Compositions and methods related to molecular conjugation

Abstract

The present invention relates to activated Michael acceptor (AMA) compounds that can be conjugated to biomolecules containing Michael donor moieties to provide plasma-stable antibody-drug conjugates (ADCs). Pharmaceutical compositions of the ADCs are also disclosed. A number of applications (e.g., therapeutic applications) in which the compositions are useful are also provided herein.

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 942,482, filed on December 2, 2019. The entire contents of this application are incorporated herein by reference. Background Art

[0003] Antibody-drug conjugates (ADCs) are emerging as a powerful class of anti-tumor agents with efficacy against a range of cancers. ADCs typically consist of three distinct structural components: a cell-binding or targeting moiety (the antibody); a linker; and a cytotoxic agent (the drug). Figure 1 ). Therefore, antibody bioconjugates play a crucial role in the development of novel bioactive conjugates for biological and medical applications. Chemoselectivity and mild processes are key to precisely install modifications without interfering with antibody structure, function, and activity. The reactivity, accessibility, and abundance of amino acid side chains are key aspects required to achieve the selective modification of a certain residue over all other proteinogenic amino acids. Among them, cysteine ​​(Cys) remains the amino acid of choice due to its low abundance and the high nucleophilicity of its thiol side chain.

[0004] Despite advances in antibody bioconjugation, maleimides remain the most commonly used reagents, primarily due to their fast kinetics and ease of synthesis. However, the sulfosuccinimide conjugates formed from these reagents undergo exchange reactions with thiols present in plasma, leading to the release of the maleimide. In the case of ADCs, this can lead to toxicity, as the product of the thiol exchange reaction is a highly potent cytotoxic drug. Therefore, there is a pressing need for methods to construct protein and antibody conjugates in a manner that allows for site-selective and irreversible installation of probes and drugs at specific sites within their sequences, resulting in plasma-stable ADCs that reliably release the drug at the intended target site.

[0005] Thiol groups on interchain cysteine ​​residues in monoclonal antibodies can be used as attachment sites for drug molecules. In human IgG1, there are four interchain disulfide bonds that can be used as conjugation sites. These four interchain disulfide bonds can be reduced by tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), thereby generating eight thiol groups that can be used for conjugation of drug molecules. In this way, conjugates with different drug-antibody ratios (DAR) can be obtained.

[0006] Classically, cysteine ​​residues can be modified by adding thiols to electrophilic reagents such as maleimides. Conjugates can therefore be prepared by reducing the disulfide bonds of the antibody and then reacting with maleimides. However, maleimide-based antibody-drug conjugates have been found to have limited stability in the blood circulation. Therefore, there is a need for an alternative method for conjugating cysteine ​​residues to active moieties that would result in ADCs that are less susceptible to the reverse Michael reaction. Summary of the Invention

[0007] In certain embodiments, the present disclosure relates to a compound of formula (I):

[0008]

[0009] or a salt thereof, wherein:

[0010] A is

[0011] M is N, CR 30 or C(-LQ);

[0012] Each L is independently selected from a spacer moiety;

[0013] Each Q is independently selected from an active moiety or a reactive group;

[0014] X is selected from -Cl, -Br and -I;

[0015] J is the targeting moiety;

[0016] R 30 and R 31 each independently selected from an electron withdrawing group, hydrogen, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a carbocyclyl group, a heterocyclyl group, and a haloalkyl group;

[0017] R 46 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl;

[0018] R 42 and R 43 Each independently selected from -OH, alkoxy, -NR 44 R 45 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic groups, wherein R 44 and R 45 Together with the nitrogen atom to which they are attached, they may form a 5- to 8-membered ring, optionally fused to an aryl or heteroaryl ring;

[0019] R 32 、R 44 and R 45are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl;

[0020] R 47 Yes O - , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl; and

[0021] n is 1 to 4.

[0022] In certain embodiments, the present disclosure relates to a method of preparing a conjugate comprising reacting a compound of formula (I) with a reagent comprising a targeting moiety covalently bound to a Michael donor, thereby producing a Michael adduct.

[0023] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0024] In certain embodiments, the present disclosure relates to a method for treating a subject in need thereof, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, or administering a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of an antibody-drug conjugate (ADC).

[0026] Figure 2 Shown are high performance liquid chromatography (HPLC) graphs demonstrating the stability of AMA-9c in an aqueous environment over time.

[0027] Figure 3 The relative reaction rates of N-Ac-cysteine ​​with compounds AMA-1, AMA-2, AMA-3, AMA-4, AMA-8, AMA-9c, AMA-10, mMPS-4, and PyrMPS-1 are illustrated.

[0028] Figure 4 The relative stability of compound N-Ac-Cys-AMA-10 compared to compound 14 is shown.

[0029] Figure 5 is a reaction scheme showing the conjugation reaction of AMA-9c with human serum albumin (1) and Thio-mAb (2).

[0030] Figure 6 is a hydrophobic interaction chromatography-HPLC (HIC-HPLC) graph showing analysis of the conjugation reaction of AMA-9c and Thio-mAb.

[0031] Figure 7 is a scheme showing the relative reaction rates of the Michael acceptor precursor Reference A compared to the AMA precursors pryMPS-1 and mMPS-4 with N-Ac-cysteine. DETAILED DESCRIPTION

[0032] Compounds of the present disclosure

[0033] In certain aspects, the present disclosure relates to activated Michael acceptor (AMA) compounds that can be conjugated to biomolecules containing Michael donor moieties. AMAs (such as vinyl aryl ketones of formula (II) and (III)) can undergo Michael addition reactions with Michael donors to provide conjugates of formula (IIa) and (IIIa) (Scheme 1). In certain aspects, the present disclosure relates to conjugates of biomolecules with AMAs, such as compounds (IIa) and (IIIa).

[0034] Solution 1.

[0035]

[0036] In the compounds of Formula (II), (III), (IIa) and (IIIa), L is a spacer moiety; Q is an active moiety (e.g., which may comprise a drug moiety, as described elsewhere herein) or a reactive group; M is N, CR 30 or C(-LQ); J is a targeting moiety (eg, which may comprise an antibody, as described elsewhere herein); R 30 and R 31 Each is independently selected from an electron withdrawing group, hydrogen, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a carbocyclyl group, a heterocyclyl group, and a haloalkyl group. For example, in certain embodiments, R 30 and R 31 At least one of is present and is an electron-withdrawing group; and R 32 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0037] In some embodiments of the invention, the Michael donor is a Cys residue of a protein, such as an antibody or a fusion protein.

[0038] In some embodiments, the active moiety Q comprises L' and Q', wherein L' is a linker and Q' is an active agent. In some aspects of the invention, L' comprises a coupling group, wherein the coupling group is coupled to L. For example, the coupling group can be selected from -C(=O)NR 32 -、-C(=O)O-、-C(=NR 32 )-、-C=NO-、-NR 32 -C(=O)-NR 32-, -OC(=O)O-, -SS-, -NR 32 S(=O)2O- and -OS(=O)2O-. Alternatively, the coupling group is selected from:

[0039]

[0040] L' may also comprise a cleavable group, wherein the cleavable group is coupled to Q'. For example, the cleavable group coupled to Q' may be selected from:

[0041] in

[0042] R 49 is hydrogen or -C(=O)R 50 ;and

[0043] R 50 It is a lower alkyl group.

[0044] Additional examples of cleavable groups are disclosed in International Patent Application Publication No. WO 2019 / 008441, which is herein incorporated by reference in its entirety.

[0045] In some embodiments, L' further comprises C6-C 100 An alkylene group containing at least one group selected from the group consisting of -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-.

[0046] In certain embodiments, the spacer moiety comprises a C6-C 100 An alkylene group containing at least one group selected from the group consisting of -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-.

[0047] Additionally or alternatively, the spacer moiety may comprise

[0048] in

[0049] a is a bond to the M-containing aromatic ring, and b is a bond to L'; and

[0050] n is 2 to 20.

[0051] In some embodiments of the present invention, Q' is a hormone, an oligonucleotide, a toxin, an affinity ligand, a probe for detection, or a combination thereof. For example, Q' can be selected from a cytokine, an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic, or a combination thereof.

[0052] In some embodiments, the targeting moiety comprises an -S- moiety.In some embodiments, the targeting moiety is coupled to the remainder of the compound of Formula (I) via the -S- moiety.

[0053] In certain embodiments, the targeting moiety comprises an antibody, such as an antibody selected from the group consisting of an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, and other modified immunoglobulin molecules comprising an antigen recognition site. For example, the targeting moiety can comprise an antibody selected from the group consisting of Muromonab-CD3, Abciximab, Rituximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab (herceptin), Etanercept, Basiliximab, Gemtuzumab ozogamicin, Alemtuzumab, Ibritu momab tiuxetan, Adalimumab, Alefacept, Omalizumab, Efalizumab, Tositumomob-I 131, Cetuximab, Bevacizumab, Natalizumab, Ranibizumab, Panitumumab, Eculizumab, Rilonacept, Certolizumab pegol, Romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, Belimumab, TACI-Ig, second-generation anti-CD20, ACZ-885, Tocili zumab, Atlizumab, Mepolizumab, Pertuzumab, Humax CD20, Tremelimumab (CP-675 206), Ticilimumab, MDX-010, IDEC-114, Inotuzumab ozo gamycin, HuMax EGFR, Aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, Catumaxomab, IGN101, MT-201, Pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, Motavizumab, MEDI-524, Efumgumab, Aurogr ab), Raxibacumab, third-generation anti-CD20, LY2469298, and Veltuzumab.

[0054] Methods of the present disclosure

[0055] The present invention relates to a method for conjugating proteins to active moieties using activated Michael acceptor (AMA) compounds. AMAs (such as vinyl aryl ketones of formula (II) and (III)) can undergo Michael addition reactions with Michael donors to provide conjugates of formula (IIa) and (IIIa) (Scheme 1).

[0056] In the compounds of formula (II) and (III), L is a spacer moiety; Q is an active moiety or reactive group; M is N, CR 30 or C(-LQ); J is the targeting moiety; R 30 and R31 are each independently selected from an electron withdrawing group, hydrogen, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a carbocyclyl group, a heterocyclyl group, and a haloalkyl group; and R 32 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0057] In some embodiments of the invention, the Michael donor is a Cys residue of a protein, such as an antibody or a fusion protein.

[0058] In certain embodiments, the Michael donor covalently bound to the targeting moiety is selected from the group consisting of: -SH, -NH2, -OH, Where R is C 1-3 Alkyl or C 1-3 Alkoxy.

[0059] Solution 1.

[0060]

[0061] Surprisingly, the inventors have found that the introduction of a nitrogen atom or an electron-withdrawing substituent (atom represented by "M") at the meta position of the aryl ring of compound (II) or (III) and / or the introduction of an electron-withdrawing substituent (group represented by "R") on the Michael acceptor itself can effectively inhibit the formation of the aryl ring of compound (II) or (III). 31 ”) greatly increases the rate of the Michael addition reaction, leading to the rapid and clean formation of conjugated compounds (IIa) and (IIIa). An example of the clean reaction of AMA compound 6 with N-Ac-Cys is shown in Scheme 2.

[0062] Option 2.

[0063]

[0064] The reaction in Scheme 3 demonstrates the chemoselectivity of the reaction of AMA with N-Ac-Cys compared to the reaction with N-Ac-Lys and N-Ac-Tyr.

[0065] Option 3.

[0066]

[0067] When exposed to a mixture containing equimolar amounts of N-Ac-Cys and N-Ac-Lys (Scheme 3, upper reaction) or a mixture of N-Ac-Cys, N-Ac-Tyr, and N-Ac-Lys (Scheme 3, lower reaction), AMA compound 6 produced a conjugate with N-Ac-Cys with over 95% chemoselectivity.

[0068] Figure 2The stability of the compound of formula (II) in an aqueous environment is demonstrated. Additional Michael acceptor compounds suitable for rapid and clean formation of protein conjugates are shown in Scheme 4.

[0069] Solution 4.

[0070]

[0071] The relative rates of Michael addition reactions of various compounds of formula (II) and other Michael acceptors with N-Ac-Cys are shown in Figure 3 As shown, placing the Michael acceptor moiety in the meta position relative to the linker substituent on the aryl ring increases the reaction rate (see Compound 3 and Compound 4). The reaction rate is further increased when electron-withdrawing substituents or nitrogen atoms are present in the meta position relative to both the Michael acceptor moiety and the linker moiety on the aryl ring (Compounds 5 and 6 and Compound 4).

[0072] In order to solve the problem of ADC instability with respect to the reverse Michael transformation, various methods for stabilizing compounds of formula (IIIa) in aqueous environments have been explored. Treatment of compounds of formula (IIIa) with a hydride source (such as NaBH4) results in the formation of alcohol-containing compounds of formula (IVa) that are stable in aqueous media (Scheme 5). In addition, the inventors have discovered that treatment of compounds of formula (IIIa) with amine-containing compounds or hydroxylamine results in the formation of imines or oximes of formula (IVb) (Scheme 5), which are stable in aqueous media (Scheme 6).

[0073] Option 5.

[0074]

[0075] R 42 Selected from -OH, alkoxy, -NR 44 R 45 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic groups, wherein R 44 and R 45 Together with the nitrogen atom to which they are attached they may form a 5- to 8-membered ring, optionally fused to an aryl or heteroaryl ring.

[0076] Option 6.

[0077]

[0078] The compound of formula (IVa) exhibited high stability for more than one week in human and mouse plasma and in PBS buffer at pH 7.4, whereas the corresponding compound of formula (IIIa) was less stable ( Figure 4 ).

[0079] In certain embodiments, the present disclosure also relates to a method of conjugating a protein to an active moiety using a precursor of an activated Michael acceptor (AMA) compound, such as compounds of formula (Va), (Vb), and (Vc) in Scheme 7, wherein

[0080] X is selected from -Cl, -Br and -I;

[0081] R 46 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl;

[0082] R 47 Yes O - , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl; and

[0083] n is 1 to 4.

[0084] Plan 7.

[0085]

[0086] Examples of compounds of Formula (Va), (Vb) and (Vc) are shown in Scheme 8. Compounds of Formula (Va), (Vb) and (Vc) can be cleanly converted into Michael acceptor reagents suitable for conjugation to Michael donor-containing molecules, such as proteins.

[0087] Plan 8.

[0088]

[0089] Further studies demonstrated that the conjugation method involving AMA can be extended to antibodies such as human serum albumin and Thio-mAb (trastuzumab) ( Figure 5 and Figure 6 Reduction of the resulting conjugate with NaBH4 afforded a plasma-stable ADC.

[0090] In certain embodiments, the present disclosure relates to a compound of formula (I):

[0091]

[0092] or a salt thereof, wherein:

[0093] A is

[0094] M is N, CR 30 or C(-LQ);

[0095] Each L is independently selected from a spacer moiety;

[0096] Each Q is independently selected from an active moiety or a reactive group;

[0097] X is selected from -Cl, -Br and -I;

[0098] J is the targeting moiety;

[0099] R 30 and R 31 each independently selected from an electron withdrawing group, hydrogen, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a carbocyclyl group, a heterocyclyl group, and a haloalkyl group;

[0100] R 46 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl;

[0101] R 42 and R 43 Each independently selected from -OH, alkoxy, -NR 44 R 45 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic groups, wherein R 44 and R 45 Together with the nitrogen atom to which they are attached, they may form a 5- to 8-membered ring, optionally fused to an aryl or heteroaryl ring;

[0102] R 32 、R 44 and R 45 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl;

[0103] R 47 Yes O - , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl; and

[0104] n is 1 to 4.

[0105] In some embodiments, M is N.

[0106] In certain embodiments, M is CR 30 , and R 30 It is an electron-withdrawing group.

[0107] In some embodiments, A is selected from

[0108]

[0109] where R 31 is an electron withdrawing group, preferably wherein L is coupled to C via an electron withdrawing group selected from an amide or an ester.

[0110] In some embodiments, M is C(-LQ), and wherein L is coupled to C through an electron withdrawing group.

[0111] In some embodiments, R 30 Yes-CO2NR 33 R 34 or -CO2R 35 , and R 33 、R 34 and R 35 Each is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0112] In some embodiments, each electron withdrawing group is independently selected from -NO2, -CN, -haloalkyl, -CO2NR 33 R 34 、-CO2R 35 、-C(=O)R 36 、-S(=O)R 37 、-S(=O)2OR 38 and -NR 39 R 40 R 41 ; and R 36 、R 37 、R 38 、R 39 、R 40 and R 41 Each is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

[0113] In certain embodiments, each electron withdrawing group is independently selected from -CN, -CONR 33 R 34 and -CO2R 35 .

[0114] In some embodiments, each electron withdrawing group is independently selected from -CN, -CONH2, and -CO2Me.

[0115] In certain embodiments, Q is an active moiety.

[0116] In some embodiments, Q comprises L' and Q', wherein L' is a linker and Q' is an active agent.

[0117] In certain embodiments, L′ comprises a coupling group, wherein the coupling group is coupled to L.

[0118] In some embodiments, the coupling group is selected from -C(=O)NR 32 -、-C(=O)O-、-C(=NR 32)-、-C=NO-、-NR 32 -C(=O)-NR 32 -, -OC(=O)O-, -SS-, -NR 32 S(=O)2O- and -OS(=O)2O-.

[0119] In certain embodiments, the coupling group is selected from

[0120]

[0121] In some embodiments, L' further comprises a cleavable group, wherein the cleavable group is coupled to Q'.

[0122] In certain embodiments, the cleavable group coupled to Q' is selected from

[0123] in

[0124] R 49 is hydrogen or -C(=O)R 50 ;and

[0125] R 50 It is a lower alkyl group.

[0126] In some embodiments, L' further comprises C6-C 100 An alkylene group containing at least one group selected from the group consisting of -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-.

[0127] In certain embodiments, L comprises C6-C 100 Alkylene, said alkylene containing at least one group selected from -NH-, -C(=O)-, -O-, -S-, -S(O)- and -S(=O)2-. For example, L contains

[0128] in

[0129] a is a bond to the M-containing aromatic ring, and b is a bond to L'; and

[0130] n is 2 to 20.

[0131] In some embodiments, Q' is a hormone, an oligonucleotide, a toxin, an affinity ligand, a probe for detection, or a combination thereof.

[0132] In certain embodiments, Q' is selected from a cytokine, an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic, or a combination thereof.

[0133] In certain embodiments, Q is a reactive group.

[0134] In some embodiments, the reactive group is selected from -N3, -C≡CH, -S(O)2Hal, -NH2, -CO2Hal, -OH, -C(O)H, -SH, -N=C=O and -N=S=C, wherein Hal is -Cl, -Br or -I.

[0135] In certain embodiments, the targeting moiety comprises an -S- moiety.

[0136] In some embodiments, the targeting moiety is coupled to the remainder of the compound of Formula (I) through an -S- moiety.

[0137] In some embodiments, A is

[0138]

[0139] In some embodiments, A is

[0140] In certain embodiments, R 31 -CN, -CO2NR 33 R 34 or -CO2R 35 .

[0141] In certain embodiments, A is

[0142] In some embodiments, R 32 is hydrogen or C 1-3 alkyl.

[0143] In some embodiments, A is

[0144] In certain embodiments, R 46 is an optionally substituted C 1-3 Alkyl, optionally substituted C6-C 12 aryl or optionally substituted heteroaryl.

[0145] In some embodiments, A is

[0146] In certain embodiments, R 47 Yes O - or C 1-3 alkyl.

[0147] In certain embodiments, A is

[0148] In some embodiments, A is

[0149] For example, A can be

[0150] Alternatively, A can be

[0151] In other embodiments, A can be

[0152] In some embodiments, A is

[0153] In certain embodiments, R 42 Is -OH or -NR 44 R 45 .

[0154] In some embodiments, the targeting moiety comprises a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment or a repebody of an antigenic polypeptide. For example, the targeting moiety may comprise an antibody, such as an antibody selected from the group consisting of a complete polyclonal antibody, a complete monoclonal antibody, an antibody fragment, a single chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, and a modified immunoglobulin molecule comprising an antigen recognition site. For example, the targeting moiety may comprise an antibody selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basilizumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I 131, cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegol, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI-Ig, second-generation anti-CD20, ACZ-885, tocilizumab, atezolizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), tesimumab, MDX-010, IDEC-114, ointuzumab, HuMax EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, prigovinomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, ifenguzumab, orolab, rixibacumab, third-generation anti-CD20, LY2469298, and veltuzumab.

[0155] In some embodiments, the compound of formula (I) is selected from

[0156]

[0157] In some embodiments, the present disclosure relates to a method of preparing a conjugate comprising reacting a compound of formula (I) with a reagent comprising a targeting moiety covalently bound to a Michael donor, thereby producing a Michael adduct.

[0158] In some embodiments, the present disclosure relates to a method further comprising reducing the Michael adduct.

[0159] In some embodiments, the Michael donor covalently bound to the targeting moiety is selected from:

[0160] -SH, -NH2, -OH, in

[0161] R is C 1-3 Alkyl or C 1-3 Alkoxy.

[0162] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0163] In some embodiments, the present disclosure relates to a method for treating a disease or condition, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0164] In some embodiments, the disease or disorder is selected from cancer, an infectious disease, or an autoimmune disease.

[0165] In certain embodiments, the disease or disorder is cancer.

