Biocatalytic synthesis of cryptophycin anticancer agents

Abstract

The present disclosure provides cryptophycin intermediates, cryptophycin analogs, and cryptophycin chimeric molecules useful in the treatment of cancer, as well as methods of making these compounds and methods of treating cancer.

Description

[Technical Field]

[0001] The present disclosure relates generally to analogs of naturally occurring compounds and their uses, such as in methods for treating, reducing the risk of, or ameliorating the symptoms of cancer, such as colon cancer. [Background technology]

[0002] Government Interest Statement This invention was made with government support under Grants GM076477 and GM118101 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and their hybrids (PKS / NRPSs) are modular proteins that generate a myriad of complex natural products. These proteins select groups of relatively simple chemical building blocks (malonates, malonate derivatives, and / or amino acids) to construct diverse scaffolds with a wide range of biological activities. 1~2 A large class of these natural products is the macrocycles 3 contain constrained ring structures that can lock them into their biologically active conformation and protect the peptide bond from degradation. 4 The biosynthesis of these important molecules is typically terminated by α,β-hydrolases that utilize a thioesterase (TE) domain, a serine, histidine, aspartate catalytic triad, resulting in regio- and stereospecific cyclization. 4~5 The molecular mechanisms by which these enzymes govern selectivity and organize linear substrates for productive macrocyclization over hydrolysis are poorly understood, which has limited our ability to predict and ultimately tailor these enzymes for use as a wide range of biocatalysts. Harnessing these distinct enzymes will be an unparalleled asset in the continued pursuit of methodologies to expand the existing synthetic toolbox for medicinal chemistry exploration. 6~8

[0004] Pioneering studies on PKS and NRPS TEs, consisting of both structural studies and in vitro biochemical analyses, have provided important insights into the complex catalytic mechanisms that mediate cyclization. The NRPS-derived tyrocidine and daptomycin TE systems utilized solid-phase peptide synthesis to formulate a series of unnatural chain-elongation intermediates that provided novel macrocyclic analogs with diverse biological activities. 9~11 This substrate tolerance is attributed to a hydrogen-bonding network that allows preorganization of the substrate. Therefore, TE was hypothesized to have minimal effect, except for amino acids used as intramolecular nucleophiles, which may be important for productive catalysis. 12 This is further supported by structural studies of NRPS TEs from the surfactin and fengycin biosynthetic pathways, each of which possesses a large, bowl-shaped active site composed primarily of nonpolar and aromatic amino acids, with specific interactions occurring primarily at the hydroxyl-bearing C-terminus of linear NRPS substrates. 13~14

[0005] Studies on PKS TEs of the erythromycin (DEBS) and pikromycin (Pik) pathways have also benefited from structural and biochemical examinations. This work shows that a complex interplay of both specific functional group interactions and the hydrophobic packing seen in more channel-type active sites is required for productive cyclization. 15~17 Despite some necessary constraints on both DEBS and PikTE, some flexibility was observed in the non-essential elements, as shown by the combinations of docking and synthetic substrates that allowed for formulating new macrocyclic products. 16、18~19 Using rational engineering, a single active site mutation was generated that altered the underlying catalytic mechanism of PikTE, promoting the productive macrocyclization of a previously inaccessible 12-membered macrolactone. 20These compelling studies continue to highlight the complexity of macrocyclization in a variety of systems. Further investigation of PKSs, NRPSs, and PKS / NRPS hybrid TEs is necessary to better understand the factors that govern macrocyclization on a large scale. Harnessing this knowledge has already facilitated protein engineering efforts that have expanded the flexibility of TE substrates, and continued efforts along these lines are expected to facilitate the generation of more active, pharmacologically relevant small molecule libraries.

[0006] Cryptophycins are a class of depsipeptide natural products produced by mixed PKS / NRPS biosynthetic systems. 21~22 They were first identified as potent antifungals in Nostoc sp. ATCC 53789. 23 , and was subsequently rediscovered as one of the most potent antiproliferative microtubule-binding agents in Nostoc sp. GSV 224. 24 Initial medicinal chemistry screening efforts demonstrated that the β-epoxide functionality was required for maximal activity, and that most modifications were detrimental to efficacy. A synthetic analog, cryptophycin 52 (Figure 1), containing a geminal-dimethyl functionality in unit C, was introduced into clinical trials for the treatment of platinum-resistant ovarian cancer. 25~27 Despite showing significant disease stabilization and overall positive results, the trial was discontinued due to dose-limiting peripheral neuropathy and lack of broad in vivo efficacy. 25、28 Although clinical trials of cryptophycin 52 have concluded, this class of metabolites remains a compelling scaffold for further lead optimization, as it is particularly effective against difficult-to-treat drug-resistant cancers. 29 Recently, cryptophycin has been used as a therapeutic agent for antibody-drug conjugates (as well as RGD peptides) because it may circumvent the systemic toxicity observed with these compounds. 30 and folic acid 31 It has been widely investigated as a potential payload for other direct targeting agents, including conjugation with . 32~36Although there remains great interest in cryptophycins as anticancer drugs, challenges in lead optimization, as well as costly synthetic efforts for their production, have hindered exploration. Based on previous indications of the potent functions of CrpTE, 21 , motivated to expand the chemoenzymatic approach for continued analog generation and biological evaluation, particularly in the unexplored area of ​​the cryptophycin scaffold.

[0007] The final step in cryptophycin biosynthesis is CrpTE-mediated macrocyclization of the chain-elongation intermediate. 21 We showed that the excised CrpTE can catalyze the facile cyclization of natural and modified substrates to varying degrees, incorporating cryptophycins 3, 51 (Figure 1), and a non-natural cryptophycin containing a terminal olefin in unit A, originally synthesized at Eli Lilly as LY404291. 37 Furthermore, CrpTE also demonstrated utility in the production of cryptophycins 24 and 51 by further engineering it in trans with the upstream NRPS module, CrpD-M2. 22 These studies demonstrated the unique intrinsic flexibility of CrpTE and its potential as a versatile biocatalyst for the generation of novel cryptophycin analogs of medical importance. Toward that end, we undertook efforts to generate a series of novel cryptophycin chain-elongation intermediates designed to probe CrpTE substrate tolerance and generate analogs that could address limitations identified in clinical trials (peripheral neuropathy and dose-limiting broad in vivo efficacy). 25、28

[0008] In view of the foregoing observations, it is apparent that a need continues to exist in the art for methods and substrates that are useful in synthesizing and using compound analogs, such as cryptophycin analogs, in treating cancer, such as colorectal cancer, reducing the risk of developing it, or ameliorating symptoms associated therewith. Summary of the Invention

[0009] The present disclosure provides compounds of formula I:

[0010] [ka]

[0011] wherein Ar is a 5-7 membered heterocyclic aryl group having 1-3 ring heteroatoms selected from N, O, and S, and optionally C 1-5 and substituted with 1 to 3 substituents independently selected from alkyl and L-R6, wherein R1 is chlorine, bromine, or iodine, R2 is OH or OC(O)CH2NHR, or R1 and R2 together (1) represent a double bond between the carbons to which they are attached, or (2) form a β-epoxide ring with the carbon to which they are attached, and each of R3 and R4 is independently selected from H, C 1-6 Alkyl, C 0-6 Alkylene-OH or C 0-6 alkylene-NH(R), or R and R together with the carbon atoms to which they are attached form a spiro C 3-5 forming a cycloalkyl or spiro 3- to 5-membered heterocycloalkyl having one nitrogen ring atom, R5 is C 1-6 Alkyl, C 0-6 Alkylene-OH, or C 0-6 alkylene-NH(R), where R is H, C 1-6 alkyl, or L-R6, where L is a linker, R6 is a reactive chemical group, and X is O, NH, or NMe, provided that the compound or salt contains 0 or 1 L-R6.

