjugate is more stable than conventional monolinked conjugates in mouse serum. CLAIM 1. A bis-linked conjugate having the structure represented by the formula (I-e), (I-f), (I-p), (I-r) - 140 044827 in which ---- represents a single bond; ----- is optionally either a single bond or a double bond, or may optionally be absent; n - integer from 1 to 20; a cell-binding molecule in frame is a molecule/agent that binds, complexes, or reacts with some portion of the cell population that is desired to be therapeutically or otherwise biologically modified; the molecule/agent that binds/binds to the cell is an antibody; wherein the molecule/agent that binds/binds to the cell contains a disulfide bond, the reduction of which results in the formation of a pair of thiols for bis-binding; the cytotoxic molecule in the box is selected from tubulisin, auristatin, amatoxin, pyrrolobenzodiazepine dimers (PBD); X and Y represent the same or different and independently a functional group that binds a cytotoxic drug, and X and Y are independently selected from NH; NHNH; N(R ₁ ); N(R ₁ )N(R ₂ ); ABOUT; C(O)NH, C(NH)NH; L ₁ and L ₂ are the same or different, and are independently selected from O; NH; NHNH; N(R ₃ ); N(R ₃ )N(R ₃ '); C ₁ -C ₈ alkyl, amides, amines, imines, hydrazines or hydrazones; ethers, esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, peptides, amides; 1~8 amino acids; polyethyleneoxy groups of the formula (OCH ₂ CH ₂ )pOR ₃ , or (OCH ₂ CH(CH,))pOR3, or NH(CH ₂ CH ₂ O)pR3, or NH(CH ₂ CH(CB)O)pR3, or N[ (CH2CH2O)pR3][(CH ₂ CH ₂ O)p'R ₃ '], or (OCH ₂ CH ₂ ) _p COOR ₃ , or CH ₂ CH ₂ (0cH ₂ CH ₂ ) _p c0oR ₃ , where p and p ' independently represent an integer selected from 0 to about 5000, or a combination thereof; R ₃ and R ₃ ' independently represent H or polyethyleneoxy groups of the formula (OCH ₂ CH ₂ ) _p or (OCH ₂ CH (CH ₃ )) _p , where p is an integer from 0 to about 5000, or the above combination; R ₁ and R 2 are independently selected from H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, heteroalkyl, alkylcycloalkyl or heterocycloalkyl; C ₃ -C ₈ aryl, Ar-alkyl, heterocyclyl, carbocyclyl, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl, or C ₂ -C ₈ esters, ether or amide; or peptides containing 1-8 amino acids; or a polyethyleneoxy group having the formula (OSH ₂ CH ₂ ) _p or (OSH ₂ CH (CH ₃ )) _p where p is an integer from 0 to about 5000, or a combination of the above groups; X ₇ and Y7 independently represent CH, CH ₂ , NH, O, S, NHNH, N(R ₁ ) and N; a chemical bond between two atoms means that it can connect any adjacent atoms. 2. The conjugate according to claim 1, wherein the disulfide bond of the internal chain of the cell binding/binding molecule/agent is reduced by a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), dithiolbutylamine (DTBA), L-glutathione ( GSH), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and/or beta mercaptoethanol (βME, 2-ME) to form a thiol pair. 3. The conjugate of claim 1 or 2, wherein the cell binding molecule/agent is capable of binding to tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated tumor cells, myeloid cells, activated T cells, B cells or melanocytes, or any cells expressing any of the following antigens or receptors: CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a , CD11b, CD11c, CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28 , CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO , CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55,CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E , CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD74, CD75, CD75s, CD76 , CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD93, CD94, CD95, CD96, CD97 , CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CDw113, CD114, CD115, CD116, CD117 ,CD118 , CD119, CDw119, CD120a, CD120b, CD121a, CD121b, CDw121b, CD122, CD123, CDw123, CD124, CD125, CDw125, CD126, CD127, CD128, CDw128, CD129, CD130, CD131, CDw131, CD132 , CD133, CD134, CD135 , CD136, CDw136, CD137, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CDw145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a , CD156b, CDw156c, CD157, CD158a, CD158b, CD159a, CD159b, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g , CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, - 141 044827 CD186, CDw186, CD187, CD188, CD189, CD190, Cd191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CDw198, CD199, CDw199, CD200, CD200a, CD200b, CD201, CD202, CD202b, CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210, CD212, CD213a1, CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD2 28, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235ab, CD235b, CD236, CD236R, CD238, CD239, CD240, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD 252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276 (B7-H3), CD277, CD278, CD279, CD280, CD281, CD282 , CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314, CD 315, CD316 , CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, CD339, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, Wnt-activated inhibitory factor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, alpha-integrin, alpha-v beta6 , aminopeptidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin-protective antigen, antitransferrin receptor, AOC3 (VAP-1), B7-H3, anthrax agent (Bacillus anthracis anthrax) , BAFF (B cell activating factor), BCMA, B cell lymphoma, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC motif, chemokine 11), CCR4 (CC chemokine receptor type 4), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3 , CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (claudin-18), agglutinating factor A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GMCSF)), CSP4, CTLA4 (cytotoxic T-lymphocyte associated protein 4), tumor antigen CTAA16.88, CXCR4, C-X-C chemokine receptor type 4, cyclic ADP-ribose hydrolase, cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3 (delta-like ligand 3), DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4) DR5 (death receptor 5), Shiga toxin type 1 Escherichia coli E .coli, Escherichia coli Shiga toxin type 2, ED-B, eGfL7 (EGF-like domain containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule ), EphA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II, beta -chain, fibronectin-B accessory domain, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-associated antigen 1, respiratory syncytial virus F protein, burn-associated receptor, fucosyl GM1, GD2 ganglioside, G-28 (glycolipid antigen cell surface), GD3 idiotype, GloboH, glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, growth differentiation factor 8, GP100, GPNMB (NMB transmembrane glycoprotein), GUCY2C (guanylate cyclase 2C, guanylyl cyclase C (GC-C ), intestinal guanylate cyclase, guanylate cyclase receptor C, heat-stable enterotoxin receptor (hSTAR)), heat shock proteins, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, chorionic human gonadotropin, HNGF, human scattering factor receptor kinase, HPV E6/E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor receptor 1), IGHE, IFN-γ , influenza virus hemagglutinin, IgE, IgE Fc region, IGHE, interleukins, in particular IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21 , IL22, IL-23, IL-27 or IL-28, IL3 IRA, ILGF2 (insulin-like growth factor 2), integrins, in particular α ₄ , a _IIb e ₃ , α _¥ β ₃ , α ₄ β ₇ , α ₅ β1, α ₆ β ₄ , α ₇ β ₇ , α11β ₃ , α ₅ β ₅ , α _Υ β ₅ ), interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, kappa Ig, LCK, Le, Legumain, Lewis antigen Y, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LFIRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1 MAD-CT-2, MAGE-1, MAGE-2 , MAGE-3, MAGE A1, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor (GIF)), MS4A1 (membrane spanning 4-domain subfamily A member 1 ), MSLN (mesothelin), MUC1 (Mucin 1 associated with cell surface (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), Melan-A/MART1 , ML-IAP, MPG, MS4A1 (membrane spanning protein 4-domain subfamily A), MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22ME), NGF , neuronal apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, nucleolin, oncogenic product Neu, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized lipoprotein - 142 044827 low density), OY-TES1, P21, non-mutant p53, P97, Page4, PAP, anti-glycolylneuraminic acid paratope), PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell protein 1), PDGF-Ra ( platelet-derived growth factor receptor alpha), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor beta, sodium phosphate cotransporter, PMEL 17, polysialic acid, proteinase 3 (PR1), prostate cancer , PS (phosphatidylserine), prostate carcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), Rh factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, respiratory -syncytial virus, RON, ROR1, sarcoma translocation initiation points, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syn-decane 1), sLe(a), somatomedin C, SIP (sphingosine-1- phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (six transmembrane prostate epithelial antigen 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor beta), TGF-e1, TGF-e2 (transforming growth factor beta 2), Tie ( CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor 10B receptor superfamily member), TNFRSF13B (tumor necrosis factor 13B receptor superfamily member), TPBG (glycoprotein trophoblast), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signaling transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP -2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR1, VEGFR2 or vimentin, WT1, XAGE 1 or cells expressing any insulin growth factor receptors or any epidermal growth factor receptors. 