[0166] definition

[0167] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0168] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0169] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0170] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, linked to oxygen. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0171] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0172] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter being an alkenyl moiety having a substituent replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may appear on one or more carbons that are or are not contained in one or more double bonds. In addition, unless stability does not allow, such substituents include all substituents contemplated for alkyl groups as discussed below. For example, it is contemplated that an alkenyl group is substituted by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups.

[0173] An "alkyl" group or "alkane" is a fully saturated straight or branched chain non-aromatic hydrocarbon. Typically, unless otherwise defined, a straight or branched chain alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10. Examples of straight and branched chain alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, amyl, and octyl. A C1-C6 straight or branched chain alkyl group is also referred to as a "lower alkyl" group.

[0174] In addition, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include "unsubstituted alkyl" and "substituted alkyl," the latter referring to an alkyl moiety having a substituent replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. If not otherwise indicated, such substituents may include, for example, halogen, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfinamido, sulfonyl, heterocyclyl, aralkyl, guanidino, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moiety substituted on the hydrocarbon chain may itself be substituted, if appropriate. For example, the substituents of the substituted alkyl group can include amino, azido, imino, amido, phosphoryl (including phosphonic acid and phosphinic acid), sulfonyl (including sulfate, sulfinamido, sulfamoyl and sulfonic acid) and silyl groups as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylic acid and ester), -CF , -CN and the like substituted and unsubstituted forms. Exemplary substituted alkyl groups are described below. Cycloalkyl groups can be further substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF , -CN and the like.

[0175] When used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C x -C y " is meant to include groups containing x to y carbons in the chain. For example, the term "C x -C y "Alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl and branched chain alkyl groups containing x to y carbons in the chain, including halogenated alkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. C0 alkyl represents a hydrogen at the terminal position of the group, or a bond if the group is internal. The term "C2-C y Alkenyl" and "C2-C y "Alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group similar in length and possible substitution to the alkyl groups described above, but containing at least one double or triple bond respectively.

[0176] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0177] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, and may be represented by the general formula alkylS-.

[0178] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond, and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter of which refers to alkynyl moieties having substituents replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are or are not contained in one or more triple bonds. Furthermore, unless stability is too high, such substituents include all substituents contemplated for alkyl groups as discussed above. For example, alkynyl groups are contemplated to be substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups.

[0179] As used herein, the term "amide" refers to a group

[0180]

[0181] Each R 10 independently represent hydrogen or a hydrocarbon group, or two R 10 Taken together with the nitrogen atom to which they are attached, they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0182] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, such as the moiety represented by the formula

[0183]

[0184] Each R 10 independently represent hydrogen or a hydrocarbon group, or two R 10 Taken together with the nitrogen atom to which they are attached, they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0185] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0186] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0187] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings, at least one of which is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Thus, the term "aryl" may include (C5-C 10 ) and (C6-C 10 ) aryl groups. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0188] The term "carbamate" is art-recognized and refers to the group

[0189]

[0190] where R 9 and R 10 independently represents hydrogen or a hydrocarbyl group, such as an alkyl group, or R 9 and R 10 Taken together with the intervening atoms, they form a heterocycle having from 4 to 8 atoms in the ring structure.

[0191] As used herein, the terms "carbocycle" and "carbocyclic" refer to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes aromatic carbocycles and non-aromatic carbocycles. Non-aromatic carbocycles include cycloalkane rings in which all carbon atoms are saturated and cycloolefin rings containing at least one double bond. "Carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring of a bicyclic carbocycle can be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with another ring. Each ring of a fused carbocycle can be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring (e.g., phenyl) can be fused to a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane, or cyclohexene). When valence permits, any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decahydronaphthalene, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions capable of carrying a hydrogen atom.

[0192] "Cycloalkyl" groups are fully saturated cyclic hydrocarbons. "Cycloalkyl" includes monocyclic and bicyclic rings. Unless otherwise defined, monocyclic cycloalkyl groups typically have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The second ring of a bicyclic cycloalkyl group can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl groups include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl group in which each ring shares two adjacent atoms with another ring. The second ring of a fused bicyclic cycloalkyl group can be selected from saturated, unsaturated, and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0193] As used herein, the term "(cycloalkyl)alkyl" refers to an alkyl group substituted with a cycloalkyl group.

[0194] The term "carbonate" is art-recognized and refers to the group -OCO2-R 10 , where R 10 represents a hydrocarbon group.

[0195] As used herein, the term "carboxyl" refers to a group represented by the formula -CO2H.

[0196] As used herein, the term "ester" refers to the group -C(O)OR 10 , where R 10 represents a hydrocarbon group.

[0197] As used herein, the term "ether" refers to a hydrocarbyl group connected to another hydrocarbyl group through an oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0198] As used herein, the terms "halo" and "halogen" mean halogen and include chlorine, fluorine, bromine, and iodine.

[0199] As used herein, the term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group.

[0200] As used herein, the term "heteroalkyl" refers to a saturated or unsaturated chain having carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.

[0201] The term "heteroaryl" includes substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems with two or more rings, wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is heteroaromatic, for example, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Thus, the term "heteroaryl" can include (C2-C 10 ) and (C2-C 10 ) heteroaryl groups. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0202] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0203] The terms "heterocycloalkyl", "heterocycle" and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocycloalkyl" and "heterocyclic" also include polycyclic ring systems having two or more rings, wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is heterocyclic, for example, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Heterocycloalkyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0204] As used herein, the term "(heterocycloalkyl)alkyl" refers to an alkyl group substituted with a heterocycloalkyl group.

[0205] As used herein, the term "alkyl" refers to a group bonded by a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a main chain that is primarily carbon, but may optionally contain heteroatoms. Thus, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be alkyls, but substituents such as acetyl (which has a =O substituent on the connecting carbon) and ethoxy (which is connected by oxygen rather than carbon) are not considered to be alkyls. alkyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0206] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0207] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, the term "lower" is meant to include groups wherein the substituent has ten or less, preferably six or less non-hydrogen atoms. For example, "lower alkyl" refers to an alkyl group containing ten or less, preferably six or less carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents as defined herein are respectively low acyl, low acyloxy, low alkyl, low alkenyl, low alkynyl or low alkoxy, whether they occur alone or in combination with other substituents, such as in the formulation of hydroxyalkyl and aralkyl (in this case, for example, when calculating the carbon atoms in the alkyl substituent, the atoms in the aryl group are not counted).

[0208] The terms "polycyclic group," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) wherein two or more atoms are shared by two adjacent rings, e.g., the rings are "fused rings." Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0209] The term "silyl" refers to a silicon moiety to which are attached three hydrocarbyl moieties.

[0210] The term "substituted" refers to a portion of a substituent having a hydrogen replacement on one or more carbons of the main chain. It should be understood that "substituted" or "substituted by ... " includes implicit prerequisites, i.e., this substitution is based on the allowed valence of the substituted atom and substituent, and the substitution produces a stable compound, for example, it will not spontaneously undergo conversions such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is considered to include all permissible substituents of an organic compound. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Permissible substituents can be one or more substituents and are the same or different for appropriate organic compounds. For purposes of the present invention, heteroatoms such as nitrogen can have any permissible substituents that satisfy the valence of a heteroatom of a hydrogen substituent and / or an organic compound as described herein. Substituents may include any substituent described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonic acid, sulfamoyl, sulfinamido, sulfonyl, heterocyclyl, aralkyl or aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the substituents themselves may be substituted if appropriate. Unless specifically stated as "unsubstituted", chemical moieties mentioned herein are understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0211] The term "sulfate" is art-recognized and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0212] The term "sulfonamide" is art-recognized and refers to a group represented by the general formula

[0213]

[0214] where R 9 and R10 independently represents hydrogen or a hydrocarbon group, such as an alkyl group, or R 9 and R 10 Taken together with the intervening atoms, they form a heterocycle having from 4 to 8 atoms in the ring structure.

[0215] The term "sulfoxide" is art-recognized and refers to the group -S(O)-R 10 , where R 10 Represents a hydrocarbon group.

[0216] The term "sulfonic acid" is art-recognized and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0217] The term "sulfone" is art-recognized and refers to the group -S(O)2-R 10 , where R 10 Represents a hydrocarbon group.

[0218] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0219] As used herein, the term "thioester" refers to the group -C(O)SR 10 or -SC(O)R 10 , where R 10 Represents a hydrocarbon group.

[0220] As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

[0221] The term "urea" is art-recognized and can be represented by the following general formula

[0222]

[0223] where R 9 and R 10 independently represents hydrogen or a hydrocarbon group, such as an alkyl group, or each occurrence of R 9 With R 10 and the intervening atoms are taken together to form a heterocycle having 4 to 8 atoms in the ring structure.

[0224] "Protecting group" refers to an atomic group that masks, reduces or prevents the reactivity of a reactive functional group in a molecule when attached to the reactive functional group in the molecule. Typically, a protecting group can be selectively removed as needed during the synthesis process. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd edition, 1999, John Wiley & Sons, NY, and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1 to 8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butyloxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), etc. Representative hydroxy protecting groups include, but are not limited to, those in which the hydroxy group is acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0225] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the incidence of the disorder or condition in treated samples relative to untreated control samples, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to untreated control samples.

[0226] The term "treatment" includes preventative and / or therapeutic treatments. The term "preventative or therapeutic" treatment is well-known in the art and includes administering one or more of the subject compositions to the host. If administered before clinical manifestation of an adverse condition (e.g., a disease or other adverse state of the host animal), the treatment is preventative (i.e., it protects the host from developing an adverse condition), while if administered after the manifestation of an adverse condition, the treatment is therapeutic (i.e., it is intended to weaken, improve, or stabilize an existing adverse condition or its side effect).

[0227] "Conjugate" refers to two or more molecules covalently linked into a larger construct. In some embodiments, the conjugate comprises one or more biomolecules (such as peptides, nucleic acids, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules (such as one or more other biomolecules or polymer linkers).

[0228] "Conjugate," "bind," "bond," "couple," or "link" are used synonymously and mean the direct or indirect binding of a first atom or molecule to another atom or molecule to form a larger molecule.

[0229] As used herein, the term "antibody-drug conjugate (ADC)" refers to a molecule in which a drug and an antibody are chemically bound to each other (eg, through a linker moiety) without reducing the biological activity of the antibody and the drug.

[0230] As used herein, the term "antibody" refers to a protein molecule comprising an immunoglobulin, an immunoglobulin chimera or an immunoglobulin-like molecule (including, by way of example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof), and similar molecules produced during an immune response in any vertebrate (e.g., mammals such as humans, goats, rabbits and mice), as well as antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. The term is intended to encompass polyclonal antibodies, monoclonal antibodies, full-length antibodies and antibody fragments containing antigen-binding domains. A full-length antibody has two full-length light chains and two full-length heavy chains, wherein each light chain is connected to a heavy chain by a disulfide bond. Full-length antibodies include IgA, IgD, IgE, IgM and IgG, and the subtypes of IgG include IgG1, IgG2, IgG3 and IgG4. The term "antibody fragment" refers to a fragment that has antigen-binding function, and is intended to include recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), disulfide-stabilized Fv proteins ("dsFv"), diabodies and tribodies (as known in the art), and camelid antibodies (see, e.g., U.S. Patent Nos. 6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808). Fab contains light and heavy chain variable regions, a light chain constant region, and the first constant domain (CH1) of the heavy chain, and has one antigen-binding site. Fab' has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies contain disulfide bonds between the cysteine ​​residues in the hinge region of Fab'. Fv refers to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. dsFv has a structure in which the heavy chain variable region and the light chain variable region are interconnected by disulfide bonds, and scFv generally has a structure in which the heavy chain variable region and the light chain variable region are covalently linked to each other by a peptide linker. These antibody fragments can be obtained using proteases (for example, Fab fragments can be obtained by digesting full-length antibodies with papain, and F(ab')2 fragments can be obtained by digesting full-length antibodies with pepsin). Preferably, these antibody fragments can be produced by genetic recombination technology.

[0231] The term "antibody" includes monoclonal antibodies produced by cells cloned from single B lymphocytes or transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies can be obtained using a variety of techniques well known to those skilled in the art, including standard hybridoma technology (see, e.g., Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988) and CA Janeway et al. (eds.), Immunobiology, 5th edition, Garland Publishing, New York, NY (2001)). Monoclonal antibodies can also be produced using other suitable techniques, including EBV-hybridoma technology (see, e.g., Haskard and Archer, J. Immunol. Methods, 74(2):361-67 (1984) and Roder et al., Methods Enzymol., 121:140-67 (1986)), phage vector expression systems (see, e.g., Huse et al., Science, 246:1275-81 (19891)), or phage display libraries containing antibody fragments such as Fab and scFv (single chain variable regions) (see, e.g., U.S. Patent Nos. 5,885,793 and 5,969,108, and International Patent Application Publication Nos. WO 92 / 01047 and WO 99 / 06587, each of which is incorporated herein by reference in its entirety). Monoclonal antibodies include humanized monoclonal antibodies. As used herein, a "humanized" antibody is one in which the complementary determining regions (CDRs) of a mouse monoclonal antibody that form the antigen-binding loop of the antibody are grafted onto the framework of a human antibody molecule. Due to the similarity of mouse and human antibody frameworks, it is generally accepted in the art that the monoclonal antibodies produced by this method are antigenically identical to human antibodies, but bind to the same antigen as the mouse monoclonal antibody from which the CDR sequences were derived. Methods for producing humanized antibodies are well known in the art and are described in detail in, for example, Janeway et al., supra, U.S. Patent Nos. 5,225,539, 5,585,089 and 5,693,761 (each of which is incorporated herein by reference in its entirety), European Patent No. 0239400B1 and British Patent No. 2188638. Humanized antibodies can also be produced using the antibody surface remodeling technology described in U.S. Patent No. 5,639,641 (which is incorporated herein by reference in its entirety) and Pedersen et al., J. Mol Biol., 235: 959-973 (1994).

[0232] The antibodies disclosed in the present invention can be natural antibodies or recombinant antibodies. As used herein, the term "natural antibody" refers to an antibody that has not been genetically modified. As used herein, the term "recombinant antibody" refers to a genetically modified antibody that may have antigen-binding activity or desired characteristics conferred by genetic modification.

[0233] As used herein, the term "cytokine" refers to small cell-signaling protein molecules secreted by numerous cells and is a class of signaling molecules widely used for intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulatory factors produced throughout the body by cells of diverse embryological origin.

[0234] As used herein, the term "hormone" refers to a chemical substance released by cells, glands, or organs in one part of the body that performs a signaling function on cells in other parts of the organism. The term includes peptide hormones, lipid and phospholipid-derived hormones, including steroid hormones, and monoamines.

[0235] "Moiety" refers to a fragment of a molecule, or a portion of a molecule, such as a conjugate.

[0236] The present invention discloses compounds having functional groups capable of undergoing Michael addition reactions. For example, RT Morrison and RN Boyd taught Michael additions in Organic Chemistry, 3rd edition, Allyn and Bacon, 1973. The reaction occurs between a molecule comprising a Michael donor moiety and a molecule comprising a Michael acceptor moiety.

[0237] Targeted therapy

[0238] The targeting portion of the conjugate can recognize cells, or be recognized by cells, thereby providing so-called targeted therapy.

[0239] In some embodiments, the conjugate comprises an active moiety Q for use in a targeted therapy for treating an autoimmune disease. In certain embodiments, the active moiety comprises an active agent selected from the group consisting of cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate cyclophosphamide, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlororambucil, DHEA, danazol, bromocriptine, meloxicam, or infliximab.

[0240] In some embodiments, the compound comprises an active moiety Q for use in a targeted therapy for treating an infectious disease. In certain embodiments, Q comprises an active agent selected from the group consisting of β-lactams (e.g., penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, becampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin), aminoglycosides (e.g., amikacin, gentamicin), styrax, ... (e.g., tamycin), kanamycin, neomycin, netilmicin, streptomycin, tobramycin), macrolides (azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin), tetracyclines (e.g., demeclocycline, doxycycline, minocycline, tetracycline), quinolones (e.g., cinoxacin, nalidixic acid), acid), fluoroquinolones (e.g., ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxicin), peptides (e.g., bacitracin, colistin, polymyxin B),B)), sulfonamides (e.g., sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, sulfacetamide), antibiotic agents (such as trimethoprim, sylfamethazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampicin, spectinomycin, or nitrofurantoin), general antiviral agents (e.g., idoxuradine, vidarabine, acyclovir, famcicyclovir, (e.g., tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delaviridine, saquinavir, ritonavir, indinavir, nelfinavir).

[0241] In some embodiments, the compounds and conjugates disclosed herein comprise an active moiety Q for use in a method for delivering an active agent to a cell to treat a tumor, wherein the targeting moiety is selected to bind to the target cell (i.e., a cancer cell). In particular, the compounds, conjugates, and compositions of the invention can be used to inhibit abnormal cell growth or treat a proliferative disease in a mammal (e.g., a human), such as where the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least preferentially associated with tumor cells rather than healthy cells).

[0242] In certain embodiments, the active moiety Q comprises a cytotoxic agent or an immunomodulatory agent, an anticancer agent, an anti-tubulin agent or a cytotoxic agent. Preferably, the cytotoxic agent or immunomodulatory agent is selected from an anti-tubulin agent, an auristatin, a DNA minor groove binder, a DNA transcription inhibitor, an alkylating agent, an anthracycline, an antibiotic, an antifolate, an antimetabolite, a calmodulin inhibitor, a chemosensitizer, a duocarmycin, an etoposide, a fluorinated pyrimidine, an ionophore, lexitropsin, maytansinoid, a nitrosourea, or a combination thereof. the anticancer agent being selected from the group consisting of methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, proocarbine, topotecan, nitrogen mustard, and thiazolinone. mustards), cyclophosphamide (cytoxan), etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etc.;Anti-tubulin agents include taxanes (e.g., paclitaxel, docetaxel), T67, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), baccatin derivatives, taxane derivatives, epothiolone (e.g., epothiolone A, epothiolone B), nocodazole, colchicine, colcimid, estramustine, crytophycins, cemadotin, maytansinoids, combrestatins, discoder molide, eleutherobin, or auristatin derivatives (e.g., AFP, MMAF, MMA E);The cytotoxic agent is selected from the group consisting of androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chloramphenicol, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, B), dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D D), hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thio-TEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16, VM-26;DNA minor groove binders (e.g., enediynes, lexitropsins, CBI compounds), duocarmycins, taxanes (e.g., paclitaxel, docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A, epothilone B, estramustine, cryptophycins, cemadotin, maytansinoids, eleutherobin, or mitoxantrone.

[0243] Cell proliferation and apoptosis

[0244] The compounds and conjugates disclosed herein can be used in methods of inducing apoptosis.

[0245] Apoptosis dysregulation is associated with a variety of diseases, including, for example, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma, or Crohn's disease), hyperproliferative disorders (e.g., breast cancer, lung cancer), viral infections (e.g., herpes, papilloma, or HIV), and other conditions such as osteoarthritis and atherosclerosis. The compounds, conjugates, and compositions described herein can be used to treat or improve any of these diseases. Such treatments typically involve administering to a subject suffering from the disease a compound, conjugate, or composition described herein in an amount sufficient to provide a therapeutic benefit. The properties of the antibody to the compound, conjugate, or composition administered will depend on the disease being treated, and thus the antibody should bind to a cell surface antigen expressed in a cell type where inhibition would be beneficial. The therapeutic benefit obtained will also depend on the specific disease being treated. In some cases, the compounds and compositions disclosed herein can treat or improve the disease itself or the symptoms of the disease when administered as a monotherapy. In other cases, the compounds and compositions disclosed herein can be part of an overall treatment regimen that includes other agents that treat or ameliorate the disease or disease symptoms being treated, along with the inhibitor or the compounds and compositions disclosed herein. Agents for treating or ameliorging specific diseases that can be administered as an adjunct to or in conjunction with the compounds and compositions disclosed herein will be readily apparent to those skilled in the art.

[0246] Although an absolute cure is always desired in any treatment regimen, achieving a cure is not required to provide a therapeutic benefit. A therapeutic benefit can include stopping or slowing the progression of a disease, causing the disease to regress but not cure, and / or improving or slowing the progression of disease symptoms. Prolonged survival and / or improved quality of life compared to the statistical mean can also be considered a therapeutic benefit.

[0247] A special class of diseases involving apoptosis disorders and being a major health burden worldwide is cancer. In certain embodiments, the compounds and compositions disclosed herein can be used to treat cancer. For example, the cancer can be a solid tumor or a hematological tumor. Cancers that can be treated with the compounds and compositions disclosed herein include, but are not limited to, bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, liver cancer, lymphocytic leukemia, follicular lymphoma, lymphoid malignancies of T cell or B cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer, small cell lung cancer and spleen cancer. The compounds and compositions disclosed herein may be particularly beneficial in treating cancer because antibodies can be used to specifically target tumor cells, thereby potentially avoiding or improving adverse side effects and / or toxicity that may be associated with systemic administration of unconjugated inhibitors. In some embodiments, the present disclosure relates to a method of treating a disease involving intrinsic apoptosis dysregulation, comprising administering to a subject suffering from a disease involving apoptosis dysregulation an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the targeting moiety of the compounds and compositions disclosed herein binds to a cell surface receptor on a cell with intrinsic apoptosis dysregulation. In some embodiments, a method of treating cancer comprises administering to a subject suffering from cancer a compound and composition disclosed herein in an amount effective to provide a therapeutic benefit, wherein the targeting moiety is capable of binding to a cell surface receptor or tumor associated antigen expressed on the surface of a cancer cell.