[0012] In some embodiments, Ar comprises pyridyl, pyrazinyl, imidazolyl, or oxazolyl optionally substituted with 1 to 3 substituents selected from methyl and isopropyl.

[0013] In various embodiments, (1) Ar is

[0014] [ka]

[0015] and R3 is CH3 and R4 is H; (2) Ar is

[0016] [ka]

[0017] wherein R3 is CH3, R4 is H or CH3, (3) Ar is

[0018] [ka]

[0019] wherein R3 is CH3, R4 is H or CH3, (4) Ar is

[0020] [ka]

[0021] where R3 is CH3, R4 is H, (5) Ar is

[0022] [ka]

[0023] wherein R3 is CH3, R4 is H, (6d) Ar is

[0024] [ka]

[0025] where R3 is CH3, R4 is H, (7) Ar is

[0026] [ka]

[0027] wherein R3 is CH3, R4 is H or CH3, (8) Ar is

[0028] [ka] wherein R3 is CH3, R4 is H or CH3, (9) Ar is

[0029] [ka]

[0030] and R3 is CH3 and R4 is H, or (10) Ar is

[0031] [ka]

[0032] where R3 is CH3 and R4 is H.

[0033] Further provided herein are methods of producing the compounds and salts described herein, comprising contacting a secocryptophycin intermediate with a cryptophycin thioesterase under conditions suitable for macrocyclization to form the compound or salt.

[0034] Also provided herein are conjugates comprising a compound or salt described herein and a peptide, protein, or antibody. In some cases, the antibody and the compound or salt are covalently bonded via a reactive chemical group on the compound or salt and a complementary reactive group on the antibody.

[0035] Other aspects of the present disclosure include pharmaceutical compositions comprising the compounds disclosed herein, the compounds disclosed herein for use in the preparation of a medicament for treating or preventing cancer in a subject, and the use of the compounds disclosed herein in a method of treating cancer in a subject.

[0036] Other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0037] [Figure 1] 1 depicts the structures of selected cryptophycin analogs. [Figure 2-1] TOF-LCMS analysis of analytical-scale CrpTE cyclization reaction of analogue 23 a–m, with starting material peaks in black, product peaks in blue, and hydrolysis by-products in green. [Figure 2-2] TOF-LCMS analysis of analytical-scale CrpTE cyclization reaction of analogue 23 a–m, with starting material peaks in black, product peaks in blue, and hydrolysis by-products in green. [Figure 3] A) Schematic diagram of the synthesis of the unit A fragment. B) Schematic diagram of the synthesis reaction of the two units. [Figure 4] Schematic of the synthesis of the unit B fragment. [Figure 5] Schematic of the synthesis of the unit AB fragment. [Figure 6] A) Schematic diagram of the synthesis of unit CD fragments. B) Schematic diagram of the unit CD synthesis reaction. [Figure 7] Schematic of the elaboration of the secocryptophycin intermediate. [Figure 8-1] Percent conversion, isolated yield, and cyclization for hydrolysis analysis of Crp TE reactions. [Figure 8-2]Percent conversion, isolated yield, and cyclization for hydrolysis analysis of Crp TE reactions. [Figure 8-3] Percent conversion, isolated yield, and cyclization for hydrolysis analysis of Crp TE reactions. [Figure 9] IC50 values ​​of cryptophycin analogs in HCT-116 human colon cancer. DETAILED DESCRIPTION OF THE INVENTION

[0038] Cryptophycins are a family of macrocyclic depsipeptides that exhibit highly potent antiproliferative activity against difficult-to-treat, drug-resistant cancers. Recently, these molecules have continued to attract attention as potential payloads for antibody-drug conjugates, highlighting the continued need for alternative synthetic strategies and analogs to facilitate the conjugation of these tubulin-binding agents to appropriate biomolecules. The unique challenges facing the synthesis and derivatization of this complex group of molecules have led us to investigate chemoenzymatic syntheses designed to access more promising analogs.

[0039] Cryptophycin thioesterase (CrpTE) is a versatile enzyme that catalyzes the macrocyclization of over 20 natural cryptophycin metabolites, enabling us to envision drug development strategies that involve its use as a unique biocatalyst for the generation of unnatural derivatives. To this end, we developed a scalable synthesis of 12 new unit-ABCD linear chain-elongation intermediates containing a heteroaromatic group as a replacement for the benzyl group of the natural unit A. The N-acetylcysteamine-activated forms of each intermediate were evaluated for turnover to macrocyclic products using wild-type, excised CrpTE, demonstrating the exceptional flexibility of this enzyme. Semi-preparative-scale reactions were performed for isolation, structural characterization, and further biological evaluation, revealing des β-epoxy analogs with low picomolar potency.

[0040] The work disclosed herein enabled the identification and biological evaluation of one of the most potent cryptophycin analogs generated to date, which contains a styrene functionality and eliminates the need for an epoxide group to achieve low pM potency. The valuable insights into the selectivity and specificity of CrpTE toward unnatural substrates from this work establish CrpTE as a powerful, broad-spectrum catalyst that can be used both in unique contexts and as part of larger enzyme cascades for the generation of biologically active molecules.

[0041] More specifically, based on the latest technology, the selectivity of CrpTE towards non-natural substrates was investigated. 37 The observed cyclization-to-hydrolysis ratios between native chain-elongation intermediates and substrates containing terminal olefins indicated that an aryl ring was required for optimal cyclization, but it remained unclear whether aryl rings of various sizes and functional group substitutions would be well tolerated. 37 This site was chosen for manipulation because it represents an unexplored area in structure-activity relationship studies (SAR) of cryptophycins and is an advantageous location for the use of tactical bioisosteres that could increase water solubility, resulting in better in vivo efficacy as well as potentially reduced toxicity. 26、28 Utilizing CrpTEs to formulate these novel cryptophycins will enable the generation of important new analogs and also provide unique insight into the enzyme's flexibility for the incorporation of non-natural starter units. A better understanding of the recognition factors employed in the selectivity of these enzymes will further expand our ability to design TEs with tunable substrate tolerance for use in biocatalytic platforms to aid the drug discovery process.