4. The conjugate according to claim 3, wherein the tumor cells are selected from the group consisting of lymphoma cells, myeloma cells, kidney cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, cells squamous cell carcinoma, small cell carcinoma cells, non-small cell carcinoma cells, ovarian cancer cells, malignant cells or any cells that grow and divide in an unregulated, accelerated manner, leading to cancer. 5. The conjugate of claim 1 or 4, wherein the cell-binding molecule/agent is an antibody selected from an IgG antibody, a monoclonal antibody, or an IgG antibody-like protein, and the cell-binding molecule/agent comprises a disulfide bond between a light chain and a heavy chain, a top disulfide bond between two heavy chains, and a bottom disulfide bond between two heavy chains, where the disulfide bond is reduced to form a thiol pair. 6. The conjugate of claim 5, wherein the cytotoxic molecules at different conjugation sites of the cell-binding molecule may differ when cytotoxic molecules containing the same or different bis-linkers are sequentially conjugated to the cell-binding molecule, or when different cytotoxic molecules containing the same or different bis-linkers are added in stages to the conjugation reaction mixture containing the cell-binding molecule. 7. Conjugate according to claim 1, having the formula 169, 177, 186, 397, 399a, 399b, 399c, 401, 449, B-21a, B-24a, B-25a, B-26a, B-28a, C- 3a, D-1a and D-2a, illustrated by the following structures: - 143 044827 399a, m ₁ =2, m ₂ =6; 399b, m ₁ =2, m ₂ =8; 399c, m ₁ =4, m ₂ =12, - 144 044827 - 145 044827 where m is 0-20, mAb is an antibody, n is defined as in claim 1. 8. A pharmaceutical composition containing a therapeutically effective amount of at least one conjugate according to claim 1 or 7, or a pharmaceutically acceptable salt thereof, as well as a carrier, diluent or excipient for the treatment or prevention of cancer, or an autoimmune disease, or an infectious disease. 9. Pharmaceutical composition according to claim 8, containing 0.1-300 g/l concentration of the conjugate according to claim 1 or 7; buffer agent with a pH from 4.5 to 7.5 at a concentration of 10-500 mM; 0-15% of one or more polyols, in particular fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, maltitol, lactitol , erythritol, threitol, sorbitol, glycerol or L-gluconate, and its metal salts; 0-1.0% surfactant selected from polysorbate, in particular including polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 or polysorbate 85), poloxamer, in particular poloxamer 188, poly(ethylene oxide)- poly(propylene oxide) or poloxamer 407, polyethylene-polypropylene glycol; Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl betaine (lauroamidopropyl); myristamido propyl, palmidopropyl or isostearamidopropyldimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate; MONAQUAT™ series (isostearylethylimidonium ethosulfate); polyethylglycol, polypropylglycol and ethylene-propylene glycol copolymers (Pluronics, PF68); 0-5 mg/ml of an antioxidant selected from ascorbic acid and/or methionine; 0-2 mM chelating agent selected from EDTA or EGTA; 0-5% preservative selected from benzyl alcohol, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl and benzyl alcohol, alkylparabens, for example methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol or m-cresol; 0-15% free amino acid; and/or a tonic agent selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate or NaCl to control the osmotic pressure from about 250 to 350 mOsm of the final formulation. 10. The pharmaceutical composition according to claim 8 or 9, contained in a vial, bottle, pre-filled syringe or pre-filled syringe with auto-injector, in the form of a solution or lyophilized solid. 11. The conjugate according to claim 1 or 7 or the pharmaceutical composition thereof according to claim 8 or 9, having cell killing activity in vitro, in vivo or ex vivo. - 146 -