[0248] In the case of tumorigenic cancers, therapeutic benefit may specifically include, in addition to the effects described above, halting or slowing the progression of tumor growth, causing regression of tumor growth, eradication of one or more tumors, and / or increasing patient survival, as compared to the statistical average for the type and stage of cancer being treated. In some embodiments, the cancer being treated is a tumorigenic cancer.

[0249] The compounds and conjugates disclosed herein can be administered as a monotherapy to provide therapeutic benefit, or can be used as an adjuvant to other chemotherapeutic agents and / or radiotherapy, or administered in combination with them. The chemotherapeutic agents for which the compounds and compositions disclosed herein can be used as adjuvant therapy can be targeted (e.g., ADC, protein kinase inhibitors, etc.) or non-targeted (e.g., non-specific cytotoxic agents such as radionucleotides, alkylating agents, and intercalating agents). Non-targeted chemotherapeutic agents with which the compounds and compositions disclosed herein can be adjunctively administered include, but are not limited to, methotrexate, paclitaxel, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, mechlorethamine, cyclophosphamide, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

[0250] The compounds and conjugates disclosed herein, which may not be effective in treating cancer as a monotherapy, can be used as an adjunct to other chemotherapeutic agents or radiotherapy, or administered together with them to provide therapeutic benefit. In some embodiments, the present disclosure relates to a method in which the compounds or compositions disclosed herein are administered in an amount effective to sensitize tumor cells to standard chemotherapy and / or radiotherapy. Thus, in the case of treating cancer, "therapeutic benefit" includes administering the compounds and compositions disclosed herein as an adjunct to chemotherapeutic agents and / or radiotherapy, or administering them together with them to patients who have not yet started such therapy or have started such therapy but have not yet shown signs of resistance, or patients who have developed signs of resistance, as a means of sensitizing the tumor to chemotherapy and / or radiotherapy.

[0251] Pharmaceutical composition

[0252] In certain embodiments, the present invention provides a solid pharmaceutical composition comprising a compound of the present invention, such as a compound of formula or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0253] In certain embodiments, the present invention provides a pharmaceutical formulation suitable for use in human patients, comprising any compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical formulation can be used to treat or prevent a condition or disease as described herein. In certain embodiments, the pharmaceutical formulation has sufficiently low pyrogenic activity to be suitable for use in human patients.

[0254] In some embodiments, the present invention relates to a pharmaceutical kit comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and, optionally, instructions on how to administer the compound.

[0255] The compositions and methods of the present invention can be used to treat individuals in need. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When applied to an animal, such as a human, the composition or compound is preferably administered in the form of a pharmaceutical composition, comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, non-aqueous vehicles, such as glycols, glycerol, oils such as olive oil, or injectable organic esters. Excipients can be selected, for example, to achieve delayed release of the agent or selectively target one or more cells, tissues or organs. Pharmaceutical compositions can be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, lyophilic colloids for reconstruction, powders, suppositories, etc. The composition can also be present in a transdermal delivery system, such as a skin patch.

[0256] Pharmaceutically acceptable carriers can contain physiologically acceptable agents that act, for example, to stabilize a compound (such as a compound of the present invention), increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) can also be a liposome or other polymer matrix, into which, for example, a compound of the present invention can be incorporated. Liposomes, for example, liposomes comprising phospholipids or other lipids, are relatively simple, non-toxic, physiologically acceptable, and metabolizable carriers to manufacture and administer.

[0257] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0258] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ethanol; and (17) other nontoxic compatible substances used in pharmaceutical formulations.

[0259] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of routes of administration, including, for example, oral routes (e.g., dips in non-aqueous solutions or suspensions for application to the tongue, tablets, capsules (including dispersible capsules and gelatin capsules), boluses, powders, granules, pastes); absorption through the oral mucosa (e.g., sublingually); anal, rectal or vaginal routes (e.g., as vaginal suppositories, creams or foams); parenteral routes (including intramuscular, intravenous, subcutaneous or intrathecal as, for example, sterile solutions or suspensions); nasal routes; intraperitoneal routes; subcutaneous routes; transdermal routes (e.g., as patches applied to the skin); and topical routes (e.g., as creams, ointments or sprays applied to the skin, or as eye drops). The compound can also be formulated for inhalation. Details of appropriate routes of administration and compositions suitable therefor can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein, each of which is incorporated herein by reference in its entirety.

[0260] The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form will vary according to the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. In general, in one hundred parts, this amount will be in the range of about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0261] Methods for preparing these formulations or compositions include the step of bringing into association the active compound (such as a compound of the present invention) with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0262] Formulations of the present invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilizates, powders, granules, or as a solution or suspension in a non-aqueous liquid, or as pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a compound of the present invention as the active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0263] To prepare solid dosage forms for oral administration (capsules (including dispersible capsules and gelatin capsules), tablets, pills, lozenges, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) moisturizers. (4) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including dispersible capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain a buffering agent. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0264] Tablets can be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0265] Other solid dosage forms of tablets and pharmaceutical compositions, such as lozenges, capsules (including dispersible capsules and gelatin capsules), pills and granules can be optionally scored or prepared with coatings and shells, such as enteric coatings or other coatings well known in the art of pharmaceutical formulation. They can also be formulated to provide slow release or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions to provide the desired release characteristics. They can be sterilized by, for example, filtering through a filter that retains bacteria or by immediately incorporating a sterilizing agent in the form of a sterile solid composition that is soluble in sterile water or some other sterile injection medium before use. These compositions may also optionally contain an opacifier and may be a composition that releases the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, with one or more of the above-mentioned excipients, as appropriate.

[0266] Formulations of pharmaceutical compositions for rectal, vaginal or urethral administration may be presented as suppositories which are solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active compound(s) by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylates.

[0267] Alternatively or additionally, the composition can be formulated for delivery via a catheter, stent, wire or other intraluminal device. Delivery via such devices may be particularly useful for delivery to the bladder, urethra, ureter, rectum or intestine.

[0268] Formulations suitable for vaginal administration also include pessaries, hemostats, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0269] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants that may be required.

[0270] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0271] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane or propane).

[0272] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of this invention to the body. Such dosage forms can also be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0273] Ophthalmic preparations, ophthalmic ointments, powders, solutions, etc. are also considered within the scope of the present invention. Exemplary ophthalmic preparations are described in U.S. Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference in their entirety. If desired, liquid ophthalmic preparations have properties similar to tears, aqueous humor, or vitreous humor, or are compatible with such liquids. The preferred route of administration is topical administration (e.g., topical administration, such as eye drops, or administration by implant).

[0274] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0275] Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders to be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0276] Examples of suitable non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0277] These compositions also can contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants.Can ensure to prevent the effect of microorganisms by comprising various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid etc..Also may need to comprise isotonic agent, such as sugar, sodium chloride etc. in the composition.In addition, can realize the delayed absorption of injectable drug form by comprising the agent (such as aluminum monostearate and gelatin) that delays absorption.

[0278] In some cases, in order to prolong the effect of the drug, it is necessary to slow down the absorption of the drug for subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the delayed absorption of the drug form for parenteral administration can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0279] Injectable reservoir forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). Depending on the ratio of drug to polymer and the properties of the specific polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions compatible with body tissues.

[0280] For use in the methods of the present invention, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) active ingredient in combination with a pharmaceutically acceptable carrier.

[0281] The introduction method can also be provided by refillable or biodegradable devices. In recent years, various slow-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs (including protein biopharmaceuticals). A variety of biocompatible polymers, including hydrogels, including biodegradable and non-degradable polymers, can be used to form implants to slowly release compounds at specific target sites.

[0282] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0283] The selected dosage level will depend upon a variety of factors, including the activity of the specific compound or combination of compounds employed, or their esters, salts or amides, the route of administration, the time of administration, the rate of excretion of the specific compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0284] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" means a concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary depending on the subject's weight, sex, age, and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the condition being treated, the stability of the compound, and, if necessary, another type of therapeutic agent administered with the compound of the present invention. A larger total dose can be delivered by multiple administrations. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th edition, 1814-1882, incorporated herein by reference).

[0285] In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0286] If desired, the effective daily dose of the active compound may optionally be administered in unit dosage form as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day. In certain embodiments of the invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

[0287] The patient receiving such treatment is any animal in need thereof, including primates, particularly humans, and other mammals such as horses, cattle, pigs, and sheep; and poultry and pets in general.

[0288] In certain embodiments, the compounds of the present invention can be used alone or in combination with another type of therapeutic agent. As used herein, the phrase "combined administration" refers to the administration of two or more different therapeutic compounds in any form, so that the second compound is administered when the previously administered therapeutic compound is still effective in vivo (for example, two compounds are effective in the patient at the same time, which may include a synergistic enhancement effect of the two compounds). For example, different therapeutic compounds can be administered simultaneously or sequentially in the same preparation or in a separate preparation. In certain embodiments, different therapeutic compounds can be administered at intervals of 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or within a week. Therefore, the individual receiving this treatment can benefit from the combined effects of different therapeutic compounds.

[0289] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, exfoliating agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0290] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0291] Example

[0292] Having generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0293] Example 1. Preparation of Compound L-1

[0294]

[0295] Preparation of compound L-1-1

[0296] A homogeneous solution of triethylene glycol (60.6 g, 403.5 mmol) in dry THF was treated with 60%-NaH (3.2 g, 80.7 mmol) at room temperature under N2 atmosphere and stirred for 15 minutes. Propargyl bromide (10 g, 67.25 mmol) was added dropwise and the resulting mixture was allowed to stand overnight. The reaction was quenched with H2O (250 mL) and extracted with DCM (250 mL × 4). The organic layer was washed with brine (500 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 3: 1) to obtain the title compound L-1-1 (12.5 g, 98%) as a liquid.

[0297] 1 H NMR (400MHz, CDCl3) δ4.18 (d, J = 2.4Hz, 2H), 3.73-3.64 (m, 10H), 3.60-3.57 (m, 2H), 2.63 (m, 1H), 2.42 (m, 1H).

[0298] Preparation of compound L-1

[0299] A homogeneous solution of L-1-1 (1.2 g, 6.22 mmol) in dry DCM was treated with TPP (2.45 g, 9.34 mmol) at 0 ° C under N2 atmosphere and stirred at 0 ° C for 5 minutes. NBS (1.66 g, 9.34 mmol) was added and the mixture was stirred at 0 ° C for 20 minutes and warmed to room temperature for 1 hour. The reaction was quenched with H2O (70 mL) and extracted with DCM (80 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 100: 1 to 200: 1) to obtain the title compound L-1 (1.3 g, 80%, purity 70%) as a liquid.

[0300] EI-MS m / z:251(M + +1).

[0301] Example 2. Preparation of Compound L-2

[0302]

[0303] Preparation of compound L-2-1

[0304] A homogeneous solution of L-1-1 (2.88 g, 15.3 mmol) in dry DCM was treated with p-TsCl (2.92 g, 15.3 mmol) and KOH (3.43 g, 61.2 mmol) at 0°C under N2 atmosphere and allowed to warm to room temperature for 3.5 hours. The reaction was quenched with H2O (50 mL) and extracted with DCM (80 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the title compound L-2-1 (crude) as a liquid.

[0305] 1 H NMR (400MHz, CDCl3) δ7.79 (d, J = 7.2Hz, 2H), 7.33 (d, J = 7.6Hz, 2H), 4.20-4.12 (m, 4H), 3.68-3.58 (m, 10H), 2.44 (s, 3H), 2.42 (m, 1H). EI-MS m / z:343(M + +1).

[0306] Preparation of compound L-2-2

[0307] A homogeneous solution of L-2-1 (5.24 g, 15.3 mmol) in anhydrous DMF was treated with NaN (1.49 g, 22.95 mmol) at room temperature under N atmosphere and heated to 60 ° C overnight. The reaction was quenched with H O (100 mL) and extracted with EA (120 mL × 2). The organic layer was washed with brine (150 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 1: 3) to obtain the title compound L-2-2 (2.39 g, 73% yield in 2 steps) as a liquid.

[0308] 1 H NMR (400MHz, CDCl3) δ4.19 (d, J = 2Hz, 2H), 3.69-3.56 (m, 10H), 3.38 (m, 2H), 2.42 (m, 1H). EI-MS m / z:236(M + +Na).

[0309] Preparation of compound L-2

[0310] A clear solution of L-2-2 (2.39 g, 11.2 mmol) in EA, diethyl ether and 5% HCl was treated with TPP (2.94 g, 11.2 mmol) at 0 ° C. under N2 atmosphere and slowly warmed to room temperature overnight. The reaction mixture was washed with diethyl ether (50 mL×2) and the H2O layer was concentrated in vacuo. The liquid was dried under high vacuum to give the title compound L-2 (2.13 g, 85%) as a colorless oil.

[0311] 1 H NMR (400MHz, DMSO-d6) δ4.14 (d, J = 1.6Hz, 2H), 3.62-3.50 (m, 10H), 2.97-2.93 (m, 2H), 2.50 (m, 1H).

[0312] Example 3. Preparation of Compounds L-3 and L-4

[0313]

[0314] Preparation of compound L-3-1

[0315] A homogeneous solution of tetraethylene glycol (10 g, 51.49 mmol) in dry DCM was treated with KOH (23.1 g, 411.88 mmol) and p-TsCl (19.6 g, 102.97 mmol) at 0°C under N2 atmosphere and allowed to warm to room temperature for 2.5 hours. The reaction was diluted with H2O (200 mL) and extracted with DCM (250 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the title compound as a liquid: L-3-1 (crude).

[0316] 1 EI-MS m / z:503(M + +1).

[0317] Preparation of compound L-3-2

[0318] A homogeneous solution of L-3-1 (25.9 g, 51.49 mmol) in anhydrous DMF was treated with NaN (10 g, 154.46 mmol) at room temperature under N atmosphere and heated to 60 ° C overnight. The reaction was quenched with H O (250 mL) and extracted with EA (250 mL × 3). The organic layer was washed with brine (350 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 1: 3) to obtain the title compound in liquid form: L-3-2 (10.64 g, 85% yield in 2 steps).

[0319] 1H NMR (400MHz, CDCl3) δ3.70-3.66(m,12H),3.41-3.37(m,4H); EI-MS m / z:267(M + +Na).

[0320] Preparation of compound L-3

[0321] A clear solution of L-3-2 (10.64 g, 43.56 mmol) in EA, diethyl ether and 5% HCl was treated with TPP (11.4 g, 43.56 mmol) at 0 ° C under N2 atmosphere and slowly warmed to room temperature overnight. The organic layer was concentrated in vacuo. The residual H2O phase was washed with DCM (150 mL×2), and the H2O layer was concentrated in vacuo. The liquid was dried under high vacuum to give the title compound as a colorless oil: L-3 (10.78 g, 78%).

[0322] EI-MS m / z:219(M + +1).

[0323] Compound L-4 was synthesized by a similar synthetic method to that of compound L-3.

[0324] Preparation of compound L-4-1

[0325] EI-MS m / z:591(M + +1).

[0326] Preparation of compound L-4-2

[0327] Yield 79%, colorless oil.

[0328] 1 H NMR (600MHz, CDCl3) δ3.69-3.66 (m, 20H), 3.39 (t, J = 4.8Hz, 4H); EI-MS m / z: 355 (M + +Na).

[0329] Preparation of compound L-4

[0330] Yield 91%, colorless oil.

[0331] 1 H NMR (600MHz, CDCl3) δ8.15 (br s, 2H), 3.93 (t, J = 4.2Hz, 2H), 3.82-3.66 (m, 18H), 3.49-3.46 (m, 2H), 3.21-3.19 (m, 2H). EI-MS m / z:307(M + +1).

[0332] Example 4. Preparation of Compound L-5

[0333]

[0334] Preparation of compound L-5-1

[0335] A homogeneous solution of tetraethylene glycol (20 g, 102.97 mmol) in dry THF was treated with t-BuOK (54.57 mL, 54.57 mmol) at room temperature under N2 atmosphere and stirred to room temperature for 30 minutes. Propargyl bromide (6.08 mL, 54.57 mmol) was added dropwise and the resulting mixture was allowed to stand for 15 hours. The reaction mixture was washed with water and purified by HPLC. Filter and wash with EA (100 mL x 2) The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (EA:HEX=5:1) to give the title compound as a liquid: L-5-1 (10.98 g, 46%).

[0336] 1 H NMR (400MHz, CDCl3) δ4.20 (d, J = 1.6Hz, 2H), 3.72-3.59 (m, 16H), 2.56 (m, 1H), 2.43 (m, 1H).

[0337] Preparation of compound L-5-2

[0338] A homogeneous solution of L-5-1 (10.98 g, 47.27 mmol) in dry DCM was treated with TEA (17.13 mL, 122.90 mmol), p-TsCl (18.02 g, 94.54 mmol) at room temperature under N2 atmosphere and stirred to room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was purified by flash chromatography (EA:HEX=1:3 to 1:1) to give the title compound as a liquid: L-5-2 (17.06 g, 93%).

[0339] 1 H NMR (400MHz, CDCl3) δ7.79-7.77(m,2H),7.33-7.30(m,2H),4.20-4.10(m,4H),3.71-3.55(m,14H),2.43-2.40(m,4H).

[0340] Preparation of compound L-5-3

[0341] A homogeneous solution of L-5-2 (8.22 g, 21.27 mmol) in anhydrous DMF was treated with NaN (2.07 g, 31.90 mmol) at room temperature under N atmosphere and heated to 60 ° C overnight. The reaction was quenched with H O (200 mL) and extracted with EA (250 mL × 3). The organic layer was washed with brine (350 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 1: 2 to 1: 1) to obtain the title compound in a liquid form: L-5-3 (2.94 g, 54%).

[0342] 1 H NMR (400MHz, CDCl3) δ4.20 (d, J = 2.4Hz, 2H), 3.72-3.60 (m, 14H), 3.40-3.37 (m, 2H), 2.42 (m, 1H).

[0343] Preparation of compound L-5

[0344] A clear solution of L-5-3 (2.94 g, 11.41 mmol) in EA, diethyl ether and 5% HCl was treated with TPP (2.99 g, 11.41 mmol) at 0 ° C under N2 atmosphere and slowly warmed to room temperature overnight. The organic layer was concentrated in vacuo. The residual H2O phase was washed with DCM (200 mL×2), and the H2O layer was concentrated in vacuo. The liquid was dried under high vacuum to give the title compound as a colorless oil: L-5 (2.55 g, 83%).

[0345] EI-MS m / z:232(M + +1).

[0346] Example 5. Preparation of Compound L-6

[0347]

[0348] Preparation of compound L-6-1

[0349] A homogeneous solution of hexaethylene glycol (7.8 g, 27.63 mmol) in dry THF was treated with t-BuOK (1.64 g, 14.64 mmol) at room temperature under N2 atmosphere and stirred to room temperature for 30 minutes. Propargyl bromide (1.63 mL, 14.64 mmol) was added dropwise and the resulting mixture was allowed to stand overnight. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: MeOH = 97: 3) to give the title compound L-6-1 (4,57 g, 52%) as a liquid.

[0350] 1H NMR (400MHz, CDCl3) δ4.20 (d, J = 2Hz, 2H), 3.72-3.59 (m, 24H), 2.65 (m, 1H), 2.42 (m, 1H).

[0351] Preparation of compound L-6-2

[0352] A homogeneous solution of hexaethylene glycol (5.6 g, 19.83 mmol) in dry DCM was treated with AgO (5.52 g, 23.80 mmol), KI (329 mg, 1.98 mmol), p-TsCl (4.16 g, 21.82 mmol) at 0 ° C under N2 atmosphere and stirred to room temperature for 3 hours. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: MeOH = 95: 5 to 90: 10) to give the title compound L-6-2 (7.71 g, 89%) as a liquid.

[0353] 1 H NMR (400MHz, CDCl3) δ7.79 (d, J = 6.8 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 4.16-4.14 (m, 2H), 3.72-3.57 (m, 22H), 2.65-2.63 (m, 1H), 2.44 (s, 3H).

[0354] Preparation of compound L-6-3

[0355] A homogeneous solution of L-6-2 (7.71 g, 17.66 mmol) in anhydrous DMF was treated with NaN (1.72 g, 26.48 mmol) at room temperature under an N atmosphere and heated to 110 ° C for 3.5 hours. The reaction mixture was concentrated in vacuo and DMF was removed under high vacuum. The residue was purified by flash chromatography (EA: MeOH = 10: 1) to give the title compound as a liquid: L-6-3 (4.74 g, 87%).

[0356] 1 H NMR (400MHz, CDCl3) δ3.74-3.58(m,22H), 3.41-3.36(m,2H), 2.67-2.62(m,1H).

[0357] Preparation of compound L-6-4

[0358] A homogeneous solution of L-6-3 (4.74 g, 15.42 mmol) in dry DCM was treated with TEA (5.59 mL, 40.10 mmol) and p-TsCl (5.88 g, 30.84 mmol) at room temperature under N2 atmosphere and stirred to room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was purified by flash chromatography (EA:HEX=5:1) to give the title compound as a liquid: L-6-4 (6.52 g, 92%).