[0042] Disclosed herein are compounds and the use of these compounds in treating cancer.One aspect of the present disclosure generally relates to the use of the compounds or pharmaceutically acceptable salts described herein, or pharmaceutically acceptable compositions comprising such compounds or pharmaceutically acceptable salts thereof, for treating cancer, such as colon, breast, leukemia, prostate, ovarian, central nervous system or non-small cell lung cancer in a subject.

[0043] Compounds of the Disclosure The present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof:

[0044] [ka]

[0045] During the ceremony, Ar is a 5-7 membered heterocyclic aryl group having 1-3 ring heteroatoms selected from N, O, and S, and optionally C 1-5 substituted with 1 to 3 substituents independently selected from alkyl and L-R6; R1 is chlorine, bromine, or iodine; R2 is OH or OC(O)CH2NHR, or R1 and R2 together (1) represent a double bond between the carbons to which they are attached, or (2) form a β-epoxide ring with the carbons to which they are attached; R3 and R4 each independently represent H, C 1-6 Alkyl, C 0-6 Alkylene-OH or C 0-6 alkylene-NH(R), or R3 and R4, together with the carbon atoms to which they are attached, form a spiro C 3-5 forming a cycloalkyl or spiro 3- to 5-membered heterocycloalkyl having one nitrogen ring atom, R5 is C 1-6 Alkyl, C 0-6 Alkylene-OH, or C 0-6 alkylene-NH(R), R is H, C 1-6 alkyl, or L-R6; L is a linker, R6 is a reactive chemical group, X is O, NH, or NMe; provided that the compound or salt contains 0 or 1 L-R6.

[0046] Ar is a 5-7 membered heterocyclic aryl group having 1-3 ring heteroatoms selected from N, O, and S, and optionally C 1-5 Heterocyclic aryl groups are substituted with 1 to 3 substituents independently selected from alkyl and L-R6. As used herein, the term "heterocyclic aryl" refers to a monocyclic aromatic ring having a total of 5 to 7 ring atoms and containing 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur atoms in the aromatic ring. Unless otherwise indicated, heterocyclic aryl groups can be unsubstituted or substituted with one or more, particularly 1 to 3, substituents. In some cases, heterocyclic aryl groups can be substituted with C, as discussed herein. 1-5 It is substituted with alkyl or L-R6 moieties. Examples of heterocyclic aryl groups include, but are not limited to, thienyl, furyl, pyridyl, pyrrolyl, oxazolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl. In some cases, heterocyclic aryl (Ar) includes pyridyl, pyrazinyl, imidazolyl, or oxazolyl, optionally substituted with 1 to 3 substituents selected from methyl and isopropyl. In some cases, Ar is substituted with L-R6.

[0047] As used herein, the term "alkyl" refers to straight-chain and branched saturated hydrocarbon groups containing 1 to 30 carbon atoms, e.g., 1 to 20 carbon atoms, or 1 to 10 carbon atoms. nThe term "n" means that the alkyl group has "n" carbon atoms. For example, C4 alkyl refers to an alkyl group having 4 carbon atoms. C1-C6 alkyl refers to alkyl groups having numbers of carbon atoms throughout the entire range (e.g., 1 to 6 carbon atoms) and all subgroups (e.g., 1 to 6, 2 to 5, 1 to 5, 3 to 6, 1, 2, 3, 4, 5, and 6 carbon atoms). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl (1,1-dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl. Unless otherwise indicated, alkyl groups can be unsubstituted or substituted alkyl groups.

[0048] As used herein, the term "alkylene" refers to an alkyl group that has a substituent. For example, the term "alkylene-OH" refers to an alkyl group that is substituted with a hydroxy group. For example, an alkylene group can be -CHCH- or -CH-. n The term alkylene means that the group has "n" carbon atoms. For example, C 1-6 Alkylene refers to an alkylene group having any number of carbon atoms, including all ranges and subgroups. The term C0 indicates a direct bond, and -C0 alkylene-OH indicates an OH substituent. Unless otherwise indicated, an alkylene group can be an unsubstituted or substituted alkylene group.

[0049] As used herein, the term "cycloalkyl" refers to an aliphatic cyclic hydrocarbon group containing 3 to 12 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms). nThe term "cycloalkyl" means that the cycloalkyl group has "n" carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group having 5 carbon atoms in the ring. C3-C5 cycloalkyl refers to a cycloalkyl group having a number of carbon atoms, including the entire range (e.g., 3 to 5 carbon atoms) and all subgroups (e.g., 3 to 4, 4 to 5, 2, 4, and 5 carbon atoms). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be unsubstituted or substituted. The cycloalkyl groups described herein can be isolated, spirocyclic, or fused to another cycloalkyl group, heterocycloalkyl group, aryl group, and / or heteroaryl group. Unless otherwise indicated, when a cycloalkyl group is fused to another cycloalkyl group, each cycloalkyl group can contain 3 to 12 carbon atoms. Unless otherwise indicated, a cycloalkyl group can be unsubstituted or substituted.

[0050] As used herein, the term "heterocycloalkyl" is defined similarly to cycloalkyl, except that the ring contains 1 to 3 heteroatoms independently selected from oxygen, nitrogen, and sulfur. Specifically, the term "heterocycloalkyl" refers to a ring containing a total of 3 to 10 atoms (e.g., 3 to 5, or 5 to 10), of which 1, 2, 3, or 3 atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur, and the remaining atoms in the ring are carbon atoms. In some cases, a heterocycloalkyl is a spiro 3- to 5-membered ring having one nitrogen ring atom. Non-limiting examples of heterocycloalkyl groups include azetidine, aziridine, pyrrolidine, piperidine, pyrazolidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and the like. In some cases, a heterocycloalkyl includes azetidine, aziridine, or pyrrolidine. A heterocycloalkyl can be substituted or unsubstituted.

[0051] As used herein, the term "substituted," when used to modify a chemical functional group, refers to the replacement of at least one hydrogen radical on the functional group with a substituent. The substituent may include, but is not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, oxy, alkoxy, heteroalkoxy, ester, thioester, carboxy, cyano, nitro, amino, amido, acetamido, and halo (e.g., fluoro, chloro, bromo, or iodo). When a chemical functional group contains multiple substituents, the substituents can be attached to the same carbon atom or to two or more different carbon atoms.