[0359] 1 H NMR (400MHz, CDCl3) δ7.79 (d, J = 8Hz, 2H), 7.34 (d, J = 8Hz, 2H), 4.15 (t, J = 5.2Hz, 2H), 3.69-3.57 (m, 20H), 3.38 (d, J = 5.2Hz, 2H), 2.44 (s, 3H).

[0360] Preparation of compound L-6-5

[0361] A homogeneous solution of L-6-1 (4.53 mg, 14.14 mmol) in dry THF was treated with 60% NaH (678 mg, 16.96 mmol) at 0 ° C under N2 atmosphere and allowed to stand for 30 minutes. L-6-4 (6.52 g, 14.14 mmol) was added, and the resulting mixture was warmed to room temperature for 7 hours. 60% NaH (678 mg, 16.96 mmol) was added and allowed to stand overnight. 60% NaH (282.7 mg, 7.07 mmol) was added and heated to 40 ° C overnight. The reaction was cooled at 0 ° C, quenched with MeOH (100 mL) and concentrated in vacuo. The residue was purified by flash chromatography (EA: MeOH = 90: 10) to obtain the title compound in liquid: L-6-5 (7.341 g, 85%).

[0362] 1 H NMR (400MHz, CDCl3) δ4.19 (m, 2H), 4.70-4.55 (m, 46H), 0.38 (m, 2H), 2.43 (m, 1H).

[0363] EI-MS m / z:610(M + +1).

[0364] Preparation of compound L-6

[0365] A clear solution of L-6-5 (906.7 mg, 1.49 mmol) in EA, diethyl ether and 5% HCl was treated with TPP (390 mg, 1.49 mmol) at 0 ° C under N2 atmosphere and slowly warmed to room temperature overnight. The organic layer was concentrated in vacuo. The residual H2O phase was washed with DCM (60 mL×3), and the H2O layer was concentrated in vacuo. The liquid was dried under high vacuum to give the title compound as a colorless oil: L-6 (495 mg, 54%).

[0366] EI-MS m / z:584(M + +1).

[0367] Example 6. Preparation of Compound L-7

[0368]

[0369] Preparation of compound L-7-1

[0370] A homogeneous solution of tetraethylene glycol (10 g, 51.48 mmol) in dry THF was treated with NaOH (3 g, 77.22 mmol) and p-TsCl (9.8 g, 51.48 mmol) at 0 ° C under N2 atmosphere and allowed to stand for 30 minutes. The reaction mixture was warmed to room temperature for 3 hours. The reaction was diluted with H2O (50 mL) and extracted with EA (50 mL×3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA:HEX=1:1 to 5:1) to obtain the title compound: L-7-1 (3.15 g, 18%) in a liquid form.

[0371] 1 H NMR(600Hz,DMSO-d6)δ7.79(d,J=8Hz,2H),7.48(d,J=8.4Hz,2H),4.57(t,J=5.6Hz,1H),4.1 2-4.09(m,2H),3.58-3.56(m,2H),3.51-3.44(m,10H),3.42-3.38(m,2H),2.42(s,3H); EI-MS m / z:349(M + +1).

[0372] Preparation of compound L-7-2

[0373] A homogeneous solution of L-7-1 (3.15 g, 9.04 mmol) in anhydrous DMF was treated with NaN (3.53 g, 54.24 mmol) at room temperature under N atmosphere and heated to 90 ° C overnight. The reaction was quenched with H O (30 mL) and extracted with EA (100 mL × 3). The organic layer was washed with brine (200 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) to give the title compound as a liquid: L-7-2 (1.8 g, 91%).

[0374] EI-MS m / z:220(M + +1).

[0375] Example 7. Preparation of compound BCN-PNP

[0376]

[0377] (1R, 8S, 9s)-bicyclo [6.1.0] nonan-4-yn-9-ylmethanol (800 mg, 5.3 mmol) was dissolved in DCM (125 mL) at room temperature under N2 atmosphere. Pyridine (1.22 mL, 15.9 mmol) and 4-nitrobenzene chloroformate (1.75 g, 8.74 mmol) were added thereto. After the mixture was stirred at the same temperature for 4 hours, the reaction was quenched by adding saturated NH4Cl solution (100 mL) and extracted with EA (100 mLx4). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (Hex:EA=10:1) to obtain BCN-PNP (1.34 g, 84%) as a white solid.

[0378] 1 H NMR (600MHz, CDCl3) δ8.29(d,J=9Hz,2H),7.39(d,J=9Hz,2H),4.41(d,J=8.4Hz,2H ),2.36-2.24(m,6H),1.62-1.55(m,2H),1.53-1.49(m,1H),1.07(t,J=10.2Hz,2H).

[0379] Example 8. Preparation of Compound L-8

[0380]

[0381] Preparation of compound L-8-1

[0382] A homogeneous solution of L-4 (740 mg, 2.16 mmol) and di-tert-butyl dicarbonate (707 mg, 3.24 mmol) in 1,4-dioxane: H2O=1:1 (10 mL) was treated with NaHCO3 (363 mg, 4.32 mmol) at room temperature under N2 atmosphere and stirred to room temperature for 2 hours. The reaction was quenched with water (10 mL) and extracted with EA (10 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Compound L-8-1 (809 mg, 96%) was used directly in the next reaction without purification.

[0383] EI-MS m / z:407(M + ).

[0384] Preparation of compound L-8

[0385] A homogeneous solution of L-8-1 (809 mg, 1.99 mmol) in anhydrous MeOH (15 mL) was treated with Pd / C (90 mg, 10 wt%) at room temperature under H2 atmosphere and stirred for 4 hours. The reaction mixture was washed with water by Filter and concentrate in vacuo.Compound L-8 (829 mg, quantitative) was used directly in the next reaction without purification.

[0386] EI-MS m / z:381(M + ).

[0387] Example 9. Preparation of Compound AMA-1

[0388]

[0389] Preparation of compound AMA-1a

[0390] A turbid mixture of 4-hydroxyacetophenone (5 g, 52.32 mmol) and glyoxylic acid monohydrate (7.4 g, 54.32 mmol) in AcOH was heated to reflux overnight at room temperature under an N atmosphere. The reaction was quenched with H O (150 mL) and extracted with EA (200 mL x 3), dried over anhydrous Na SO , filtered, and concentrated in vacuo. The residue was purified by flash chromatography (EA:HEX=3:1 to EA:MeOH=97:3) to give the title compound AMA-1a (1.17 g, 11%, mixture 6.61 g) as a brown solid.

[0391] 1H NMR (400MHz, DMSO-d6) δ7.94 (d, J = 8Hz, 2H), 7.86 (d, J = 15.6Hz, 1H), 6.90 (d, J = 8.8Hz, 2H), 6.63 (d, J = 15.6Hz, 1H).

[0392] Preparation of compound AMA-1b

[0393] A homogeneous solution of AMA-1a (457.9 mg, 2.38 mmol) in anhydrous DMF was treated with DMAP (43.7 mg, 0.36 mmol), EDCI (456.8 mg, 2.38 mmol), DIPEA (2.08 mL, 11.91 mmol) and NHCl (1.25 g, 23.83 mmol) at 0 ° C. under N atmosphere and slowly warmed to room temperature overnight. The reaction was quenched with saturated citric acid (35 mL), saturated NaHCO (35 mL) and brine (30 mL) and washed with EA (50 mL), then dried over anhydrous NaSO, filtered and concentrated in vacuo. The residue was purified by flash chromatography (100% DCM to EA:MeOH=97:3) to give the title compound AMA-1b (24.4 mg, 5%) as a brown solid.

[0394] 1 H NMR (400MHz, DMSO-d6) δ7.94-7.90 (m, 3H), 7.75 (d, J = 15.2Hz, 1H), 7.48 (br s, 1H), 6.88 (d, J = 15.2Hz, 1H), 6.85 (d, J = 8.4Hz, 2H).

[0395] Preparation of compound AMA-1

[0396] A homogeneous solution of AMA-1b (12 mg, 0.063 mmol) and L-1 (31 mg, 0.126 mmol) in anhydrous DMF was treated with KCO (8.7 mg, 0.063 mmol) at 0 ° C under N atmosphere and heated to 40 ° C overnight. The reaction was quenched with H O (20 mL) and extracted with EA (25 mL). The organic layer was washed with brine (20 mL), dried over anhydrous Na SO, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 18: 1) to give the title compound AMA-1 (10 mg, 44%) as a light yellow gum.

[0397] 1H NMR (400MHz, CDCl3) δ8.05-7.95(m,3H),7.02-6.94(m,3H),5.73(br s,1H),5.60(br s,1H),4.24-4.19(m,4H),3.92-3.88(m,2H),3.77-3.67(m,8H),2.43-2.42(m,1H). EI-MS m / z:362(M + +1).

[0398] Example 10. Preparation of Compounds AMA-2 and AMA-3

[0399]

[0400] Preparation of compound AMA-2a

[0401] A homogeneous solution of 4-acetylbenzoic acid (306 mg, 1.86 mmol) and L-2 (500 mg, 2.24 mmol) in anhydrous DMF was treated with EDCI (428 mg, 2.24 mmol), DIPEA (0.81 mL, 4.66 mmol), HOBt (342 mg, 2.24 mmol) at room temperature under N2 atmosphere and stirred to room temperature overnight. The reaction was quenched with saturated citric acid (35 mL). The mixture was then extracted with saturated NaHCO3 (35 mL), EA (40 mL) and brine (30 mL) and dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) to give the title compound AMA-2a (105 mg, 17%) as a light yellow gum.

[0402] 1 H NMR (400MHz, CDCl3) δ8.01(d,J=8.4Hz,2H),7.90(d,J=8.8Hz,2H),6.90(brs,1H),4.13(m,2H),3.68(m,12H),2.64(s,3H),2.42(m,1H). EI-MS m / z:334(M + +1).

[0403] Preparation of compound AMA-2b

[0404] A uniform solution of AMA-2a (105 mg, 0.31 mmol) and glyoxylic acid monohydrate (58 mg, 0.63 mmol) in AcOH was heated to reflux for 6.5 hours at room temperature under N2 atmosphere. A solution of another glyoxylic acid monohydrate (58 mg, 0.63 mmol) in AcOH was added, and the resulting mixture was allowed to stand overnight. A third portion of glyoxylic acid monohydrate (58 mg, 0.63 mmol) in AcOH and the resulting mixture were added and stirred for 6 hours. The mixture was concentrated in a vacuum. The residue was purified by flash chromatography (DCM: MeOH = 15: 1 to 9: 1 to 7: 1) to obtain the title compound as a light yellow jelly: AMA-2b (39 mg, 32% yield, 35 mg starting material recovered).

[0405] EI-MS m / z:390(M + +1).

[0406] Preparation of compound AMA-2

[0407] A homogeneous solution of AMA-2b (14 mg, 0.036 mmol) in dry THF was treated with NMM (5 μL, 0.043 mmol) at -15 ° C under N atmosphere and i-BuCO Cl (7 μL, 0.054 mmol) was added dropwise and allowed to stand for 40 minutes. 0.5 M ammonia in THF (1 mL) was added and stirred for 40 minutes. The reaction was quenched with H O (10 mL) and extracted with EA (15 mL). The organic layer was washed with brine (10 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 9: 1) to give the title compound AMA-2 (11 mg, 79%) as a light yellow gum.

[0408] EI-MS m / z:389(M + +1).

[0409] Compound AMA-3 was synthesized by a similar synthetic method to that of compound AMA-2.

[0410] Preparation of compound AMA-3a

[0411] Yield 40%, light yellow gum.

[0412] 1H NMR (400MHz, CDCl3) δ8.38(s,1H),8.09(d,J=7.6Hz,1H),8.03(d,J=8.4Hz,1H),7.57-7.53(m,1H),6.95(br s,1H),4.12(m,2H),3.70-3.68(m,12H),2.66(s,3H),2.40(m,1H). EI-MS m / z:334(M + +1).

[0413] Preparation of compound AMA-3b

[0414] Yield 65%, light yellow gum.

[0415] EI-MS m / z:390(M + +1).

[0416] Preparation of compound AMA-3

[0417] Yield: 36%, pale yellow gum.

[0418] 1 H NMR (400MHz, CDCl3) δ8.47(s,1H),8.14(d,J=7.6Hz,2H),8.02(d,J=15.2Hz,1H),7.62-7.58(m,1H),7.40(br s,1H),7.00(d,J=14.8Hz,1H),6.10(br s,1H),5.91(brs,1H),4.11(m,2H),3.72-3.67(m,12H),2.41(m,1H). EI-MS m / z:389(M + +1).

[0419] Example 11. Preparation of compound AMA-4

[0420]

[0421] Preparation of compound AMA-4a

[0422] A solution of methyl 5-bromonicotinate (3 g, 13.89 mmol), PdCl2(PPh3)2 (487 mg, 0.96 mmol) and tributyl(1-ethoxyvinyl)tin (5.86 mL, 17.36 mmol) in anhydrous toluene was heated to reflux at room temperature under N2 atmosphere for 3 hours. The mixture was washed with water by The product was filtered and washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (30 mL) and 10M HCl (30 mL) was added at room temperature under N2 atmosphere. The resulting mixture was allowed to stand for 2 hours. The reaction was quenched with saturated Na2CO3 (120 mL) and extracted with EA (150 mL × 3). The organic layer was washed with brine (250 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (EA: HEX = 1: 2) to obtain compound AMA-4a (2.22 g, 89%) as a white solid.

[0423] 1 H NMR (400MHz, CDCl3) δ9.37(s,1H),9.31(s,1H),8.79(s,1H),4.00(s,3H),2.69(s,3H).

[0424] Preparation of compound AMA-4b

[0425] A homogeneous solution of AMA-4a (636 mg, 3.55 mmol) in MeOH was treated with 1N NaOH (10.64 mL) at room temperature under N atmosphere and stirred for 1 hour. After the mixture was concentrated under reduced pressure, the reaction was quenched with 1N HCl (pH 2) and extracted with EA (80 mL x 3). The organic layer was washed with brine (150 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give the title compound AMA-4b as a white solid. Compound AMA-4b was used directly in the next reaction without purification.

[0426] 1 H NMR (400MHz, DMSO-d6) δ9.31(s,1H),9.25(s,1H),8.64(s,1H),2.69(s,3H).

[0427] Preparation of compound AMA-4c

[0428] A homogeneous solution of AMA-4b (606 mg, 3.67 mmol) and L-2 (985 g, 4.40 mmol) in anhydrous DMF was treated with EDCI (1.06 g, 5.50 mmol), DIPEA (1.92 mL, 11.01 mmol), HOBt (843 mg, 5.50 mmol) at room temperature under N2 atmosphere and stirred to room temperature overnight. The reaction was extracted and washed with EA (100 mL x 2) and brine (80 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) to give the title compound AMA-4c (880 mg, 74% yield over 2 steps) as a light yellow gum.

[0429] 1 H NMR (400MHz, CDCl3) δ9.22(s,1H),9.19(s,1H),8.61(s,1H),4.08(m,2H),3.67-3.60(m,12H),2.66(s,3H),2.38(m,1H). EI-MS m / z:335(M + +1).

[0430] Preparation of compound AMA-4d

[0431] A solution of di-tert-butyl tartrate (500 mg, 1.91 mmol) in MeOH was treated with a solution of NaIO (489 mg, 2.29 mmol) in H2O at 0°C under an N2 atmosphere and allowed to stand for 1.5 hours. The reaction was quenched with H2O (40 mL) and extracted with ether (45 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the title compound AMA-4d as a colorless oil. Compound AMA-4d was used directly in the next reaction without purification.

[0432] Preparation of compound AMA-4e

[0433] A homogeneous solution of AMA-4c (48 mg, 0.14 mmol) and AMA-4d (56 mg, 0.43 mmol) in AcOH was heated to reflux overnight at room temperature under an N atmosphere. The mixture was concentrated in vacuo. The residue was purified by flash chromatography (DCM:MeOH = 15:1 to 9:1 to 7:1) to give the title compound AMA-4e (27 mg, 48%) as a light yellow gum.

[0434] EI-MS m / z:391(M + +1).

[0435] Preparation of compound AMA-4

[0436] A homogeneous solution of AMA-4e (27 mg, 0.069 mmol) in dry THF was treated with NMM (9.1 uL, 0.083 mmol) at -15 ° C under N atmosphere and i-BuCO Cl (13.5 μL, 0.104 mmol) was added dropwise and stirred for 40 minutes. 0.5 M ammonia in THF (2 mL) was added and the resulting mixture was stirred for 30 minutes. The reaction was quenched with H O (30 mL) and extracted with EA (35 mL). The organic layer was washed with brine (30 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) to give the title compound AMA-4 (1.2 mg, 4%) as a light yellow gum.

[0437] EI-MS m / z:390(M + +1).

[0438] Example 12. Preparation of compound pyrMPS-1

[0439]

[0440] Preparation of compound pyrMPS-1a

[0441] A turbid mixture of AMA-4c (2.67 g, 16.19 mmol), piperidine HCl (1.97 g, 16.19 mmol) and paraformaldehyde (1.46 g, 48.57 mmol) in EtOH (25 mL) was treated with concentrated HCl (178 μL) at room temperature under N2 atmosphere and heated to reflux overnight. After the reaction was cooled to room temperature, the mixture was quenched with acetone (25 mL) and cooled to 0 ° C and stirred for 1 hour. The solid was collected by filtration, washed with ether (50 mL × 2) and dried under high vacuum to obtain the title compound pyrMPS-1a (1.45 g, 30%, mixture 3.21 g) as a white solid.

[0442] EI-MS m / z:264(M + +1).

[0443] Preparation of compound pyrMPS-1b

[0444] A turbid mixture of pyrMPS-1a (1.45 g, 4.85 mmol) and 4-toluene thiophenol (603 mg, 4.85 mmol) in EtOH (15 mL) and MeOH (10 mL) was treated with piperidine (72 μL, 0.73 mmol) at room temperature under N2 atmosphere and heated to reflux overnight. The reaction was cooled to 0 ° C for 1 hour. The solid was collected by filtration, washed with ether (50 mL × 2) and dried under high vacuum to obtain the title compound Int-5-3 (154 mg, 11%, mixture 1.31 g) as a white solid.

[0445] 1 H NMR(400Hz,DMSO-d6)δ9.26(d,J=2Hz,1H),9.23(d,J=2Hz,1H),8.58(m,1H),7.26(d,J=8 Hz, 2H), 7.13 (d, J = 8.4Hz, 2H), 3.46 (t, J = 7.2Hz, 2H), 3.24 (d, J = 7.2Hz, 2H), 2.26 (s, 3H). EI-MS m / z:302(M + +1).

[0446] Preparation of compound pyrMPS-1c

[0447] A turbid mixture of pyrMPS-1b (154 mg, 0.51 mmol) in MeOH (8 mL) and H2O (8 mL) was treated with potassium monopersulfate (691 mg, 1.12 mmol) at 0°C under N2 and allowed to warm to room temperature for 6.5 hours. The reaction was quenched with H2O (30 mL) and extracted with CHCl3 (50 mL x 4). The organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to afford the title compound pyrMPS-1c (74 g, 43%) as a white solid.

[0448] EI-MS m / z:334(M + +1).

[0449] Preparation of compound pyrMPS-1

[0450] A homogeneous solution of pyrMPS-1c (74 mg, 0.22 mmol) and L-4 (91 mg, 0.27 mmol) in anhydrous DMF (5 mL) was treated with HBTU (106 mg, 0.27 mmol) and DIPEA (77.4 μL, 0.44 mmol) at room temperature under N2 atmosphere and stirred at room temperature for 3 hours. The reaction was quenched with H2O (40 mL) and extracted with DCM (50 mL x 4). The organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC to give the title compound pyrMPS-1 (18 mg, 13%, mixture 43 mg) as a light yellow gum.

[0451] EI-MS m / z:622(M + +1).

[0452] Example 13. Preparation of compound AMA-5

[0453]

[0454] Preparation of compound AMA-5a

[0455] A homogeneous solution of trimethyl-1,3,5-benzenetricarboxylate (34.28 g, 135.91 mmol) in dry THF was treated with 4M LiBH in THF (16.99 mL, 67.95 mmol) at 0 ° C under N2 atmosphere and heated to reflux overnight. After the reaction was cooled to room temperature, the mixture was acidified with 4N HCl (pH 2) and quenched with H2O (800 mL). The mixture was extracted with EA (800 mL × 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 1: 5 to 1: 2) to obtain the title compound AMA-5a (16.75 g, 55%, 6.07 g starting material recovered) as a white solid.

[0456] 1 H NMR (400MHz, CDCl3) δ8.59 (s, 1H), 8.23 ​​(s, 2H), 4.81 (d, J = 6Hz, 2H), 3.95 (s, 6H), 1.97 (t, J = 5.6Hz, 1H).

[0457] Preparation of compound AMA-5b

[0458] A homogeneous solution of AMA-5a (16.75 g, 74.72 mmol) in dry DCM was treated with DCC (80.53 g, 373.58 mmol) at room temperature under N2 atmosphere and heated to reflux overnight. The reaction mixture was purified by flash chromatography (EA:HEX=1:3) to give the title compound AMA-5b (13.84 g, 83%) as a white solid.

[0459] 1 H NMR (400MHz, CDCl3) δ10.13(s,1H),8.92(s,1H),8.72(s,2H),4.00(s,6H).