[0052] As used herein, the term "linker" refers to a moiety that connects two portions of a molecule (e.g., compounds described herein and salts thereof). Non-limiting examples of linkers include peptides having 1 to 10 amino acids, polyethylene glycol having 1 to 15 ethylene glycol monomers, and β-glucuronic acid. In some cases, L comprises a peptide having 1 to 10 amino acids. In some cases, the amino acids are selected from the group consisting of alanine (Ala), citrulline (Cit), glutamine (Gln), glycine (Gly), lysine (Lys), acetyl-lysine (AcLys), proline (Pro), phenylalanine (Phe), and valine (Val). In some cases, L comprises polyethylene glycol having 1 to 15 (e.g., 5 to 15, 3 to 10, 3 to 15, 5 to 10) ethylene glycol monomers. In some cases, L comprises β-glucuronic acid. In some cases, the β-glucuronic acid comprises a furanose. In some cases, the β-glucuronic acid comprises a pyranose.

[0053] In some cases, L is the structure

[0054] [ka]

[0055] It has.

[0056] In some cases, L is attached to the compound of Formula (I) via a single (i.e., direct covalent) bond, an ester bond, an amide bond, a sulfide bond, a disulfide bond, a para-aminobenzyl (PAB) group, or a para-aminobenzyloxycarbonyl (PABC) group. In some cases, L is attached via a single bond. In some cases, L is attached via an ester bond. In some cases, L is attached via an amide bond. In some cases, L is attached via a para-aminobenzyl (PAB) group.

[0057] As used herein, the term "reactive chemical group" refers to a chemical functional group that can react with another complementary functional group to form a covalent bond. The reactive group can be an amine, such that in the presence of a complementary functional group (e.g., a carboxylic acid, an acyl chloride, etc.), an amide functional group can be formed. Some specific, non-limiting examples of reactive chemical groups are maleimide groups, maleimidocaproyl groups, maleimidoPEG groups, bromoacetamide groups, N-hydroxysuccinimide esters, and O-alkylhydroxylamines. In some cases, R6 comprises a maleimide group. In some cases, R6 comprises a maleimidocaproyl group. In some cases, R6 comprises a maleimidoPEG group. In some cases, R6 comprises an N-hydroxysuccinimide ester. In some cases, R6 comprises an O-alkylhydroxylamine.

[0058] In some cases, (1) Ar is

[0059] [ka]

[0060] and R3 is CH3 and R4 is H; (2) Ar is

[0061] [ka]

[0062] wherein R3 is CH3, R4 is H or CH3, (3) Ar is

[0063] [ka]

[0064] wherein R3 is CH3, R4 is H or CH3, (4) Ar is

[0065] [ka]

[0066] where R3 is CH3, R4 is H, (5) Ar is

[0067] [ka]

[0068] where R3 is CH3, R4 is H, (6) Ar is

[0069] [ka]

[0070] where R3 is CH3, R4 is H, (7) Ar is

[0071] [ka]

[0072] wherein R3 is CH3, R4 is H or CH3, (8) Ar is

[0073] [ka]

[0074] wherein R3 is CH3, R4 is H or CH3, (9) Ar is

[0075] [ka]

[0076] and R3 is CH3 and R4 is H, or (10) Ar is

[0077] [ka]

[0078] and R3 is CH3 and R4 is H. In some cases, Ar is

[0079] [ka]

[0080] and R3 is CH3 and R4 is H. In some cases, Ar is

[0081] [ka]

[0082] and R is CH and R is H or CH. In some cases, Ar is

[0083] [ka]

[0084] and R is CH and R is H or CH. In some cases, Ar is

[0085] [ka]

[0086] and R3 is CH3 and R4 is H. In some cases, Ar is

[0087] [ka]

[0088] and R3 is CH3 and R4 is H. In some cases, Ar is

[0089] [ka]

[0090] and R3 is CH3 and R4 is H. In some cases, Ar is

[0091] [ka]

[0092] and R is CH and R is H or CH. In some cases, Ar is

[0093] [ka]

[0094] and R is CH and R is H or CH. In some cases, Ar is

[0095] [ka]

[0096] and R3 is CH3 and R4 is H. In some cases, Ar is

[0097] [ka]

[0098] where R3 is CH3 and R4 is H.

[0099] In some cases, R1 and R2 together represent a double bond between the carbons to which they are attached. In some cases, R1 and R2 together form a β-epoxide ring with the carbons to which they are attached. In some cases, R1 is Cl and R2 is OH. In some cases, R1 is Cl and R2 is OC(O)CH2NH2.

[0100] In some cases, (1) Ar is

[0101] [ka]

[0102] wherein R3 is CH3, R4 is H or CH3, R5 is CH(CH3)2, and (2) Ar is

[0103] [ka]

[0104] and R3 is CH3 、 R4 is H or CH3, R5 is CH(CH3)2, or (3) Ar is

[0105] [ka]

[0106] and R3 is CH3 and R4 is H or CH3 、 R5 is CH(CH3)2. In some cases, Ar is

[0107] [ka]

[0108] wherein R3 is CH3, R4 is H or CH3, and R5 is CH(CH3)2. In some cases, Ar is

[0109] [ka]

[0110] wherein R3 is CH3, R4 is H or CH3, and R5 is CH(CH3)2. In some cases, Ar is

[0111] [ka]

[0112] wherein R3 is CH3, R4 is H or CH3, and R5 is CH(CH3)2.

[0113] In some cases, R3 is NH2, NHMe, CH2-NH2, or CH2-NHMe. In some cases, R3 is NH2. In some cases, R3 is NHMe. In some cases, R3 is CH2-NH2. In some cases, R3 is CH2-NHMe. In some cases, R3 is OH or CH2-OH. In some cases, R3 is OH. In some cases, R3 is CH2-OH.

[0114] In some cases, R5 is NH2, NHMe, (CH)CH3NH2, or (CH)CH3NHMe. In some cases, R5 is NH2. In some cases, R5 is NHMe. In some cases, R5 is (CH)CH3NH2. In some cases, R5 is (CH)CH3NHMe.

[0115] In some cases, X is O. In some cases, X is NH or NMe. In some cases, X is NH. In some cases, X is NMe.

[0116] In some cases, the compound is selected from the group consisting of:

[0117] [ka]

[0118] In some cases, Ar is

[0119] [ka]

[0120] In some cases, R2 is OC(O)CH2NHL(R6) and R1 is Cl.

[0121] In some cases, R3 is NH-LR6 and R4 is H. In some cases, (1) Ar is

[0122] [ka] where R1 and R2 together represent a double bond or a β-epoxide, R5 is CH(CH3)2, and (2) Ar is

[0123] [ka]

[0124] wherein R1 and R2 together represent a double bond or a β-epoxide, R5 is CH(CH3)2, or (3) Ar is

[0125] [ka]

[0126] wherein R1 and R2 together represent a double bond or a β-epoxide, and R5 is CH(CH3)2, CH(CH3)2. In some cases, Ar is

[0127] [ka]

[0128] wherein R1 and R2 together represent a double bond or a β-epoxide, and R5 is CH(CH3)2. In some cases, Ar is

[0129] [ka]

[0130] wherein R1 and R2 together represent a double bond or a β-epoxide, and R5 is CH(CH3)2. In some cases, Ar is

[0131] [ka]

[0132] where R1 and R2 together represent a double bond or a β-epoxide, and R5 is CH(CH3)2, CH(CH3)2.

[0133] In some cases, R5 is NH-LR6. In some cases, (1) Ar is

[0134] [ka] wherein R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3; (2) Ar is

[0135] [ka]

[0136] wherein R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3, or (3) Ar is

[0137] [ka]

[0138] wherein R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3. In some cases, Ar is

[0139] [ka]

[0140] wherein R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3. In some cases, Ar is

[0141] [ka]

[0142] wherein R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3. In some cases, Ar is

[0143] [ka]

[0144] where R1 and R2 together represent a double bond or a β-epoxide, R3 is CH3, and R4 is H or CH3.

[0145] The compounds disclosed herein may be in the form of pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salts" refers to salts that are suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanoate, and the like. The salts include benzoate, benzoyl peroxide ...Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such salts include alkali metal, alkaline earth metal, aluminum, ammonium, N, and Zn salts. + (C 1-4 Examples of suitable salts include, but are not limited to, salts of alkyl (alkyl) 4 salts and salts of organic bases, such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine. The present invention also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water- or oil-soluble or dispersible products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0146] Also provided herein is a conjugate comprising a compound or salt described herein and a peptide, protein, or antibody.In some cases, the conjugate comprises a compound or salt described herein and an antibody.In some cases, the antibody and the compound or salt are covalently bonded via a reactive chemical group of the compound or salt and a complementary reactive group on the antibody.In some cases, the complementary reactive group on the antibody comprises an amine.In some cases, the amine is the ε-amine of a lysine on the antibody.

[0147] In some cases, the antibody is a monoclonal antibody or a nanobody. In some cases, the antibody is a monoclonal antibody. In some cases, the antibody is a nanobody. In some cases, the monoclonal antibody is brentuximab, cetuximab, gemtuzumab, panitumumab, ofatumumab, rituximab, or trastuzumab. In some cases, the nanobody is a single domain antibody or a camelid antibody.

[0148] Synthesis of Compounds of the Present Disclosure The compounds disclosed herein can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or readily prepared intermediates by employing standard synthetic methods and procedures known to those skilled in the art or in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or standard textbooks in the field. While not limited to any one or several sources, established works such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001, and Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, are useful and recognized reference texts on organic synthesis known to those skilled in the art. For example, the compounds disclosed herein may be synthesized by methods described in Merrifield, J. Am. Chem. Soc. 1963;85:2149; Davis et al., Biochem. Intl. 1985;10:394-414; Larsen et al., J. Am. Chem. Soc. 1993;115:6247; Smith et al., J. Peptide Protein Res. 1994;44:183; O'Donnell et al., J. Am. Chem. Soc. 1996;118:6070; Stewart and Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al., The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al., The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976).The following descriptions of synthetic methods are intended to illustrate general procedures for the preparation of compounds of the present disclosure and are not intended to be limiting.

[0149] The synthetic processes disclosed herein can tolerate a wide variety of functional groups and, therefore, can employ a variety of alternative starting materials. While the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt, ester, or prodrug.

[0150] Also provided are methods of producing the compounds or salts described herein, comprising contacting a secocryptophycin intermediate with a cryptophycin thioesterase under conditions suitable for macrocyclization to form the compound or salt. In some cases, the macrocyclization is macrolactonization.

[0151] In some cases, the cryptophycin thioesterase is derived from a polyketide synthase protein complex, a non-ribosomal protein synthetase protein complex, or a hybrid polyketide synthase / non-ribosomal peptide synthetase protein complex. In some cases, the cryptophycin thioesterase is derived from a hybrid polyketide synthase / non-ribosomal peptide synthetase protein complex.

[0152] In some cases, the method further includes contacting the secocryptophycin with a cryptophycin P450 to form a β-epoxide ring between R1, R2, and the carbon to which they are attached. In some cases, the cryptophycin P450 is a cryptophycin epoxidase.

[0153] Pharmaceutical Compositions, Dosages, and Routes of Administration Further provided is a pharmaceutical composition (sometimes interchangeably referred to herein as a formulation) comprising a compound described herein (e.g., a compound of Formula I or a pharmaceutically acceptable salt of that compound) and a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable" means that a reference substance, such as a compound of the present disclosure, or a composition comprising the compound or a particular excipient, is safe and suitable for administration to a patient or subject. The term "pharmaceutically acceptable excipient" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered. An excipient can be any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API).

[0154] The compound described herein can be administered to a subject in a therapeutically effective amount (for example, an amount sufficient to prevent or alleviate the symptoms of cancer).The compound can be administered alone or as part of a pharmaceutically acceptable composition or preparation.In addition, the compound can be administered once, multiple times, or delivered substantially uniformly over a period of time.It should also be noted that the dose of the compound can vary over time.

[0155] The specific dosing regimen for a particular subject will depend in part on the compound, the amount of compound administered, the route of administration, and the cause and extent of any side effects. The amount of compound administered to a subject (e.g., a mammal such as a human) according to the present disclosure should be sufficient to produce the desired response over a reasonable time frame. The term "therapeutically effective amount" refers to the amount of a compound or combination of therapeutically active compounds that ameliorates, attenuates, or eliminates one or more symptoms of a particular disease or condition (e.g., cancer), or prevents or delays the onset of one or more symptoms of a particular disease or condition. Dosage typically depends on the route, timing, and frequency of administration. Thus, clinicians titrate dosages and modify the route of administration to achieve optimal therapeutic effects; conventional range-finding techniques are known to those skilled in the art.

[0156] By way of example only, this method may involve administering, for example, from about 0.1 mg / kg up to about 100 mg / kg of the compound or more, depending on the factors described above. In other embodiments, the dosage ranges from 1 mg / kg up to about 100 mg / kg, or from 5 mg / kg up to about 100 mg / kg, or from 10 mg / kg up to about 100 mg / kg. In some situations, long-term treatment may be required, with or without the administration of multiple lower doses of the compound. If desired, the compound dose may be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. The duration of treatment depends on the patient's particular condition and type of disease and may last from one day to several months.