[0460] Preparation of compound AMA-5c

[0461] A homogeneous solution of AMA-5b (13.84 g, 62.30 mmol) in dry THF was treated with 3M MeMgBr in diethyl ether (20.77 mL, 62.30 mmol) at 0 ° C under N2 atmosphere and stirred for 2.5 hours. After the reaction was quenched with saturated NH4Cl (150 mL), the mixture was extracted with EA (200 mL). The organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: HEX = 1: 3 to 1: 2) to give the title compound AMA-5c (6.6 g, 44%) as a white solid.

[0462] 1 H NMR (400MHz, CDCl3) δ8.57 (s, 1H), 8.24 (s, 2H), 5.05-4.99 (m, 1H), 3.95 (s, 6H), 2.01 (d, J = 3.6Hz, 1H), 1.53 (t, J = 6.8Hz, 3H).

[0463] Preparation of compound AMA-5d

[0464] A homogeneous solution of AMA-5c (6.6 g, 27.70 mmol) in dry DCM was treated with DCC (29.86 g, 138.52 mmol) at room temperature under N2 atmosphere and heated to reflux overnight. The reaction mixture was purified by flash chromatography (EA:HEX=1:5) to give the title compound AMA-5d (4.75 g, 73%, mixture 1.25 g) as a white solid.

[0465] 1 H NMR (400MHz, CDCl3) δ8.87(m,1H),8.78(m,2H),4.00(s,6H),2.70(s,3H).

[0466] Preparation of compound AMA-5e

[0467] A homogeneous solution of AMA-5d (4.75 g, 20.11 mmol) in acetone was treated with a solution of NaOH in MeOH (845 mg, 21.11 mmol) at 0 ° C under N2 atmosphere and warmed to room temperature. After the reaction mixture was stirred overnight, the mixture was concentrated in vacuo and dried under high vacuum. The residue was dissolved in H2O and acidified with 4N HCl (pH 1-2). The H2O phase was extracted with EA (100 mLx4) and dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 20: 1) to obtain the title compound AMA-5e (2.34 g, 52%, mixture 1.72 g) as a light yellow solid.

[0468] 1 H NMR (400MHz, CDCl3) δ8.94(s,1H),8.85(s,1H),8.83(s,1H),4.00(s,3H),2.72(s,3H).

[0469] Preparation of compound AMA-5f

[0470] AMA-5e (200 mg, 0.9 mmol) and L-5 (337 mg, 1.26 mmol) in a homogeneous solution of dry DCM were treated with EDCI (259 mg, 1.35 mmol), TEA (376 uL, 2.7 mmol), HOBt (207 mg, 1.35 mmol) at 0 ° C under N atmosphere and warmed to room temperature. After the reaction mixture was stirred overnight, the reaction was quenched with H o (30 mL) and extracted with DCMC (35 mLx6). The organic layer was washed with brine (150 mL), dried over anhydrous Na sO , filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA=100%) to obtain the title compound AMA-5f (248 mg, 63%) as a light yellow oil.

[0471] 1 H NMR(400MHz, CDCl3)δ8.71(s,1H),8.66(s,1H),8.61(s,1H),7.17(br s,1H),4.13(d,J=2.4Hz,2H),3.98(s,3H),3.71-3.63(m,16H),2.70(s,3H),2.40(m,1H).

[0472] Preparation of compound AMA-5g

[0473] A homogeneous solution of AMA-5f (248 mg, 0.57 mmol) and glyoxylic acid monohydrate (410 mg, 4.56 mmol) in AcOH was heated to reflux at room temperature under N2 atmosphere for 8 hours. Five additional glyoxylic acid monohydrates (210 mg, 2.28 mmol) in AcOH were added to the reaction mixture at intervals of 4 hours. After the mixture was concentrated in vacuo, the residue was purified by flash chromatography (EA=100% to DCM:MeOH=9:1) to obtain the title compound AMA-5g (124 mg, 44%) as a light yellow oil.

[0474] EI-MS m / z:492(M + +1).

[0475] Preparation of compound AMA-5

[0476] A homogeneous solution of AMA-5g (124 mg, 0.25 mmol) in dry THF was treated with NMM (33 μL, 0.30 mmol) at -15 ° C under N2 atmosphere and i-BuCO2Cl (49 μL, 0.38 mmol) was added dropwise and stirred for 40 minutes. After 0.5 M ammonia in THF (1 mL) was added thereto, the mixture was stirred for 30 minutes. The reaction mixture was concentrated in vacuo. The residue was dissolved in DMSO and acidified with acetic acid. The mixture was purified by preparative HPLC to obtain the title compound AMA-6 (28 mg, 23%) as a light yellow jelly.

[0477] 1 H NMR (400MHz, CDCl3) δ8.77(s,1H),8.75(s,1H),8.70(s,1H),8.03(d,J=15.6Hz,1H),7.79(br s,1H),7.03(d,J=15.6Hz,1H),6.40(br s,1H),6.13(br s,1H),4.12-4.09(m,2H),3.96(s,3H),3.75-3.61(m,16H),2.39(m,1H). EI-MS m / z:491(M + +1).

[0478] Example 14. Preparation of Compound AMA-6

[0479]

[0480] A homogeneous solution of phenol (24.9 mg, 0.13 mmol) and L-7 (43.4 mg, 0.15 mmol) in anhydrous DMF was treated with KCO (27 mg, 0.20 mmol) at 0 ° C under N2 atmosphere and warmed to room temperature. After the reaction mixture was stirred for 2.5 hours, a second portion of KCO (9 mg, 0.07 mmol) was added to the reaction mixture, and the mixture was stirred at 40 ° C overnight. The reaction was quenched with H2O (15 mL) and extracted with DCM (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) and preparative HPLC to give the title compound AMA-6 (5.9 mg, 12%) as a light yellow oil.

[0481] EI-MS m / z:393(M + +1).

[0482] Example 15. Preparation of compound AMA-7

[0483]

[0484] Preparation of compound AMA-7a

[0485] A homogeneous solution of 3-acetylbenzoic acid (5 g, 30.46 mmol) and L-3 (7.76 g, 30.46 mmol) in anhydrous DMF was treated with TBTU (19.56 g, 60.92 mmol) and TEA (21.2 mL, 152.3 mmol) at 0 ° C under N2 atmosphere. The reaction was warmed to room temperature and stirred overnight. The mixture was extracted with EA (500 mL), 1N HCl (350 mL) and saturated NaHCO3 (350 mL). The organic layer was washed with brine (350 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was dried under high vacuum to give the title compound AMA-7a (7.77 g, 70%) as a dark brown oil.

[0486] 1 H NMR (400MHz, CDCl3) δ8.38-8.37(m,1H),8.10-8.07(m,1H),8.05-8.02(m,1H),7.57-7.53(m,1H),6.94(br s, 1H), 3.71-3.61 (m, 14H), 3.34 (d, J = 5.2Hz, 2H), 2.66 (s, 3H). EI-MS m / z:365(M + +1).

[0487] Preparation of compound AMA-7b

[0488] A solution of SeO2 (365.4 mg, 3.29 mmol) in 1,4-dioxane and H2O was heated to 50°C at room temperature under N2 atmosphere for 30 minutes. A solution of AMA-7a (300 mg, 0.82 mmol) in 1,4-dioxane and H2O was slowly added thereto. The reaction mixture was refluxed for 4 hours. Additional solution of SeO2 (182.7 mg, 1.65 mmol) in 1,4-dioxane and H2O was added, and the resulting mixture was stirred overnight. After cooling to room temperature, the mixture was passed through The mixture was filtered and concentrated in vacuo. The residue was extracted with DCM (50 mL x 2) and washed with H2O (35 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was dried under high vacuum to give the title compound AMA-7b (crude) as a light yellow oil.

[0489] EI-MS m / z:397(M + +H2O).

[0490] Preparation of compound AMA-7

[0491] A homogeneous solution of AMA-7b (97.6 mg, 0.26 mmol) in dry DCM was treated with (triphenylphosphoranylidene)acetonitrile (77.7 mg, 0.26 mmol) at room temperature under N2 atmosphere and stirred overnight. The reaction was quenched with H2O (20 mL) and extracted with DCM (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA:HEX=2:1 to 3:1 to 5:1 to DCM:MeOH=20:1) and preparative HPLC to give the title compound AMA-7 (9.5 mg, 9%) as a white gum.

[0492] EI-MS m / z:402(M + +1).

[0493] Example 16. Preparation of compound AMA-8

[0494]

[0495] Preparation of compound AMA-8a

[0496] A homogeneous solution of 4-hydroxyacetophenone (87 mg, 0.64 mmol) and L-1 (242 mg, 0.96 mmol) in anhydrous DMF (3 mL) was treated with KCO (177 mg, 1.28 mmol) at room temperature under N2 atmosphere and stirred at room temperature overnight. The reaction was quenched with water (10 mL) and extracted with EA (10 mL × 2). The organic layer was washed with brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: Hex = 1: 2) to obtain the title compound AMA-8a (120 mg, 62%).

[0497] EI-MS m / z:307(M + ).

[0498] Preparation of compound AMA-8b

[0499] A homogeneous solution of AMA-8a (120 mg, 0.39 mmol) in AcOH (4 mL) was treated with glyoxylic acid monohydrate (47 mg, 0.51 mmol) under N2 atmosphere. The mixture was refluxed overnight. Another five portions of glyoxylic acid monohydrate (210 mg, 2.28 mmol) in AcOH were added to the reaction mixture at intervals of 4 hours. After the mixture was concentrated in vacuo, the residue was purified by flash chromatography (DCM: MeOH = 12: 1) to give the title compound AMA-8b (28 mg, 20%) as a light yellow gum.

[0500] EI-MS m / z:363(M + ).

[0501] Preparation of compound AMA-8

[0502] A homogeneous solution of AMA-8b (24 mg, 0.08 mmol) and iodomethane (14 μL, 0.23 mmol) in anhydrous DMF (2 mL) was treated with KCO (21 mg, 0.15 mmol) at room temperature under N2 atmosphere. The reaction mixture was stirred to room temperature for 3 hours. The reaction was quenched by adding water (5 mL) and extracted with EA (5 mL x 2). The organic layer was washed with brine (8 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (EA: Hex = 1: 1) to obtain the title compound AMA-8 (16 mg, 65%).

[0503] 1H-NMR (400MHz, CDCl3) δ8.00(d,J=8.8Hz,2H),7.94(d,J=15.6Hz,1H),7.01(d,J=8.8Hz,2H),6.88(d,J=15.6H EI-MS m / z:377(M + ).

[0504] Example 17. Preparation of Compound AMA-9

[0505]

[0506] Compounds AMA-9a, AMA-9b, and AMA-9c were synthesized in a manner similar to the preparation method of compound AMA-5 of Example 13.

[0507] Preparation of compound AMA-9a

[0508] Yield 77%. 1 H NMR(600MHz, CDCl3)δ8.71(s,1H),8.65(s,1H),8.60(s,1H),7.05(s,1H),4. 12(s,2H),3.99(s,3H),3.72-3.69(m,12H),2.70(s,3H),2.38(s,1H); EI-MS m / z:392(M + ).

[0509] Preparation of compound AMA-9b

[0510] Yield: 27%. EI-MS m / z: 448 (M + ).

[0511] Preparation of compound AMA-9c

[0512] Yield: 27%. EI-MS m / z: 447 (M + ).

[0513] Compound AMA-9 was synthesized in a manner similar to the preparation method of compound AMA-7 of Example 15.

[0514] Yield 8%. EI-MS m / z: 429 (M + ).

[0515] Example 18. Preparation of compound AMA-10

[0516]

[0517] Preparation of compound AMA-10a

[0518] A homogeneous solution of AMA-5d (1.05 g, 4.44 mmol) in anhydrous MeOH (50 mL) was treated with NaOH (356 mg, 8.88 mmol) at 0°C under an N2 atmosphere. The mixture was warmed to room temperature and refluxed overnight. The mixture was quenched by the careful addition of dilute HCl (10 mL) and concentrated under reduced pressure. The aqueous layer was extracted with EA (20 mL), and the resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. After concentration, compound AMA-10a (984 mg, 100%) was used directly in the next reaction without purification.

[0519] EI-MS m / z:209(M + ).

[0520] Compounds AMA-10b, AMA-10c, and AMA-10 were synthesized in a manner similar to the preparation method of compound AMA-5 of Example 13.

[0521] Preparation of compound AMA-10b

[0522] Yield 84%.

[0523] EI-MS m / z:547(M + ).

[0524] Preparation of compound AMA-10c

[0525] Yield 22%.

[0526] EI-MS m / z:603(M + ).

[0527] Preparation of compound AMA-10c

[0528] Yield 32%.

[0529] EI-MS m / z:602(M + ).

[0530] Example 19. Preparation of compounds pyrMPS-2 and PyrMPS-3

[0531]

[0532] Compounds pyrMPS-2 and pyrMPS-3 were synthesized in a manner similar to the preparation method of compound pyrMPS-1 of Example 12.

[0533] Preparation of compound pyrMPS-2

[0534] Yield 22%.

[0535] EI-MS m / z:547(M + ).

[0536] Preparation of compound pyrMPS-3

[0537] Yield 26%.

[0538] EI-MS m / z:900(M + ).

[0539] Example 20. Preparation of compounds mMPS-1 and mMPS-2

[0540]

[0541] Compounds mMPS-1 and mMPS-2 were synthesized in a manner similar to the preparation method of compound pyrMPS-1 in Example 12.

[0542] Preparation of compound mMPS-1a

[0543] Yield 51%, white solid.

[0544] EI-MS m / z:262(M + ).

[0545] Preparation of compound mMPS-1b

[0546] Yield 72%, white solid

[0547] 1 H NMR(600Hz,DMSO-d6)δ8.40(s,1H),8.18-8.15(m,2H),7.66-7.63(m,1H),7.26(d,J=7.8 Hz,2H),7.14(d,J=7.8Hz,2H),3.40-3.37(m,2H),3.26-3.23(m,2H),2.27(s,3H); EI-MS m / z:301(M + +1).

[0548] Preparation of compound mMPS-1c

[0549] Yield 47%, light yellow solid.

[0550] 1H NMR(600Hz,DMSO-d6)δ8.37-8.36(m,1H),8.20-8.16(m,2H),7.81(d,J=7.8Hz,2H),7.68-7 .65(m,1H),7.46(d,J=8.4Hz,2H),3.66-3.63(m,2H),3.44-3.41(m,2H),2.41(s,3H); EI-MS m / z:333(M + +1).

[0551] Preparation of compound mMPS-1

[0552] Yield 60%, white solid.

[0553] EI-MS m / z:546(M + +1).

[0554] Preparation of compound mMPS-2

[0555] Yield 326%, white solid.

[0556] EI-MS m / z:898(M + ).

[0557] Example 21. Preparation of compound mMPS-3

[0558]

[0559] Preparation of compound mMPS-3a

[0560] A homogeneous solution of mMPS-1c (158 mg, 0.48 mmol) and L-8 (297 mg, 0.57 mmol) in anhydrous DMF (3 mL) was treated with DIPEA (0.25 mL, 1.43 mmol) and HBTU (270 mg, 0.71 mmol) at room temperature under an N atmosphere and stirred for 3 hours. The reaction was quenched with water (10 mL) and extracted with EA (15 mL x 2). The organic layer was washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by flash chromatography (DCM:MeOH = 15:1) to give the title compound mMPS-3a (377 mg, quantitative).

[0561] EI-MS m / z:695(M + ).

[0562] Preparation of compound mMPS-3b

[0563] A homogeneous solution of mMPS-3a (100 mg, 0.14 mmol) in dry DCM (3 mL) was treated with 4N HCl in dioxane (360 μL, 1.44 mmol) at 0° C. under N 2 atmosphere and stirred for 2 hours. After the reaction mixture was concentrated in vacuo, mMPS-3b (91 mg, quantitative) was used directly in the next reaction without purification.

[0564] EI-MS m / z:595(M + ).

[0565] Preparation of compound mMPS-3

[0566] A homogeneous solution of mMPS-3b (91 mg, 0.144 mmol) and BCN-PNP (55 mg, 0.173 mmol) in anhydrous DMF (2 mL) was treated with DIPEA (75 μL, 0.433 mmol) at room temperature under N2 atmosphere and stirred at room temperature for 3 hours. The reaction was quenched with water (5 mL) and extracted with EA (8 mL × 2). The organic layer was washed with brine (8 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM:MeOH=15:1) to give the title compound mMPS-3 (44 mg, 38%).

[0567] EI-MS m / z:771(M + ).

[0568] Example 22. Preparation of compound mMPS-4

[0569]

[0570] Compound mMPS-4 was synthesized in a manner similar to the preparation method of compound mMPS-1 in Example 20.

[0571] Preparation of compound mMPS-4a

[0572] Yield 99%.

[0573] EI-MS m / z:511(M + ).

[0574] Preparation of compound mMPS-4b

[0575] Yield 99%.

[0576] EI-MS m / z:608(M + ).

[0577] Preparation of compound mMPS-4c

[0578] Yield 50%.

[0579] EI-MS m / z:647(M + ).

[0580] Preparation of compound mMPS-4

[0581] Yield 8%.

[0582] EI-MS m / z:679(M + ).

[0583] Example 23. Preparation of compounds Mal-1 and Mal-2

[0584]

[0585] A homogeneous solution of N-succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (30 mg, 0.09 mmol) and L-2 (18 mg, 0.096 mmol) in dry DCM was treated with DIPEA (16 μL, 0.09 mmol) at room temperature under N atmosphere and stirred to room temperature for 45 minutes. The reaction was diluted with DCM (15 mL) and washed with 1N HCl (10 mL), brine (10 mL), dried over anhydrous NaSO, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM: MeOH = 15: 1) and preparative HPLC to give the title compound Mal-1 (14.3 mg, 39) as a white gum.

[0586] EI-MS m / z:407(M + +1).

[0587] Compound MaI-2 was synthesized by a similar synthetic route as described above for compound MaI-1.

[0588] Yield 61%, white jelly.

[0589] EI-MS m / z:4519(M + +1).

[0590] Example 24. Preparation of Int-TG

[0591]

[0592] β-D-galactose pentaacetate (5.0 g, 12.81 mmol) was dissolved in 33% HBr in AcOH (20 mL) at 0 ° C under N2 atmosphere. The mixture was warmed to room temperature. After stirring at room temperature for 4 hours, the mixture was concentrated under reduced pressure, and EA (1000 mL) and saturated sodium bicarbonate (1000 mL) were added. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG (5.2 g, 99%).

[0593] 1 H NMR(400Hz, CDCl3) δ6.70(d,J=4.0Hz,1H),5.52(d,J=2.4Hz,1H),5.41(dd,J=7.6,2.8Hz,1H ), 5.05 (dd, J = 6.4, 4.0Hz, 1H), 4.49 (t, J = 6.4Hz, 1H), 4.22-4.09 (m, 2H), 2.16-2.01 (m, 12H).

[0594] Example 25. Preparation of Compounds L-9 and L-10

[0595]

[0596] Preparation of compound L-9-1

[0597] At -78 ℃ under N2 atmosphere, LAH (3.6 g, 95.15 mmol) was slowly added to a solution of 5-hydroxyisophthalic acid dimethyl ester (5 g, 23.79 mmol) in dry THF (300 mL). The reaction mixture was stirred at room temperature for 17 hours. After the reaction was complete, 15% NaOH solution (4 mL), H2O (8 mL) and EA (100 mL) were added, and the reaction mixture was then stirred for 1 hour. The mixture was filtered and concentrated under reduced pressure. Residue was purified by column chromatography to obtain compound L-9-1 (3.02 g, 82%).

[0598] 1 H NMR (400MHz, DMSO-d6) δ9.21 (s, 1H), 6.66 (s, 1H), 6.58 (s, 2H), 5.07 (t, J = 6.0Hz, 2H), 4.38 (d, J = 4.6Hz, 4H).

[0599] Preparation of compound L-9-2

[0600] Under N2 atmosphere, compound L-9-1 (2g, 12.97mmol) was dissolved in HBr (5.0mL, 33% in AcOH). After stirring at 60°C for 18 hours, the reaction was quenched by adding NaHCO3 solution (pH about 8). Distilled water (50mL) and EA (100mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-9-2 (2.9g, 80%).

[0601] 1 H NMR (400MHz, CDCl3) δ6.99(s,1H),6.81(s,2H),4.85(s,1H),4.41(s,2H).

[0602] Preparation of compound L-9

[0603] At room temperature under N2 atmosphere, TEA (0.45 mL, 3.21 mmol) was added to a solution of compound L-1-2 (1.0 g, 3.57 mmol) in DCM (35 mL). SO2F2 gas was introduced through a balloon, and the mixture was stirred at room temperature for 1 hour. The mixture was washed with DCM (50 mL) and water (30 mL). The organic layer was washed with NaHCO3 aqueous solution, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-2 (941.7 mg, 73%).

[0604] 1 H NMR (400Hz, CDCl3) δ7.47(s,1H),7.32(s,2H),4.46(s,4H).

[0605] Preparation of compound L-10

[0606] At room temperature under N2 atmosphere, imidazole (27 mg, 0.39 mmol) and TBDMS-Cl (59 mg, 0.39 mmol) were added to a solution of compound L-9-2 (100 mg, 0.36 mmol) in dry DCM (3 mL). After stirring for 16 hours, distilled water (50 mL) and EA (100 mL) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-10 (110 mg, 79%).

[0607] 1H NMR (400MHz, CDCl3) δ7.00 (s, 1H), 6.80 (s, 2H), 4.41 (s, 4H), 0.99 (s, 9H), 0.21 (s, 6H).

[0608] Example 26. Preparation of Compound L-11

[0609]

[0610] Preparation of compound L-11-1

[0611] To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was added KI (294 mg, 1.77 mmol), Ag2O (4.92 g, 19.48 mmol) and p-TsCl (3.7 g, 19.48 mmol) under N2 atmosphere. The mixture was stirred at room temperature overnight. After the reaction was complete, the mixture was washed with water and then dried over a slurry of water. Filter and wash with DCM (100 mL) The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-11-1 (5.98 g, 73%).

[0612] 1 H NMR (400Hz, CDCl3) δ7.80 (d, J = 8.4Hz, 2H), 7.35 (d, J = 8.4Hz, 2H), 4.16 (t, J = 4.8Hz, 2H), 3.71-3.58 (m, 22H), 2.88 (br, 1H), 2.45 (s, 3H).

[0613] Preparation of compound L-11-2

[0614] Under N2 atmosphere, to a solution of compound L-11-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was added NaN3 (1.34 g, 20.55 mmol). The mixture was stirred at 110 ° C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-11-2 (4.1 g, 97%).

[0615] 1 H NMR (400Hz, CDCl3) δ3.72-3.60 (m, 22H), 3.39 (t, J = 4.8Hz, 2H), 2.78 (br, 1H).

[0616] Preparation of compound L-11-2a

[0617] Under N2 atmosphere, compound L-11-2 (1.9 g, 6.18 mmol) was dissolved in DCM (20 mL), and triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added thereto. The mixture was stirred at room temperature overnight. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-11-2a (2.58 g, 91%).

[0618] 1 H NMR (400Hz, CDCl3) δ7.80 (d, J = 8.4Hz, 2H), 7.35 (d, J = 8.4Hz, 2H), 4.16 (t, J = 4. 8Hz, 2H), 3.70-3.61 (m, 16H), 3.56 (s, 1H), 3.39 (t, J = 4.8Hz, 2H), 2.45 (s, 3H).

[0619] EI-MS m / z:462(M + +1).

[0620] Preparation of compound L-11-3

[0621] To a solution of compound L-11-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) was added 5% Pd / C (1.04 g, 0.49 mmol) under H2 atmosphere. The mixture was stirred at room temperature for 4 hours. The mixture was washed with water. Filter to remove Pd / C and concentrate under reduced pressure. The residue is dissolved in DCM (25 mL). Boc2O (852.1 mg, 3.9 mmol) is added, and the resulting mixture is stirred at room temperature for 3 hours. The mixture is concentrated under reduced pressure. The residue is purified by column chromatography to obtain compound L-11-3 (330 mg, 28%).

[0622] 1 H NMR(400Hz, CDCl3)δ5.19(br s,1H),3.73(t,J=4.8Hz,2H),3.67(s,12H),3.63-3.60(m,6H),3.54(t,J=5.2Hz,2H),3.34-3.27(m,1H),1.44(s,9H).

[0623] EI-MS m / z:382(M + +1).

[0624] Preparation of compound L-11-4

[0625] By the homogeneous solution of compound L-11-3 (450mg, 1.18mmol) in anhydrous THF (10mL) at 0 DEG C under N2 atmosphere with NaH (60% dispersion in mineral oil, 47.2mg, 1.18mmol) process.After stirring the mixture at 0 DEG C for 20 minutes, L-11-2a (544.5mg, 1.18mmol) is added thereto. The reaction is warmed to room temperature and stirred overnight. The reaction is cooled, quenched with MeOH (5mL), and concentrated under reduced pressure. The residue is purified by column chromatography to obtain compound L-11-4 (582.9mg, 74%).

[0626] Preparation of compound L-11

[0627] To a solution of compound L-11-4 (582.9 mg, 0.87 mmol) in DCM (3 mL) was added 4M HCl (in 1,4-dioxane, 1 mL) at 0°C under N2 atmosphere. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to obtain compound L-11 (527.6 mg, quantitative).

[0628] EI-MS m / z:571(M + +1).

[0629] Example 27. Preparation of compounds Int-TG1 and Int-TG2

[0630]

[0631] Preparation of compound Int-TG1-1

[0632] To a solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL) was added benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO (2.53 g, 43.06 mmol) at room temperature under N atmosphere. The mixture was stirred overnight at room temperature under N atmosphere. The reaction was extracted with EA (200 mL × 2) and distilled water (100 mL). The organic layer was dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1-1 (2.56 g, 39%).

[0633] 1 H NMR (400Hz, CDCl3) δ11.41 (s, 1H), 9.95 (s, 1H), 8.34 (d, J = 2.0Hz, 1H), 8.23 ​​(d d, J=6.4Hz, 2.4Hz, 1H), 7.46-7.35 (m, 5H), 7.04 (d, J=9.2Hz, 1H), 5.37 (s, 2H).

[0634] Preparation of compound Int-TG1-2

[0635] To a solution of compound Int-TG1-1 (1.0 g, 3.90 mmol) and compound Int-TG (1.6 g, 3.90 mmol) in anhydrous MeCN (30 mL) was added molecular sieves (8 g) and Ag2O (3.62 g, 15.61 mmol) at room temperature under N2 atmosphere. The mixture was stirred for 1 hour at room temperature and then The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1-2 (2.1 g, 92%).

[0636] 1 H NMR (400Hz, CDCl3) δ10.34(s,1H),8.55(d,J=2.0Hz,1H),8.26(dd,J=6.8,2.0Hz,1H),7.45-7.35(m,5H),7.17(d,J=8.8Hz,1H),5.63-5.60(m, 1H), 5.50 (d, J = 3.6Hz, 1H), 5.37 (s, 2H), 5.23 (d, J = 8.0Hz, 1H), 5.16 (dd, J = 7.2, 3.6Hz, 1H) 4.24-4.10 (m, 4H), 2.20 (s, 3H), 2.10-2.03 (m, 9H).

[0637] Preparation of compound Int-TG1-3

[0638] To a solution of compound Int-TG1-2 (2.1 g, 3.58 mmol) in DCM (30 mL) was added m-CPBA (2.65 g, 10.74 mmol) at 0 ° C under N2 atmosphere. After stirring at 0 ° C for 7 hours, the mixture was quenched by adding saturated sodium bicarbonate (40 mL × 2). The mixture was separated, and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. At 0 ° C under N2 atmosphere, the residue was dissolved in DCM (5 mL), and hydrazine hydrate (261 μL, 5.37 mmol) was added to the mixture. After stirring at 0 ° C for 1 hour, EA (30 mL × 2) and 1M HCl aqueous solution (10 mL) were added. The resulting organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain compound Int-TG1-3 (1.1 g, 55%).

[0639] EI-MS m / z:574(M + +Na)

[0640] Preparation of compound Int-TG1-4

[0641] At 0 ° C under N2 atmosphere, TBDMS-OTf (224 μ L, 0.97 mmol) and Et3N (207 μ L, 1.46 mmol) were added to a solution of compound Int-TG1-3 (280 mg, 0.49 mmol) in DCM (5 mL). The mixture was stirred at room temperature for 1.5 hours and then quenched by adding citric acid (20 ml). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1-4 (246.3 mg, 68%).

[0642] 1 H NMR (400Hz, CDCl3) δ7.67 (d, J = 8.4Hz, 1H), 7.57 (s, 1H), 7.44-7.34 (m, 5H), 7 .02(d,J=8.4Hz,1H),5.49-5.44(m,2H),5.30(s,2H),5.19(d,J=7.6Hz,1H),5 .10(dd,J=6.8,3.2Hz,1H)4.20-4.11(m,2H),4.05(t,J=6.8Hz,2H),2.19(s, 3H), 2.04 (s, 3H), 2.01 (d, J = 6.0Hz, 6H), 1.02 (s, 9H), 0.20 (d, J = 15.6Hz, 6H).

[0643] Preparation of compound Int-TG1-5

[0644] To a solution of compound Int-TG1-4 (283.2 mg, 0.41 mmol) in EA (5 mL) was added Pd / C (5%, 87.5 mg, 0.04 mmol) at room temperature under H2. The mixture was stirred for 1 hour and then filtered. The mixture was filtered and then concentrated under reduced pressure. Compound Int-TG1-5 was used directly in the next reaction without further purification (246 mg, quantitative).

[0645] 1H NMR (400Hz, CDCl3) δ7.67(d,J=8.8Hz,1H),7.57(s,1H),7.05(d,J=8.4Hz,1H),5.49-5.45(m,2H),5.22(d,J=7.6Hz,1H),5.12( dd,J=7.2,3.6Hz,1H)4.20-4.06(m,4H),2.19(s,3H),2.05(s,3H),2.02(d,J=7.6Hz,6H),1.01(s,9H),0.21(d,J=15.2Hz,6H).

[0646] Preparation of compound Int-TG1

[0647] At room temperature under N2 atmosphere, to a solution of compound Int-TG1-5 (243.2 mg, 0.41 mmol) and 11-azido-3,6,9-trioxaundecane-1-amine (Aldrich, CAS 134179-38-7, 89.5 mg, 0.41 mmol) in DMF (5 mL) was added PyBOP (275 mg, 0.53 mmol) and DIPEA (176 μL, 1.02 mmol). The mixture was stirred at room temperature under N2 atmosphere for 2 hours. The reaction was extracted with EA (30 mL×2) and distilled water (10 mL). The resulting organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1 (272.8 mg, 84%).

[0648] 1 H NMR (400Hz, CDCl3) δ7.34 (s, 1H), 7.31 (d, J = 9.2Hz, 1H), 7.02 (d, J = 8.0Hz, 1H) ,6.73(s,1H),5.48-5.44(m,2H),5.19(d,J=7.6Hz,1H),5.10(dd,J=6.4,3.6Hz ,1H),4.20-4.10(m,2H),4.06(t,J=6.4Hz,2H),3.66(s,14H),3.38(t,J=4.4Hz ,2H),2.19(s,3H),2.02(t,J=8.4Hz,9H),1.00(s,9H),0.20(d,J=14.4Hz,6H).

[0649] EI-MS m / z:799(M + +1).

[0650] Preparation of compound Int-TG2

[0651] To a solution of compound Int-TG1-5 (246 mg, 0.41 mmol) and L-9 (249.5 mg, 0.41 mmol) in DMF (3 mL) was added PyBOP (278 mg, 0.53 mmol) and DIPEA (179 μL, 1.02 mmol) at room temperature under N2 atmosphere. After the mixture was stirred for 2 hours, the reaction mixture was subjected to preparative HPLC to obtain compound Int-TG2 (384.6 mg, 81%). EI-MS m / z: 1152 (M + +1).

[0652] Example 28. Preparation of compound Int-TG3

[0653]

[0654] Preparation of compound Int-TG3a

[0655] At room temperature under N2 atmosphere, to a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 mL) was added Et3N (2.28 mL, 16.38 mmol). SO2F2 gas was introduced via a balloon, and the mixture was stirred at room temperature for 2 hours. The mixture was washed with DCM (30 mL × 3) and brine (30 mL), and the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG3a (790 mg, 63%).

[0656] 1 H NMR (400Hz, CDCl3) δ10.06 (s, 1H), 8.05 (d, J = 8.0Hz, 2H), 7.55 (d, J = 8.8Hz, 2H).

[0657] Preparation of compound Int-TG3-1

[0658] To a solution of compound Int-TG1 (100 mg, 0.13 mmol) and compound Int-TG3a (26 mg, 0.13 mmol) in anhydrous MeCN (3 mL) was added DBU (4 μL, 25 μmol). The mixture was stirred at room temperature for 1 hour and washed with distilled water (10 mL) and EA (15 mL × 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG3-1 (103 mg, 94%).

[0659] EI-MS m / z:869(M + +1).

[0660] Preparation of compound Int-TG3-2

[0661] To a solution of compound Int-TG3-1 (103 mg, 0.12 mmol) in THF (8 mL) was added NaBH 4 (9 mg, 0.24 mmol) at 0° C. under N 2 atmosphere. After stirring at room temperature for 2 hours, distilled water (10 mL) and EA (10 mL×2) were added. The organic layer was dried over anhydrous Na 2 SO 4, filtered and concentrated under reduced pressure to obtain compound Int-TG3-2 (101 mg, 98%).

[0662] EI-MS m / z:871(M + +1).

[0663] Preparation of compound Int-TG3-3

[0664] To a solution of compound Int-TG3-2 (320.5 mg, 0.0.37 mmol) in DCM (3 ml) was added 1 M PBr3 in DCM (165 μL, 0.19 mmol) at 0 ° C under N2 atmosphere. After stirring for 2 hours at 0 ° C. The mixture was quenched by adding saturated sodium bicarbonate (8 mL×2). The mixture was separated, and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG3-3 (202.6 mg, 59%)

[0665] EI-MS m / z:934(M + +1).

[0666] Preparation of compound Int-TG3

[0667] To a solution of compound Int-TG3-3 (10 mg, 0.01 mmol) in DMF (1 mL) was added dimethylamine (0.1 mL) at room temperature under N2 atmosphere. After stirring at room temperature for 10 minutes, the reaction mixture was purified by preparative HPLC to obtain compound Int-TG3 (6 mg, 63%). EI-MS m / z: 898 (M + +1).

[0668] Example 29. Preparation of Compound L-12

[0669]

[0670] Preparation of compound L-12-1

[0671] To a solution of vanillic acid (50.0 g, 0.30 mol) in MeOH (700 mL) was added dropwise SOCl2 (207 mL, 2.85 mol) and the resulting mixture was stirred at 0 ° C under N2 atmosphere, and then stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The reaction was adjusted to pH 7 to 8 with saturated NaHCO3 aqueous solution, and then diluted with distilled water (100 mL) and EA (200 mL × 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-12-1 (54.2 g, quantitative)

[0672] 1 H NMR (400MHz, CDCl3) δ7.64(dd,J=6.4,1.6Hz,1H),7.55(s,1H),6.94(d,J=8.4Hz,1H),6.05(s,1H),3.95(s,3H),3.89(s,3H).

[0673] Preparation of compound L-12-2

[0674] To a solution of compound L-12-1 (54.2 g, 0.30 mol) in DMF (200 mL) was added KCO (61.6 g, 0.45 mol) and benzyl bromide (39.0 mL, 0.33 mol) under N atmosphere. After stirring at 100 ° C for 6 hours, the mixture was cooled to room temperature and diluted with distilled water (100 mL) and EA (200 mL × 2). The organic layer was dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-12-2 (79.8 g, 98%).

[0675] 1 H NMR (400MHz, CDCl3) δ7.60 (dd, J=6.4, 2.0Hz, 1H), 7.56 (d, J=2.0Hz, 1H), 7.4 4-7.31(m,5H),6.89(d,J=8.4Hz,1H),5.22(s,2H),3.94(s,3H),3.88(s,3H).

[0676] Preparation of compound L-12-3

[0677] Under N2 atmosphere, compound L-12-2 (79.8 g, 0.29 mol) was dissolved in acetic anhydride (550 mL) and then cooled to 0°C. Copper (II) nitrate hemi-(pentahydrate) (75.0 g, 0.32 mol) was added in portions. After stirring at 0°C for 6 hours, the reaction was quenched with ice water (800 mL). The solid was filtered and washed with distilled water (100 mL) and hexane (200 mL×2) to obtain compound L-12-3 (85.5 g, 92%).

[0678] 1 H NMR (400MHz, CDCl3) δ7.52(s,1H),7.45-7.35(m,5H),7.08(s,1H),5.22(s,2H),3.98(s,3H),3.91(s,3H).

[0679] Preparation of compound L-12-4

[0680] To a solution of compound L-12-3 (85.5 g, 0.27 mol) in THF (800 mL) and MeOH (300 mL) was added 2N NaOH (404 mL, 0.81 mol). After stirring at 65 ° C for 5 hours, the reaction was cooled to room temperature and adjusted to pH 2 by adding 2N HCl solution, then extracted with distilled water (100 mL) and EA (300 mL × 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue solid was collected and washed with hexane to obtain compound L-12-4 (79.2 g, 97%).

[0681] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.47-7.35(m,5H),7.03(s,1H),5.24(s,2H),3.91(s,3H).

[0682] Preparation of compound L-12

[0683] At 0 ° C under N2 atmosphere, to a solution of compound L-12-4 (100 mg, 0.33 mmol) in anhydrous THF (500 μL) and anhydrous DCM (1.5 mL) was slowly added dropwise oxalyl chloride (42.4 μL) and 1 drop of DMF. After stirring for 30 minutes, the reaction mixture was concentrated under reduced pressure. Compound L-12 was used directly in the next reaction without further purification.

[0684] Example 30. Preparation of compound Mono-1

[0685]

[0686] Preparation of compound Mono-1-1

[0687] To a solution of L-2-thienylalanine (500 mg, 2.92 mmol) in distilled water (5.0 mL) was added concentrated HCl (206 μL) dropwise and stirred at 0 ° C under N2 atmosphere, and then formaldehyde (37%, 261 μL, 3.5 mmol) was added thereto. The mixture was refluxed overnight. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (3.0 mL) and 4M HCl (in 1,4-dioxane, 1.0 mL) was added thereto. After stirring for 2 hours, the solid was filtered and washed with IPA (5 mL) and ether (20 mL) to obtain compound Mono-1-1 (495.7 mg, 77%).

[0688] 1 H NMR(400MHz,DMSO-d6)δ9.95(br s,1H),7.48(d,J=5.2Hz,1H),6.94(d,J=5.2Hz,1H),4.48-4.44(m,1H),4.28(d,J=1 5.6Hz, 1H), 4.18 (d, J = 16.0Hz, 1H), 3.39 (dd, J = 11.6, 5.2Hz, 1H), 3.17-3.10 (m, 1H). EI-MS m / z:184(M + +1).

[0689] Preparation of compound Mono-1-2

[0690] Under N2 atmosphere, compound Mono-1-1 (495.7 mg, 2.25 mmol) was dissolved in MeOH (10.0 mL) and then cooled to 0°C. SOCl2 (491.3 μL, 6.76 mmol) was added dropwise at 0°C. The reaction mixture was then refluxed for 3 hours. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL × 2) to obtain compound Mono-1-2 (521.5 mg, 99%).

[0691] 1 H NMR(400MHz,DMSO-d6)δ10.22(br s,2H),7.49(d,J=5.2Hz,1H),6.94(d,J=5.2Hz,1H),4.65-4.61(m,1H),4.30(d,J=15.6Hz, 1H), 4.19 (d, J=15.6Hz, 1H), 3.80 (s, 3H), 3.60 (dd, J=11.6, 5.2Hz, 1H), 3.21-3.14, (m, 1H). EI-MS m / z:198(M + +1).

[0692] Preparation of compound Mono-1-3

[0693] To a solution of compound L-12 (856.5 mg, 2.66 mmol) in anhydrous THF (3.0 ml) was added compound Mono-1-2 (518.5 mg, 2.22 mmol) dissolved in DMF (3.0 mL) and DIPEA (772.8 μL, 4.44 mmol) at 0 ° C, and the resulting reaction mixture was stirred at room temperature overnight. After the reaction was completed, distilled water (20 mL) and EA (50 mL × 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-1-3 (888.5 mg, 89%)

[0694] EI-MS m / z:483(M + +1).

[0695] Preparation of compound Mono-1-4

[0696] At -78 ° C under N2 atmosphere, DIBAL (3.6 mL, 3.6 mmol, 1.0 M in toluene) was added dropwise to a solution of Mono-1-3 (880 mg, 1.82 mmol) in anhydrous DCM (5.0 mL) and toluene (15.0 mL). The reaction mixture was stirred at -78 ° C for 3 hours. The reaction was quenched with MeOH (5 mL) and 2N HCl (20.0 mL) at -78 ° C. Distilled water (20 mL) and EA (50 mL × 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-1-4 (701.9 mg, 85%).

[0697] EI-MS m / z:453(M + +1).

[0698] Preparation of compound Mono-1-5

[0699] To a solution of Mono-1-4 (700mg, 1.55mmol) in THF (15.0mL) and distilled water (3.0mL) was added Na2S2O4 (2.2g, 12.4mmol) at room temperature for 4 hours. After the reaction was complete, the reaction was quenched with MeOH (5mL). The reaction mixture was concentrated under reduced pressure. The residue was suspended in toluene (20mL) and evaporated to help remove any remaining water. The white solid obtained was completely dried by placing it under high vacuum overnight. The residue was suspended in anhydrous MeOH (10mL), and acetyl chloride (1.1mL, 15.5mmol) was then added. After 15 minutes, the turbid solution was filtered and the solid was washed with anhydrous MeOH (5mL×2). The filtrate was stirred for 2 hours. The reaction mixture was quenched with NaHCO3 solution (pH about 7), and then distilled water (20mL) and EA (50mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-1-5 (701.9 mg, 85%)

[0700] 1 H NMR (400MHz, CDCl3) δ7.55(d,J=5.6Hz,1H),7.47(m,5H),7.22(d,J=5.2Hz,1H),6.95(d,J=5.2Hz,1H),6.85(s,1H),5 .26-5.14(m,2H),4.98(d,J=16.4Hz,1H),4.44(d,J=16.8Hz,1H),4.08-4.02(m,1H),3.98(s,3H),3.32-3.26(m,1H).

[0701] EI-MS m / z:453(M + +1).