[0157] Suitable methods for administering physiologically acceptable compositions, such as pharmaceutical compositions containing compounds disclosed herein (e.g., compounds of Formula I), are well known in the art. Compounds can be administered using more than one route, although certain routes may provide a more rapid and effective response than others. Depending on the situation, pharmaceutical compositions containing compounds are applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and / or introduced into the circulation. For example, in certain situations, it may be desirable to deliver pharmaceutical compositions containing agents orally, by injection intravenously, intraperitoneally, intracerebrally (intraparenchymal), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, intralesionally, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, intestinal, topical, sublingually, urethrally, intravaginally, or rectally, by sustained release systems, or by implanted devices. If desired, compound is locally administered by intrathecal administration, intracerebral (intraparenchymal) administration, intracerebroventricular administration, or intraarterial or intravenous administration, which is delivered to the target region.Alternatively, composition is locally administered by implanting a membrane, sponge, or other suitable material in which the desired compound is absorbed or encapsulated.When using implantation device, this device, in one aspect, is implanted in any suitable tissue or organ, and the delivery of the desired compound is, for example, via diffusion, sustained release bolus, or continuous administration.

[0158] To facilitate administration, compounds are formulated in various embodiments into physiologically acceptable compositions containing carriers (e.g., vehicles, adjuvants, or diluents). The specific carriers employed are limited only by chemical and physical considerations, such as solubility and lack of reactivity with the compound, and the route of administration. Physiologically acceptable carriers are well known in the art. Exemplary pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions (see, for example, U.S. Patent No. 5,466,468). Injectable formulations are further described, for example, in Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Pharmaceutical compositions containing compounds are, in one aspect, placed in a container along with packaging material that provides instructions for use of such pharmaceutical compositions. Generally, such instructions include specific wording describing reagent concentrations and, in certain embodiments, the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be required to reconstitute the pharmaceutical composition.

[0159] Suitable compositions for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into injectable sterile solutions or dispersions.Suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicle examples include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate.Appropriate fluidity can be maintained, for example, by using coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0160] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Microbial contamination can be prevented by adding various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0161] Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) a filler or extender, such as starch, lactose, sucrose, mannitol, and silicic acid; (b) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) a humectant, such as glycerol; (d) a disintegrant, such as agar, calcium carbonate, potato or The granules are mixed with tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (a) solution retarders such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage form may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0162] Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art.Solid dosage forms can also contain opacifying agents.In addition, solid dosage forms can be embedded compositions, so that they release active compound(s) in a delayed manner to a certain part of the intestinal tract.Examples of embedding compositions that can be used are polymeric substances and waxes.Active compound can also be in a microencapsulated form, optionally containing one or more excipients.

[0163] The liquid dosage form for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.In addition to active compound, liquid dosage form can contain the inert diluent commonly used in this field, such as water or other solvent, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, or the mixture of these substances.

[0164] In addition to such inert diluents, compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents. Suspensions can contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances.

[0165] Compositions for rectal administration are preferably suppositories, which can be prepared by mixing a compound of the present disclosure with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol, or a suppository wax which is solid at ordinary room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.

[0166] The compositions used in the methods of the present invention can be formulated in micelles or liposomes. Such formulations include sterically stabilized micelles or liposomes and sterically stabilized mixed micelles or liposomes. Such formulations can facilitate intracellular delivery because the lipid bilayer of liposomes and micelles is known to condense with the plasma membrane of cells and deliver the entrapped contents to intracellular compartments.

[0167] Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The formulation can be easily administered in a variety of dosage forms, such as injection solutions, drug-release capsules, etc.For example, when parenterally administered in aqueous solution, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0168] The frequency of administration depends on the pharmacokinetic parameters and the route of administration of the drug. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990) Mack Publishing Co., Easton, PA, pages 1435-1712, which is incorporated herein by reference. Such formulations may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, appropriate doses are calculated according to body weight, body surface area, or organ size. Further refinement of the calculations required to determine appropriate therapeutic doses can be routinely performed by those skilled in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein and the pharmacokinetic data observed in animal or human clinical trials.

[0169] The exact dose employed will depend on several factors, including the host, whether veterinary or human, the nature and severity of the condition, e.g., the disease or disorder being treated, the mode of administration, and the specific active substance used. The compound can be administered by any conventional route, particularly enterally, and in one embodiment, orally in tablet or capsule form. The administered compound can be in free form or in the form of a pharmaceutically acceptable salt, as appropriate for pharmaceutical use, particularly for the prophylactic or therapeutic treatment of the disease of interest. These measures slow the progression of the disease state and help the body reverse the process in a natural way.

[0170] It will be appreciated that the pharmaceutical compositions and treatment methods of the present invention are useful in human and veterinary medicine. Thus, the subject to be treated is, in one embodiment, a mammal. In a representative embodiment, the mammal is a human.

[0171] In jurisdictions that prohibit patents on methods performed on the human body, the meaning of "administering" a composition to a human subject shall be limited to formulating a controlled substance that the human subject would self-administer by any means (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation consistent with any statute or regulation defining patentable subject matter is intended. In jurisdictions that do not prohibit patents on methods performed on the human body, "administering" a composition includes both the method and the aforementioned activities performed on the human body.

[0172] How to use The compounds described herein (e.g., compounds of Formula I and their conjugates) can be used to treat cancer. As used herein, terms such as "treating," "treat," or "treatment" include preventative (e.g., prophylactic) and palliative treatment. The compounds disclosed herein are particularly advantageous for treating difficult-to-treat or drug-resistant cancers. Non-limiting examples of cancers for which the compounds described herein are useful for treating include colon, breast, leukemia, prostate, ovarian, central nervous system, or non-small cell lung cancer.

[0173] In some cases, the cancer is colon cancer. In some cases, the cancer is breast cancer. In some cases, the cancer is leukemia. In some cases, the cancer is prostate cancer. In some cases, the cancer is ovarian cancer. In some cases, the cancer is central nervous system cancer. In some cases, the cancer is brain cancer. In some cases, the cancer is lung cancer. In some cases, the cancer is non-small cell lung cancer.

[0174] The compounds described herein can be used, for example, to reduce or prevent cancer in human subjects with ovarian cancer. In certain examples, the compound or mixture is administered orally, such as by mixing with distilled water. In another example, the test compound or mixture is administered intravenously, such as with saline or distilled water. In some examples, treatment with the test compound can be a single dose or multiple doses. The test compound can be administered about every 6 hours, about every 12 hours, about every 24 hours (daily), about every 48 hours, about every 72 hours, or about weekly. Treatment with multiple doses can continue for a period of time, for example, from about 1 week to 12 months, for example, from about 1 week to about 6 months, or from about 2 weeks to about 3 months, or from about 1 to 2 months. Administration of the compound can also be continued indefinitely. The dose of the test compound is about 0.1 mg / kg to about 400 mg / kg, for example, about 1 mg / kg to about 300 mg / kg, about 2 mg / kg to 200 mg / kg, about 10 mg / kg to about 100 mg / kg, about 20 mg / kg to about 75 mg / kg, or about 25 mg / kg to about 50 mg / kg.