[0702] Preparation of compound Mono-1

[0703] To a solution of Mono-1-5 (60 mg, 0.15 mmol) in anhydrous DCM (3 mL) was added methanesulfonic acid (700 μL) in DCM (2.0 mL) at 0 ° C, and the resulting mixture was stirred at ° C for 2 hours. The reaction was quenched with NaHCO solution (pH about 7), and then distilled water (5 mL) and EA (20 mL × 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-1 (38.3 mg, 82%).

[0704] 1H NMR (400MHz, CDCl3) δ7.58(d,J=5.6Hz,1H),7.54(s,1H),7.23(d,J=5.2Hz,1H),6.95(d,J=5.2Hz,1H),6.89(s,1H),6.06 (s,1H),5.30(s,1H),4.99(d,J=16.4Hz,1H),4.44(d,J=16.4Hz,1H),4.10-4.04(m,1H),3.99(s,3H),3.32-3.26(m,1H).

[0705] EI-MS m / z:315(M + +1).

[0706] Example 31. Preparation of Compound Mono-2

[0707]

[0708] Preparation of compound Mono-2-1

[0709] At -13 ° C, to a solution of Fmoc-His (Trt) -OH (15.0 g, 24.2 mmol) and HOBT (5.0 g, 24.2 mmol) in anhydrous THF (200 mL) was added dropwise DCC (1.15 g, 8 mmol) in THF (40 mL) and MeOH (20 mL) over 30 minutes. The reaction mixture was slowly warmed to room temperature while stirring for 5 hours. After the reaction was completed, distilled water (50 mL) and DCM (200 mL × 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-1 (13.0 g, 84%).

[0710] 1 H NMR(400MHz, CDCl3)δ7.75(d,J=7.6Hz,2H),7.62(t,J=7.6Hz,2H),7.41-7.28(m,14H),7.15-7.06(m,7H),6 .54(s,1H),6.52(d,J=7.6Hz,1H),4.66-4.59(m,1H),4.38-4.22(m,2H),3.63(s,3H),3.07(t,J=6.4Hz,1H). EI-MS m / z:634(M + +1).

[0711] Preparation of compound Mono-2-2

[0712] To a solution of compound Mono-2-1 (13 g, 20.51 mmol) in DMF (50 mL) was added iodomethane (3.8 mL, 61.54 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-2 (11 g, 83%).

[0713] 1 H NMR (400MHz, CDCl3) δ8.42 (s, 1H), 7.76 (d, J = 7.6Hz, 2H), 7.70-7.60 (m, 2H), 7.46-7.20 (m, 19h), 6.89 (s, 1H ), 6.60 (d, J = 7.2Hz, 1H), 4.70-4.62 (m, 1H), 4.30-4.12 (m, 3H), 4.01 (s, 3H), 3.67 (s, 3H), 3.50-3.28 (m, 2H). EI-MS m / z:648(M + +1).

[0714] Preparation of compound Mono-2-3

[0715] To a solution of compound Mono-2-2 (11 g, 16.95 mmol) in DCM (150 mL) was added TFA (40 mL) and triethylsilane (8.12 mL, 50.86 mmol) at 0 ° C under N2 atmosphere. The reaction was allowed to warm to room temperature and stirred for 6 hours. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-3 (6.25 g, 91%).

[0716] 1 H NMR(400MHz, CDCl3)δ8.87(s,1H),7.78(d,J=7.2Hz,2H),7.59(d,J=7.6Hz,2H),7.45-7.30(m,4H),7.09(s,1H), 5.69(d,J=6.0Hz,1H),4.64-4.50(m,2H),4.48-4.38(m,1H),3.79(s,6H),3.51-3.44(m,1H),3.29-3.10(m,2H). EI-MS m / z:407(M + +1).

[0717] Preparation of compound Mono-2-4

[0718] At 0 ° C under N2 atmosphere, piperidine (3.0 mL, 30.74 mmol) was added to a solution of compound Mono-2-3 (6.25 g, 15.37 mmol) in DCM (150 mL). The reaction mixture was allowed to warm to room temperature and stirred for 7 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain compound Mono-2-4 (2.65 g, 95%).

[0719] 1 H NMR (400MHz, CDCl3) δ7.51(s,1H),6.87(s,1H),3.78-3.69(m,4H),3.63(s,3H),3.09-2.84(m,2H). EI-MS m / z:184(M + +1).

[0720] Preparation of compound Mono-2-5

[0721] Compound Mono-2-4 (2.65 g, 14.46 mmol) was dissolved in distilled water (100 mL) under N2 atmosphere, and the reaction mixture was cooled to 0 ° C. Concentrated HCl (2.5 mL) was added dropwise at 0 ° C, followed by formaldehyde (37%, 2.2 mL, 28.93 mmol). The reaction mixture was refluxed overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (20 mL) and 4M HCl (in 1,4-dioxane, 4.0 mL) was added. The reaction mixture was stirred for 2 hours. The solid was filtered and washed with IPA (5 mL) and ether (10 mL × 2) to obtain compound Mono-2-5 (3.14 g, 99%)

[0722] EI-MS m / z:182(M + +1).

[0723] Preparation of compound Mono-2-6

[0724] To a solution of Mono-2-5 (3.14 g, 14.43 mmol) in MeOH (100 mL) was added dropwise SOCl (2.5 mL, 35.15 mmol) at 0°C. After the reaction mixture was refluxed for 5 hours, the mixture was concentrated under reduced pressure. The residue was washed with ether (25 mL × 2) to obtain compound Mono-2-6 (2.18 g, 65%).

[0725] EI-MS m / z:196(M + +1).

[0726] Preparation of compound Mono-2-7

[0727] To a solution of compound L-12 (3.93 g, 12.23 mmol) and compound Mono-2-6 (2.18 g, 9.41 mmol) in anhydrous THF (30 ml) and DMF (30 mL) was added DIPEA (4.9 mL, 28.22 mmol) at 0 ° C. After stirring at room temperature for 2 hours, the mixture was quenched with distilled water (200 mL) and EA (1000 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-7 (2.81 g, 62%).

[0728] EI-MS m / z:481(M + +1).

[0729] Preparation of compound Mono-2-8

[0730] To a solution of compound Mono-2-7 (2.5 g, 5.20 mmol) in anhydrous DCM (12.5 mL) and toluene (37.5 mL) was added dropwise DIBAL (10.4 mL, 10.41 mmol, 1.0 M in toluene) at -78 ° C under N2 atmosphere. After stirring for 5 hours at -78 ° C, the mixture was quenched with MeOH (1.0 mL) and 2N HCl (100 mL) at the same temperature. The mixture was diluted with water (100 mL) and DCM (200 mL) in succession, and the organic layer was then dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-8 (1.21 g, 52%).

[0731] EI-MS m / z:451(M + +1).

[0732] Preparation of compound Mono-2-9

[0733] At room temperature, Na2S2O4 (3.7g, 21.31mmol) is added to a solution of compound Mono-2-8 (1.2g, 2.66mmol) in THF (100mL) and distilled water (70mL). After stirring for 6 hours, the reaction is quenched with MeOH (20mL). By using toluene as a cosolvent, the mixture is concentrated three times under reduced pressure to remove water. The gained yellow solid is suspended in anhydrous MeOH (200mL), and acetyl chloride (1.9mL, 26.64mmol) is added thereto. After stirring for 15 minutes, the pH value of the reaction mixture is adjusted to 8 by adding saturated NaHCO3 solution, and diluted with distilled water (250mL), MeOH (250mL) and DCM (200mL). The organic layer is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue is purified by column chromatography to obtain compound Mono-2-9 (918mg, 78%).

[0734] 1 H NMR (400MHz, CDCl3) δ7.58(d,J=5.6Hz,1H),7.57(s,1H),7.52(s,1H),7.49-7.27(m,5H),6.84(s,1H) ,5.26-5.15(m,2H),4.66(s,2H),4.16(t,J=6.0Hz,1H),3.97(s,3H),3.67(s,3H),3.22-2.94(m,2H). EI-MS m / z:403(M + +1).

[0735] Preparation of compound Mono-2

[0736] To a solution of compound Mono-2-9 (50 mg, 0.12 mmol) in anhydrous DCM (2 mL) was added methanesulfonic acid (0.1 mL) in DCM (0.2 mL) at 0° C. After stirring at room temperature for 1 hour, the pH of the mixture was adjusted to 8 by adding saturated NaHCO 3 solution. The residue was purified by preparative HPLC to obtain compound Mono-2 (27 mg, 71%)

[0737] 1H NMR(400MHz,CD3OD)δ8.34(br s,1H),7.68(s,1H),7.28(s,1H),6.42(s,1H),4.77(d,J=16.0Hz,1H),4.56(d,J=16.0Hz,1 H), 4.33 (d, J = 7.6Hz, 1H), 4.10-4.02 (m, 1H), 3.84 (s, 3H), 3.66 (s, 3H), 3.02-2.82 (m, 2H). EI-MS m / z:313(M + +1).

[0738] Example 32. Preparation of compound Mono-3

[0739]

[0740] Preparation of compound M-3-1

[0741] To a solution of (s)-(-)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5.0 g, 28.22 mmol) in MeOH (140 mL) was added dropwise SOCl (2.30 mL, 31.04 mmol) at 0°C under N2 atmosphere. After stirring at 40°C for 21 hours, the mixture was concentrated under reduced pressure. The residue was washed with diethyl ether (25 mL x 2) to obtain compound M-3-1 (6.42 g, 99% yield).

[0742] 1 H NMR (400MHz, DMSO-d6) δ10.02(s,2H),7.27(s,4H),4.60-4.56(m,1H),4.39-4.29(m,2H),3.82(s,3H),3.19-3.12(m,2H); EI-MS m / z:192(M + +1).

[0743] Preparation of compound M-3-2

[0744] To a solution of compound L-12 (9.07 g, 28.22 mmol) in anhydrous THF (50 ml) was added compound M-3-1 (6.42 g, 28.22 mmol) in THF (100 mL) and TEA (7.9 mL, 56.43 mmol) at 0 ° C. After stirring at room temperature for 2 hours, the reaction was diluted with distilled water (500 mL) and EA (800 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound M-3-2 (12.01 g, 90%). EI-MS m / z: 477 (M + +1).

[0745] Preparation of compound M-3-3

[0746] To a solution of compound M-3-2 (4 g, 8.39 mmol) in anhydrous DCM (18 mL) and toluene (52 mL) was added dropwise DIBAL (16.8 mL, 16.79 mmol, 1.0 M in toluene) at -78 ° C under N2 atmosphere. After stirring at -78 ° C for 4 hours, the reaction was quenched with MeOH (0.4 mL) and 2N HCl (25 mL) at -78 ° C. Distilled water (100 mL) and EA (500 mL) were added thereto. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound M-3-3 (3.07 g, 82%).

[0747] EI-MS m / z:447(M + +1).

[0748] Preparation of compound M-3-4

[0749] At room temperature, Na2S2O4·2H2O (11.3 g, 53.76 mmol) is added to a solution of compound M-3-3 (3 g, 6.72 mmol) in THF (130 mL) and distilled water (86 mL). After stirring for 5 hours, the reaction is concentrated four times under reduced pressure by using toluene as a cosolvent, thereby removing water. The gained yellow solid is dissolved in anhydrous MeOH (220 mL), and acetyl chloride (4.8 mL, 67.19 mmol) is added thereto. After stirring for 15 minutes, the pH value of the reaction mixture is adjusted to 7 by adding saturated NaHCO3 solution, and diluted with distilled water (100 mL) and EA (250 mL × 2). The organic layer is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue is purified by column chromatography to obtain compound M-3-4 (2.48 g, 93%).

[0750] 1 H NMR(400MHz, CDCl3)δ7.55(s,1H),7.45-7.27(m,10H),6.84(s,1H),5.24-5.15(m,2H),5.00(d ,J=15.2,1H),4.56(d,J=15.6,1H),3.97(s,3H),3.93-3.92(m,1H),3.31-3.12(m,2H).; EI-MS m / z:399(M + +1).

[0751] Preparation of compound M-3

[0752] To a solution of compound M-3-4 (1 g, 2.51 mmol) in anhydrous DCM (10 mL) was added methanesulfonic acid (5 mL) in DCM (10 mL) at 0 ° C. After stirring for 3 hours at 0 ° C, the mixture was quenched with NaHCO solution and then diluted with distilled water (100 mL) and EA (400 mL). The organic layer was dried over anhydrous Na SO, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound M-3 (703 mg, 91%).

[0753] 1 H NMR (400MHz, CDCl3) δ7.54 (s, 1H), 7.48 (d, J = 4.8Hz, 1H), 7.37-7.26 (m, 4H), 6.88 (s, 1H), 6.03 (s, 1H) ), 5.00 (d, J = 15.6Hz, 1H), 4.56 (d, J = 15.6Hz, 1H), 3.98 (s, 3H), 3.95-3.90 (m, 1H), 3.30-3.13 (m, 2H).

[0754] EI-MS m / z:309(M + +1).

[0755] Example 33. Preparation of Compound D-101

[0756]

[0757] To a solution of compound Mono-2 (2.0 mg, 0.005 mmol) and compound L-10 (3.3 mg, 0.010 mmol) in DMF (1.0 mL) was added KCO (2.0 mg, 0.012 mmol) at room temperature under N2 atmosphere. After stirring for 3 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-101 (1.2 mg, 34%).

[0758] EI-MS m / z:743(M + +1).

[0759] Example 34. Preparation of Compound D-102

[0760]

[0761] Preparation of compound D-102a

[0762] To a solution of compound Mono-1 (100 mg, 0.318 mmol) and 1,3,5-tribromomethylbenzene (57 mg, 0.159 mmol) in DMF (3 mL) was added KCO (66 mg, 0.477 mmol) at room temperature under N2 atmosphere. After stirring for 3 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-102a (62 mg, 48%).

[0763] EI-MS m / z:824(M + +1).

[0764] Preparation of compound D-102

[0765] To a solution of compound D-102a (62 mg, 0.075 mmol) in DMF (1 mL) was added 1 M dimethylamine in THF (0.5 mL) at room temperature under N2 atmosphere. After stirring for 1 hour, the reaction mixture was purified by preparative HPLC to obtain compound D-102 (39 mg, 60%).

[0766] EI-MS m / z:788(M + +1).

[0767] Example 35. Preparation of Compound D-103

[0768]

[0769] Compound D-103 was synthesized by a similar synthetic route described in Example 34.

[0770] Preparation of compound D-103a

[0771] Yield 42%.

[0772] EI-MS m / z:812(M + +1).

[0773] Preparation of compound D-103

[0774] Yield 17%.

[0775] EI-MS m / z:776(M + ).

[0776] Example 36. Preparation of compound MMAF-OMe

[0777]

[0778] MMAF-OMe was synthesized by similar synthetic methods to those described in US Patents 7,423,116 and 7,498,298 and International Patent Application Publication No. WO 2002 / 088172, each of which is incorporated herein by reference in its entirety.

[0779] Example 37. Preparation of compound Int-TG4

[0780]

[0781] Preparation of compound Int-TG4-1

[0782] To a solution of compound Int-TG1 (100 mg, 0.13 mmol) and compound Int-TG3a (26 mg, 0.13 mmol) in anhydrous MeCN (3 mL) was added DBU (4 μL, 25 μmol). The mixture was stirred at room temperature for 1 hour and washed with distilled water (10 mL) and EA (10 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG4-1 (103 mg, 94%).

[0783] EI-MS m / z:869(M + ).

[0784] Preparation of compound Int-TG4-2

[0785] To a solution of compound Int-TG4-1 (103 mg, 0.12 mmol) in THF (8 mL) was added NaBH 4 (9 mg, 0.24 mmol) at 0° C. under N 2 atmosphere. After stirring at room temperature for 2 hours, distilled water (10 mL) and EA (10 mL×2) were added. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain compound Int-TG4-2 (101 mg, 98%).

[0786] EI-MS m / z:871(M + ).

[0787] Preparation of compound Int-TG4

[0788] To a solution of compound Int-TG4-2 (47 mg, 54 μmol) in DMF (2 mL) was added bis(4-nitrophenyl) carbonate (25 mg, 81 μmol) and DIPEA (14 μL, 81 μmol) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature overnight. Distilled water (10 mL) and EA (10 mL × 2) were added, and the organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG4 (53 mg, 94%).

[0789] EI-MS m / z:1036(M + ).

[0790] Example 38. Preparation of Compound T-Int-1 (B-3)

[0791]

[0792] Preparation of compound T-Int-1a

[0793] To a solution of compound Int-TG4 (65 mg, 0.063 mmol) and MMAF-OMe (52 mg, 0.069 mmol) in DMF (1 mL) was added HOBt (2 mg, 0.013 mmol), DIPEA (12 μL, 0.069 mmol) and pyridine (330 μL) at room temperature under N2 atmosphere. After stirring overnight, the mixture was adjusted to pH 2 to 3 with 1N HCl and extracted with EA (8 mL×2). The organic layer was washed with distilled water (8 mL) and brine (12 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain compound T-Int-1a (73 mg, 71%).

[0794] EI-MS m / z:1644(M +1 ).

[0795] Preparation of compound T-Int-1

[0796] A homogeneous solution of T-Int-1a (73 mg, 0.044 mmol) in anhydrous MeOH (1.5 mL) was treated with LiOH (14 mg, 0.333 mmol) and distilled water (1.5 mL) at 0° C. under N 2 atmosphere and stirred to room temperature for 2 hours. The reaction was quenched with 1N HCl (2 mL), and the reaction mixture was purified by preparative HPLC chromatography to give the title compound T-Int-1 (45 mg, 69%).

[0797] EI-MS m / z:1462(M + ).

[0798] Example 39. Preparation of Compound T-Int-2

[0799]

[0800] Preparation of compound T-Int-2-1

[0801] To a solution of compound Mono-2 (14 mg, 0.04 mmol) and compound L-9 (8.0 mg, 0.02 mmol) in DMF (0.6 mL) was added KCO (9.3 mg, 0.07 mmol) at 30°C under N2 atmosphere. After stirring for 3 hours, the reaction mixture was purified by preparative PLC to obtain compound T-Int-2-1 (5.4 mg, 29%). EI-MS m / z: 825 (M + +1).

[0802] Preparation of compound T-Int-2-2

[0803] To a solution of compound T-Int-2-1 (7.5 mg, 0.01 mmol) and compound Int-TG1 (14 mg, 0.02 mmol) in MeCN (0.5 mL) and DMF (0.5 mL) was added BEMP (1 μL, 0.004 mmol) at room temperature under N2 atmosphere. After stirring at room temperature for 5 hours, the reaction mixture was purified by HPLC to obtain compound T-Int-2-2 (67 mg, 83%). EI-MS m / z: 1490 (M + +1).

[0804] Preparation of compound T-Int-2

[0805] To a solution of compound T-Int-1-2 (8.1 mg, 0.01 mmol) in MeOH (1 mL) and DCM (0.1 mL) was added KCO (5.6 mg, 0.04 mmol) under N2 atmosphere. After stirring at 0°C for 1 hour, the reaction mixture was purified by HPLC to obtain compound T-Int-2 (5.5 mg, 76%). EI-MS m / z: 1322 (M + +1).

[0806] Example 40. Preparation of Compound T-Int-3

[0807]

[0808] Preparation of compound T-Int-3-1

[0809] To a solution of compound D-103 (22.8 mg, 0.03 mmol) and compound Int-TG3-3 (27.4 mg, 0.03 mmol) in DMF (2 mL) was added DIPEA (12 uL, 0.07 mmol) at 40° C. under N 2 atmosphere. After stirring at room temperature for 5 hours, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-3-1 (28.9 mg, 71%).

[0810] EI-MS m / z:1630(M + +1).

[0811] Preparation of compound T-Int-3

[0812] To a solution of compound T-Int-3-1 (28.9 mg, 0.02 mmol) in MeOH (2 mL) was added KCO (12.2 mg, 0.09 mmol) under N atmosphere. After stirring at 0° C. under N atmosphere for 1 hour, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-3 (18.4 mg, 71%).

[0813] EI-MS m / z:1462(M + +1).

[0814] Example 41. Preparation of Compounds T-1 and T-2

[0815]

[0816] To a homogeneous solution of T-Int-1 (2 mg, 1.37 μmol) and AMA-5 (2 mg, 4.11 μmol) in DMSO (4.5 mL) was added (BimC4A)3 (4.5 mg, 5.48 μmol) and CuBr (6.42 mg, 44.8 μmol) at room temperature under nitrogen atmosphere, and the resulting mixture was stirred for 10 minutes. The reaction mixture was purified by preparative HPLC chromatography to give the title compound T-1 (0.4 mg, 15%). EI-MS m / z: 954 (M + 1 / 2).

[0817] Compound T-2 was synthesized in a manner similar to the preparation method of compound T-1.

[0818] Yield 33%, white solid. EI-MS m / z: 976 (M / 2 + +1).

[0819] Example 42. Preparation of Compound T-3

[0820]

[0821] Compound T-3 was synthesized in a similar manner to the preparation method of Compound T-2 of Example 41.

[0822] Yield 40%, white solid. EI-MS m / z: 906 (M + +1).

[0823] Example 43. Preparation of Compound T-4

[0824]

[0825] Compound T-4 was synthesized in a manner similar to the preparation method of Compound T-2 of Example 41.

[0826] Yield 77%, white solid. EI-MS m / z: 977 (M + +1).