[0175] Methods for evaluating the effectiveness of test compounds for treating such diseases in cells, suitable animal models, or affected subjects are known to those skilled in the art.As used herein, the terms "patient" and "subject" are used interchangeably and refer to animals such as dogs, cats, cows, horses, and sheep (e.g., non-human animals) and humans.Particular patients or subjects are mammals (e.g., humans).The terms patient and subject include males and females.

[0176] Also provided herein is the use of the compounds disclosed herein in the preparation of a medicament for treating cancer. [Example]

[0177] The present disclosure will be more readily understood by reference to the following examples.

[0178] Example 1 material and method Unless otherwise stated, chemical reagents and solvents were purchased from EMD Millipore, Sigma-Aldrich, Oakwood, Combi blocks, Chem impex, and Thermo-Fisher Scientific. Kanamycin sulfate and isopropyl-β-D-thiogalactopyranoside (IPTG) were obtained from Gold Biotechnology. Lysozyme was purchased from RPI. Imidazole was purchased from AK Scientific. Amicon Ultra centrifugal filters used for protein concentration were purchased from GE Healthcare.

[0179] Deionized water was obtained from a Milli-Q system (EMD Millipore) using a Q-Gard 2 / Quantum Ex Ultrapure organex cartridge. Media components for E. coli growth were purchased from EMD Millipore, Sigma-Aldrich, and Thermo-Fisher Scientific. Glycerol was purchased from BDH through VWR. LB broth and LB agar (Miller) were purchased in pre-made granular form from EMD Millipore. TB broth was made from separately purchased components and consisted of 4% v / v glycerol. Media and solutions were autoclaved or sterile filtered before use. pH was monitored for all solutions using a VWR symphony SB70P pH meter calibrated according to the manufacturer's specifications.

[0180] Chemically competent E. coli Bl21(DE3) was used for protein overexpression. These cells were prepared according to the instructions provided with the MixandGo buffer kit purchased from Zymo Research. Optical density (OD 600 ) was measured using an Eppendorf BioPhotometer.

[0181] Unless otherwise noted, all reactions were carried out in flame-dried glassware under a dry nitrogen atmosphere. Reactions performed at elevated temperatures were controlled by an IKA RET control visc (model RS 232 C), and reactions were carried out at room temperature (rt), approximately 21–23 °C. Reactions performed below room temperature were carried out in an ice bath (0 °C), dry ice / acetone (–78 °C), or isopropanol / ThermoNESLAB (model CC100) for extended periods and / or other temperatures. Commercially available starting materials and reagents were used as received unless otherwise noted. Dichloromethane was purchased as HPLC grade from Fisher and used directly. Tetrahydrofuran, N,N-dimethylformamide, and pyridine were purchased in anhydrous, unstable forms (EMD Millipore DriSolv). Acetone was dried over Na2SO4 before use in reactions. Diisopropylamine and trimethylamine were distilled from calcium hydride immediately before use.

[0182] 1 H NMR spectra were recorded on either a Varian 600 NMR System (600 MHz) or a Varian 400-MR (400 MHz) spectrometer. Chemical shifts are reported in parts per million (ppm) using the solvent resonance as the internal standard (CDCl3 7.26 and CD3OD 3.31 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, h = hextet), coupling constant (Hz), and integral. Proton decoupled. 13C NMR spectra were recorded on a Varian 600 NMR System (150 MHz) or a Varian 400-MR (100 MHz) spectrometer. Chemical shifts are reported in ppm relative to the residual solvent peak (CDCl3, 77.0 ppm or CD3OD, 49.0 ppm). High-resolution mass spectra were obtained on an Agilent Technologies 6500 or 6545 Q-TOF LC / MS. Analytical reactions were evaluated on an Agilent 1290 Infinity II HPLC module (monitoring at 254 nM) connected to a 6230 TOF LC / MS.

[0183] Analytical thin-layer chromatography was performed on EMD Millipore 0.25 mm silica gel. F254 The analysis was performed on plates. Visualization was achieved using a 254 nm UV lamp in combination with either potassium permanganate (KMNO4), cerium ammonium molybdate (CAM), or p-anisaldehyde. Purification was performed by forced-air flash chromatography using EMD Millipore Silica Gel 60 (40-63 μm) or a Biotage Isolera one-flash purification system. Columns used with the Biotage Isolera one purification system include Biotage SNAP Ultra, SiliaSep, SiliaSepHP, and SiliaSep amine cartridges.

[0184] Example 2 chemical synthesis 1. Unit A Synthesis Compound 2 was synthesized over two steps from commercially available S1, as shown in Figure 3B. All spectra were in accordance with previous literature reports. 1、2

[0185] (R)-4-Benzyl-3-((2R,3S)-3-hydroxy-5-((4-methoxybenzyl)oxy)-2 vinylpentanoyl)oxazolidin-2-one (S3). A three-neck flask equipped with an internal temperature probe was charged with a solution of 1 (7.502 g, 30.58 mmol, 1 equiv) in CHCl (305 mL, 0.1 M) and cooled to −78 °C. This was treated with dibutylboron trifluoromethanesulfonate (1 M in DCM, 33.64 mL, 33.64 mmol, 1.1 equiv) and EtN (6.02 mL, 42.8 mmol, 1.4 equiv). After 1 h at −78 °C, the reaction was warmed to 0 °C and stirred for 30 min. The solution was recooled to −78 °C and treated with a solution of aldehyde 2 (8.31 g, 42.81 mmol, 1.4 equiv) in CHCl (25 mL), stirred for 1 h, and warmed to 0 °C. After 1 h at 0 °C, sodium phosphate buffer (pH 7, 500 mM, 30 mL) was added, followed by methanol (30 mL). After 20 min, 30% HO (30 mL) was added, keeping the temperature below 10 °C, and the mixture was stirred at 0 °C for 1 h. The organics were removed under reduced pressure, and the remaining aqueous layer was extracted with ethyl acetate (EtOAc) (3 × 100 mL). The combined organics were washed with 1 N HCl, 5% aqueous NaHCO, brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (33% EtOAc / hexanes) to give 3 (11.98 g, 89% yield) as a clear, colorless oil: R f =0.2 (33% EtOAc / hexane); 1H NMR (600MHz, CDCl3) δ 7.31(ddd, J=7.4, 6.4, 1.3Hz, 2H), 7.28-7.25(m, 2H), 7.24-7.21(m, 2H), 7.20-7.15(m, 2H), 6.04(dddd, J=17.6, 10.0, 9.0, 1.1Hz, 1H), 5.39-5.36(m, 1H), 5.36-5.34(m, 1H), 4.68(ddt, J=11.4, 6.3, 3.0Hz, 1H), 4.58-4.51(m, 1H) ), 4.42(s, 2H), 4.25-4.19(m, 1H), 4.18-4.11(m, 2H), 3.78(d, J=1.1Hz, 3H), 3.69-3.64(m, 1H), 3.64-3.56(m, 1H) ), 3.28-3.17(m, 1H), 2.74(dd, J=13.4, 9.5Hz, 1H), 1.93-1.78(m, 1H), 1.74(dt, J=14.5, 7.0Hz, 1H), 1.55(s, 2H); 13 C NMR (150MHz, CDCl3) δ 173.46, 159.19, 152.90, 135.04, 131.67, 130.14, 129.42, 129.30, 128.91, 127.35, 121.0 0, 113.78, 72.87, 70.54, 67.74, 65.95, 55.25, 55.17, 52.53, 37.58, 33.87;HRMS(ES) calculated value C 25 H 29 NO6[M+Na] 462.1887, actual value 462.1885