[0827] Example 44. Preparation of Compound T-5

[0828]

[0829] At room temperature under N2 atmosphere, a homogeneous solution of T-Int-1 (2.5 mg, 0.0017 mmol) and Mal-1 (2.3 mg, 0.0051 mmol) in DMSO (3461 μ L) was treated with (BimC4A)3 in DMSO (1368 μ L, 0.0068 mmol) and stirred for 10 minutes. CuBr (171 μ L, 0.017 mmol) in DMSO was added to the reaction mixture and the resulting mixture was stirred for 5 minutes. The product was purified by preparative HPLC (column: InnovaL ODS-2 10 μ m, The reaction mixture was purified by column chromatography (21.2 x 250 mm; flow rate: 15 mL / min, A buffer 0.1% formic acid in water / B buffer 0.1% formic acid in ACN, method gradient, solvent A: solvent B 95:5 to 5:95, 1 hour, wavelength 214 nm) to obtain compound 11 (2.1 mg, 64%) as a white solid.

[0830] EI-MS m / z:957(M / 2 + +1).

[0831] Example 45. Preparation of Compound T-6

[0832]

[0833] Compound T-6 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0834] Yield 70%, white solid. EI-MS m / z: 1007 (M / 2 +1 ).

[0835] Example 46. Preparation of Compound T-7

[0836]

[0837] Compound T-7 was synthesized in a similar manner to the preparation method of compound T-5 of Example 44.

[0838] Yield 53%, white solid. EI-MS m / z: 1181 (M / 2 +1 ).

[0839] Example 47. Preparation of Compound T-8

[0840]

[0841] Compound T-8 was synthesized in a similar manner to the preparation method of compound T-5 of Example 44.

[0842] Yield 15%, white solid. EI-MS m / z: 935 (M / 2 +1 ).

[0843] Example 48. Preparation of Compound T-9

[0844]

[0845] Compound T-9 was synthesized in a manner similar to the preparation of compound T-5 in Example 44. Yield: 61%, white solid. EI-MS m / z: 957 (M / 2 +1 ).

[0846] Example 49. Preparation of Compound T-10

[0847]

[0848] Compound T-10 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0849] Yield 65%, white solid. EI-MS m / z: 963 (M / 2 +1 ).

[0850] Example 50. Preparation of compound "BG-SIG"

[0851]

[0852] BG-SIG was synthesized by a similar route as described in US Patent 10,383,949, which is incorporated herein by reference in its entirety.

[0853] Example 51. Preparation of Compound T-Int-4

[0854]

[0855] T-Int-4-1, T-Int-4-2, and T-Int-4-3 were synthesized by a similar route to that described in US Patent 10,383,949, which is herein incorporated by reference in its entirety.

[0856] Preparation of compound T-Int-4-1

[0857] Yield 82%; EI-MS m / z: 1357 (M +1 ).

[0858] Preparation of compound T-Int-4-2

[0859] Yield 76%; EI-MS m / z: 1257 (M +1 ).

[0860] Preparation of compound T-Int-4-3

[0861] Yield 75%; EI-MS m / z: 1457 (M +1 ).

[0862] Preparation of compound T-Int-4

[0863] Compound T-Int-4 was synthesized in a manner similar to the preparation method of compound T-Int-1 of Example 38.

[0864] Yield 88X%; EI-MS m / z: 1303 (M +1 ).

[0865] Example 52. Preparation of Compound T-11

[0866]

[0867] Compound T-11 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0868] Yield 80%, white solid. EI-MS m / z: 846 (M / 2 +1 ).

[0869] Example 53. Preparation of Compound T-12

[0870]

[0871] Compound T-12 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0872] Yield 71%, white solid. EI-MS m / z: 925 (M / 2 +1 ).

[0873] Example 54. Preparation of Compound T-13

[0874]

[0875] Compound T-13 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0876] Yield 56%, white solid. EI-MS m / z: 877 (M / 2 +1 ).

[0877] Example 55. Preparation of Compound T-14

[0878]

[0879] Compound T-14 was synthesized in a manner similar to the preparation method of compound T-5 of Example 44.

[0880] Yield 70%, white solid. EI-MS m / z: 897 (M / 2 +1 ).

[0881] Example 56. Preparation of compound "MPS-1"

[0882]

[0883] Compound MPS-1 was synthesized in a manner similar to that described in Example 20.

[0884] 1 H NMR (400Hz, CDCl3) δ 8.04-7.99 (m, 4H), 7.81 (d, J = 8.4Hz, 2H), 7.46 (d, J = 8.4Hz, 2H), 3.63 (t, J = 7.2Hz, 2H), 3.41 (t, J = 7.2Hz, 2H), 2.44 (s, 3H). EI-MS m / z:333(M + +1).

[0885] Example 57. Preparation of compound "Reference A"

[0886]

[0887] Reference A was synthesized by a similar synthetic route described in Example 20.

[0888] EI-MS m / z:621(M + +1).

[0889] Example 58. Preparation of compound "N-Ac-Cys-AMA-9c"

[0890]

[0891] A homogeneous solution of AMA-9c (11 mg, 0.025 mmol) in PBS buffer pH 7.4 (2 mL) and DMSO (0.2 mL) was treated with N-Ac-cysteine ​​(4.2 mg, 0.026 mmol) and stirred for 1.5 hours at room temperature under N2 atmosphere. After checking LC-Mass, the reaction mixture was used in situ for the next step.

[0892] EI-MS m / z: 610 (M+).

[0893] Example 59. Preparation of compound "N-Ac-Cys-AMA-10"

[0894]

[0895] Compound N-Ac-Cys-AMA-10 was synthesized in a manner similar to the preparation method of compound N-Ac-Cys-AMA-9c of Example 59.

[0896] Biological testing

[0897] Example 60. Preparation of conjugate

[0898] Reduction of antibodies for conjugation / Oxidation: Cysteine ​​engineered monoclonal antibodies were reduced with approximately 20-50 fold excess TCEP (tris(2-carboxyethyl)phosphine hydrochloride) or DTT (dithiothreitol) in 4 mM Tris pH 7.3 and 1 mM EDTA at 37° C. for 1 hour. The reduced thiomab was diluted and loaded onto a PD-10 column in PBS. The column was eluted with 10 mM PBS pH 7.3. The eluted reduced thiomab was reconstituted by air oxidation. The thiol / Ab value was checked by measuring the reduced antibody concentration by the absorbance of the solution at 280 nm and the thiol concentration by reaction with DTNB (Aldrich, CAS No D8130) and measuring the absorbance at 412 nm.

[0899] Conjugation method 1 :

[0900] Compound T-1 (3.80 μL, 3.0 mmol, as a linker-toxin intermediate) obtained in Example 41 in DMSO was treated with reduced reoxidized antibody (45 μL, 0.053 mmol) and gently stirred at room temperature for 3 hours. Sodium borohydride (3.80 μL, 300 mmol) was added to the solution of the reaction mixture and incubated at 37 ° C for 1 hour to block the reversible deconjugation reaction. The conjugated mixture was loaded and eluted through a PD-10 column to remove excess drug-linker intermediate and other impurities.

[0901] Conjugation method 2 :

[0902] After the reduction and reoxidation reaction, the antibody was dissolved in PBS. A solution of compounds T-3 and T-4 (8.86 μL, 3.0 mmol, as a joint-toxin intermediate) obtained in Examples 42 and 43 in DMSO was treated with reduced reoxidized antibody (70 μL, 0.053 mmol) and gently stirred at room temperature for 3 hours. Hydroxylamine (8.86 μL, 1,500 mmol) was added to the solution of the reaction mixture and incubated at 37 ° C for 8 hours to block the reversible deconjugation reaction. The conjugated mixture was loaded and eluted through a PD-10 column to remove excess drug-joint intermediates and other impurities.

[0903] According to the method proposed in the reference literature, compounds T-1, T-3, and T-4 obtained in Examples 41, 42, and 43 were conjugated to the thiol group of the engineered cysteine ​​of trastuzumab (anti-HER2) to prepare T-1-AB, T-3-AB, and T-4-AB as thiomab drug conjugates (TDCs), respectively. [See Nature Biotechnology, 2008, 26, 925-932, Bioconjugate Chem., 2013, 24, 1256-1263, Bioconjugate Chem., 2016, 27, 1324-1331, Bioconjugate Chem. 2014, 25, 460-469.] The DAR (drug-to-antibody ratio) of the conjugated antibodies was analyzed by HIC, and the results of the analysis are shown in Table 1.

[0904] Conjugation method 3 : Maleimide conjugation protocol.

[0905] After the reduction and reoxidation reactions, the resulting antibody was treated with 3.5 equivalents of compound T-10 as a 3 mM stock solution in DMSO to achieve 7.5% (volume / volume) total organic matter. The reaction was allowed to stand at 40°C for 1 hour, and then excess drug-linker intermediate and other impurities were removed by using a PD-10 column. The final sample was concentrated to approximately 5 mg / ml protein.

[0906] Table 1. Antibody-drug conjugates (ADCs)

[0907] ADCs DAR Conjugation method Linker-Toxin, Example T-1-AB 1.76 1 T-1, Example 41 T-3-AB 0.5 2 T-3, Example 42 T-4-AB 0.42 2 T-4, Example 43

[0908] Example 61. Cytotoxicity of Antibody-Drug Conjugates

[0909] NCI-N87 cancer cells were seeded in 96-well plates at a density of 5,000 cells per well in 100 μL of culture medium and cultured for 24 hours. T-DM1 was treated with a 1:5 serial dilution from 50 nM to 0.000128 nM. After 72 hours of incubation, 0.2 mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye dissolved in PBS buffer (5 mg / mL) was added to each well of the plate. The formazan formed by the reduction of MTT dye by mitochondrial oxidoreductase in living cells was dissolved in DMSO and measured using absorbance at 550 nm.

[0910] Example 62. Chemoselectivity study of the reaction of AMA-9c with N-Ac-Cys, N-Ac-Lys and N-Ac-Tyr.

[0911]

[0912] To a homogeneous solution of AMA-9c (10 μL, stock solution 10 mmol) in PBS buffer (870 μL) and DMSO (90 uL) were added N-acetyl-L-lysine (10 μL, stock solution 10 mmol), N-acetyl-L-tyrosine (10 μL, stock solution 10 mmol), and N-acetyl-L-cysteine ​​(10 μL, stock solution 10 mmol) and stirred for 1 hour. The chemoselectivity of the AMA-9c reaction was examined using LC-MS. The reaction of AMA-9c in an equimolar mixture of N-acetyl-L-lysine, N-acetyl-L-tyrosine, and N-acetyl-L-cysteine ​​showed complete chemoselectivity for the thiol group of cysteine.

[0913] Example 63. Chemical Stability (Hydration Stability) Study of AMA-9c

[0914] This study was conducted to examine the stability of AMA-9c. Compound AMA-9c was dissolved in DMSO and mixed with a PBS (pH 7.4) buffer solution to prepare a solution with a concentration of 500 μM (5% DMSO). MPS, used as a standard substance, was prepared as a solution with a concentration of 500 μM in a PBS buffer solution. 420 μL of the buffer solution and 140 μL of the compound AMA-6 solution and 140 μL of the MPS solution were mixed to prepare a reaction mixture with a total amount of 700 μL. The reaction mixture was incubated at room temperature while shielding from light. Aliquots of the reaction mixture were taken on day 0 (before the reaction) and 1 day, 2 days, 4 days, and 7 days after the reaction, with each aliquot having a volume of 70 μL. The remaining compound AMA-9c and MPS were then quantified by HPLC analysis, indicating the stability of AMA-9c in PBS buffer (see Figure 2 ).

[0915] Example 64. Plasma Stability Study of N-Ac-Cys-AMA-10

[0916] The compounds N-Ac-Cys-AMA-10 and methyl phenyl sulfone (used as standard substances) were dissolved in DMSO to a concentration of 30 mM. Then, each of human plasma (Biochemed 752PR-SC-PMG) and mouse plasma (Biochemed 029-APSC-MP) was mixed with N-Ac-Cys-AMA-10 and MPS to give a final concentration of N-Ac-Cys-AMA-10 and methyl phenyl sulfone of 300 μM. The resulting plasma mixture was incubated in a water bath at 37°C. Aliquots were taken before the reaction and 1 day, 2 days, 4 days and 7 days after the reaction, each aliquot being 200 μL. To complete the reaction, two volumes of acetonitrile were added, followed by brief vortexing and centrifugation to obtain a plasma protein precipitate. Each supernatant obtained after centrifugation was collected and analyzed by HPLC. As Figure 4 As shown, the compound was detected and quantified in mouse and human plasma for up to 7 days. The study demonstrated that N-Ac-Cys-AMA-10 has high stability in plasma over the course of 7 days.

[0917] Example 65. Relative Reaction Rates of "Reference A", PyrMPS-1, and mMPS-4 with N-Ac-Cysteine

[0918]

[0919] To a homogeneous solution of "reference A", PyrMPS-1 and mMPS-4 (10 μL, stock solution 10 mmol) in PBS buffer (870 μL) and DMSO (90 μL) was added N-acetyl-L-cysteine ​​(10 μL, stock solution 10 mmol) and stirred at room temperature for 1 hour. The reaction rate of the resulting sample was checked using LC / MS.

[0920] Compound name Relative reaction rate (Reference A) 1 pyrMSP-1 Increased 11.5 times mMPS-4 Increased 7 times

[0921] The introduction of the meta-substituent improves the solubility of the compound and provides a rapid reaction with the thiol group.

[0922] Incorporated by reference

[0923] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0924] Equivalent solutions

[0925] While specific embodiments of the present invention have been discussed, the above description is illustrative and not restrictive. Many variations of the present invention will be apparent to those skilled in the art after reading this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, their full scope of equivalents, and the specification, along with such variations.

Claims

1. A compound of formula (I): or a salt thereof, wherein: A is M is N, CR 30 or C(-LQ), where L is coupled to C via an electron-withdrawing group; Each L is independently selected from a spacer moiety; Each Q is independently selected from active moieties and reactive groups; X is selected from -Cl, -Br and -I; J is the targeting moiety; R 30 It is an electron-withdrawing group; R 31 is selected from electron withdrawing groups, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic, heterocyclic and haloalkyl groups, wherein R 30 and R 31 At least one of the is present and is an electron-withdrawing group; R 46 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 42 and R 43 Each independently selected from -OH, alkoxy, -NR 44 R 45 , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic and heterocyclic groups, wherein R 44 and R 45 Together with the nitrogen atom to which they are attached, they may form a 5- to 8-membered ring, optionally fused to an aryl or heteroaryl ring; R 32 、R 44 and R 45 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl; R 47 Selected from O - , alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, and heterocyclyl; and n is 1 to 4.

2. The compound of claim 1, wherein M is N.

3. The compound of claim 1, wherein M is CR 30 , and where R 30 It is an electron-withdrawing group.

4. The compound of claim 1, wherein A is selected from And where R 31 It is an electron-withdrawing group.

5. The compound of claim 1, wherein M is C(-LQ), and wherein L is coupled to C via an electron withdrawing group selected from an amide or an ester.

6. The compound of claim 1, wherein R 30 Yes-CONR 33 R 34 or -CO2R 35 , and R 33 、R 34 and R 35 Each is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

7. The compound of claim 1, wherein: Each electron withdrawing group is independently selected from -NO2, -CN, -haloalkyl, -CONR 33 R 34 、-CO2R 35 、-C(=O)R 36 、-S(=O)R 37 、-S(=O)2OR 38 and -NR 39 R 40 R 41 ;and R 36 、R 37 、R 38 、R 39 、R 40 and R 41 Each is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, and haloalkyl.

8. The compound of claim 7, wherein each electron withdrawing group is independently selected from -CN, -CONR 33 R 34 and -CO2R 35 .

9. The compound of claim 8, wherein each electron withdrawing group is independently selected from -CN, -CONH2 and -CO2Me.

10. The compound of claim 1, wherein Q is an active moiety.

11. The compound of claim 1, wherein Q comprises L' and Q', wherein L' is a linker and Q' is an active agent. The compound of claim 11 , wherein L′ comprises a coupling group, wherein the coupling group is coupled to L.

13. The compound of claim 12, wherein the coupling group is selected from -C(=O)NR 32 -、-C(=O)O-、-C(=NR 32 )-、-C=NO-、-NR 32 -C(=O)-NR 32 -, -OC(=O)O-, -SS-, -NR 32 S(=O)2O- and -OS(=O)2O-.

14. The compound of claim 12, wherein the coupling group is selected from 15. The compound of claim 12, wherein L' further comprises a cleavable group, wherein the cleavable group is coupled to Q'.

16. The compound of claim 15, wherein the cleavable group coupled to Q' is selected from in R 49 is hydrogen or -C(=O)R 50 ;and R 50 It is hydrogen or lower alkyl.

17. The compound of claim 12, wherein L' further comprises C6-C 100 An alkylene group containing at least one group selected from the group consisting of -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-.

18. The compound of claim 12, wherein L comprises C6-C 100 An alkylene group containing at least one group selected from the group consisting of -NH-, -C(=O)-, -O-, -S-, -S(O)-, and -S(=O)2-.

19. The compound of claim 12, wherein L comprises in a is a bond to the M-containing aromatic ring, and b is a bond to L'; and n is 2 to 20.

20. The compound of claim 11, wherein Q' is a hormone, an oligonucleotide, a toxin, an affinity ligand, a probe for detection, or a combination thereof.

21. The compound of claim 11, wherein Q' is selected from a cytokine, an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic, or a combination thereof.

22. The compound of claim 1, wherein Q is a reactive group.

23. The compound of claim 22, wherein the reactive group is selected from -N3, -C≡CH, -S(O)2Hal, -NH2, -CO2Hal, -OH, -C(O)H, -SH, -N=C=O and -N=S=C, wherein Hal is -Cl, -Br or -I.

24. The compound of claim 1, wherein the targeting moiety comprises an -S- moiety.

25. The compound of claim 24, wherein the targeting moiety is coupled to the remainder of the compound of formula (I) via the -S- moiety.

26. The compound of claim 1, wherein A is 27. The compound of claim 26, wherein A is 28. The compound of claim 27, wherein R 31 -CN, -CO2NR 33 R 34 or -CO2R 35 .

29. The compound of claim 26, wherein A is 30. The compound of claim 29, wherein R 32 is hydrogen or C 1-3 alkyl.

31. The compound of claim 26, wherein A is 32. The compound of claim 31, wherein R 46 is an optionally substituted C 1-3 Alkyl, optionally substituted C6-C 12 aryl or optionally substituted heteroaryl.

33. The compound of claim 26, wherein A is 34. The compound of claim 33, wherein R 47 Yes O - or C 1-3 alkyl.

35. The compound of claim 26, wherein A is 36. The compound of claim 1, wherein the compound is selected from 37. The compound of claim 1, wherein A is 38. The compound of claim 37, wherein A is 39. The compound of claim 37, wherein A is 40. The compound of claim 37, wherein A is 41. The compound of claim 40, wherein A is 42. The compound of claim 41, wherein R 42 Is -OH or -NR 44 R 45 .

43. The compound of claim 37, wherein the targeting moiety comprises a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment or a repeat of an antigenic polypeptide.

44. The compound of claim 43, wherein the targeting moiety comprises an antibody, such as an antibody selected from the group consisting of complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments, single-chain Fv (scFv) mutants, multispecific antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant portion of an antibody, and modified immunoglobulin molecules comprising an antigen recognition site.

45. The compound of claim 44, wherein the targeting moiety comprises an antibody selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I 131 , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegol, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI-Ig, second-generation anti-CD20, ACZ-885, tocilizumab, atezolizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), tesimumab, MDX-010, IDEC-114, ointuzumab, HuMax EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, prigovinomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, ifenguzumab, orolab, rixibacumab, third-generation anti-CD20, LY2469298, and veltuzumab.

46. ​​A method of preparing a conjugate, the method comprising reacting a compound as claimed in any one of claims 26 to 36 with a reagent comprising a targeting moiety covalently bound to a Michael donor, thereby producing a Michael adduct.

47. The method of claim 46, further comprising reducing the Michael adduct to produce the compound of any one of claims 40 to 42.

48. The method of claim 46, wherein the Michael donor covalently bound to the targeting moiety is selected from the group consisting of: -SH, -NH2, -OH, in R is C 1-3 Alkyl or C 1-3 Alkoxy.

49. The method of claim 46, wherein the targeting moiety comprises a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment or a repeat of an antigenic polypeptide.

50. The method of claim 49, wherein the targeting moiety comprises an antibody selected from the group consisting of an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, and a modified immunoglobulin molecule comprising an antigen recognition site.

51. The method of claim 50, wherein the targeting moiety comprises an antibody selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I 131 , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegol, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI-Ig, second-generation anti-CD20, ACZ-885, tocilizumab, atezolizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), tesimumab, MDX-010, IDEC-114, ointuzumab, HuMax EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, prigovinomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, ifenguzumab, orolab, rixibacumab, third-generation anti-CD20, LY2469298, and veltuzumab.

52. A pharmaceutical composition comprising a compound according to any one of claims 37 to 45 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

53. Use of the compound of any one of claims 37 to 45 or a pharmaceutically acceptable salt thereof, or the composition of claim 52, in the manufacture of a medicament for the treatment of a disease or condition.

54. The use of claim 53, wherein the disease or condition is selected from cancer, infectious disease or autoimmune disease.

55. The use of claim 54, wherein the disease or condition is cancer.

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