[0186] (R)-4-Benzyl-3-((2R,3S)-3-((tert-butyldimethylsilyl)oxy)-5-((4-methoxybenzyl)oxy)-2-vinylpentanoyl)oxazolidin-2-one (3). To a solution of S3 (17.56 g, 39.95 mmol, 1 equiv.) and 2,6-lutidine (46.28 mL, 79.91 mmol, 2 equiv.) in CHCl (135 mL, 0.3 M) was added tert-butylsilyl trifluoromethanesulfonate (11.6 mL, 59.9 mmol, 1.5 equiv.) and stirred. After 18 h at room temperature, HO (100 mL) was added and stirred for 30 min. The organic layer was separated, and the aqueous layer was extracted with CHCl (2 × 100 mL). The combined organics were washed with 1 N HCl, saturated aqueous NaHCO, brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (20% EtOAc / hexanes) to give 3 (18.08 g, 84% yield) as a pale yellow oil: R f =0.6 (33% EtOAc / hexane); 1 H NMR (400MHz, CDCl3) δ 7.33-7.26(m, 3H), 7.25(d, J=8.8Hz, 2H), 7.18(d, J=6.9Hz, 2H), 6.85(d, J=8.6Hz, 2H), 6.00(ddd, J=9, 9.3, 18.2Hz, 1H), 5.27(d, J=10 .2Hz, 1H), 5.26(d, J=18.1Hz, 1H), 4.58(dd, J=6.6Hz, 8.8Hz, 1H), 4.56-4.52(m, 1H), 4.41(d, J=11.4Hz, 1H), 4.36(d, J=11.5Hz, 1H), 4. 21(td, J=5.2Hz, 6.4Hz, 1H), 4.05(dd, J=2Hz, 9.2Hz, 1H), 3.86(t, J=8.2Hz, 1H), 3.78(s, 3H), 3.59(td, J=6.5, 9.2Hz, 1H), 3.48(dt, J= 6.2, 9.4Hz, 1H), 3.23(dd, J=3.0, 13.4Hz, 1H), 2.70(dd, J=9.7, 13.4Hz, 1H), 1.95-1.85(m, 2H), 0.86(s, 9H), 0.02(s, 3H), 0.01(s, 3H); 13C NMR (100MHz, CDCl3) δ 172.62, 159.24, 153.02, 135.58, 134.24, 130.86, 129.68, 129.49, 129.10, 127.48, 119.70, 113.86, 72. 77, 71.29, 66.03, 65.94, 55.66, 55.48, 53.39, 37.73, 35.47, 26.02, 18.22, -4.23, -4.38; HRMS (ES) calculated value C 31 H 43 NO6Si[M+Na] 576.2752, measured value 576.2767.

[0187] (2S,3S)-3-((tert-butyldimethylsilyl)oxy)-5-((4-methoxybenzyl)oxy)-2-vinylpentan-1-ol (4). To a solution of 3 (17.22 g, 31.14 mmol, 1 equiv) in tetrahydrofuran (THF) (625 mL, 0.05 M) cooled to 0 °C was added a solution of NaBH (5.891 g, 155.7 mmol, 5 equiv) in HO (240 mL). After 10 min at 0 °C, the solution was warmed to room temperature and stirred for 5 h. The reaction was quenched by adding saturated aqueous NH Cl (200 mL), and the mixture was stirred at room temperature for 1 h. The THF was removed under reduced pressure, the aqueous layer was extracted with EtOAc (3 × 75 mL), and the organics were combined, washed with brine, dried over Na SO , filtered, and concentrated. The residue was purified by flash chromatography (18% EtOAc / hexanes) to give 4 (10.04 g, 85% yield) as a colorless oil: R f =0.25 (20% EtOAc / hexane); 1H NMR (400MHz, CDCl3) δ 7.25(d, J=8.8Hz, 2H), 6.88(d, J=8.6Hz, 2H), 5.71(ddd, J=17.3, 10.4, 8.6Hz, 1H), 5.17(d, J=10.4Hz, 1H), 5.10(d, J=17.4Hz, 1H), 4.43(d, J=11.5Hz, 1H), 4.38(d, J=11.5Hz, 1H), 4.04-3.94(m, 1H), 3.80(s , 3H), 3.75(dt, J=10.8, 6.6Hz, 1H), 3.61(ddd, J=11.0, 7.0, 5.0Hz, 1H), 3.46(t, J=6.3Hz, 2H), 2.43(q d, J=7.6, 2.7Hz, 1H), 2.15(t, J=5.7Hz, 1H), 1.88-1.68(m, 2H), 0.88(s, 9H), 0.09(s, 3H), 0.06(s, 3H). 13 C NMR (100MHz, CDCl3) δ HRMS(ES) calculated value C 21 H 36 O4Si[M+Na] 403.2275, measured value 403.2271.

[0188] (5S,6S)-5-(2-((4-methoxybenzyl)oxy)ethyl)-2,2,3,3,9,9,10,10-octamethyl-6-vinyl-4,8-dioxa-3,9-disilaundecane (5). 3、4 To a stirred solution of 4 (9.81 g, 25.8 mmol, 1 equiv) in dry pyridine (250 mL, 0.1 M) cooled to 0 °C was added p-toluenesulfonyl chloride (7.372 g, 38.66 mmol, 1.5 equiv). The mixture was stirred for 30 min and allowed to warm to room temperature. After 4 h, the mixture was recooled to 0 °C and 0.5 N HCl (300 mL) was slowly added. The aqueous layer was extracted with diethyl ether (3 × 200 mL), and the organics were combined, washed with 1 M HCl, brine, dried over NaSO, filtered, and concentrated. The residue was purified using a chromatographic plug (18% EtOAc / hexanes) to give 5 (11.67 g, 84% yield) as a colorless oil: R f =0.4 (20% EtOAc / hexane); 1 H NMR (400MHz, CDCl3) δ 7.75(d, J=8.4Hz, 2H), 7.30(d, J=8.4Hz, 2H), 7.21(d, J=8.4Hz, 2H), 6.86(d, J=8.4Hz, 2H), 5.58(ddd, J=8.7 , 10.3, 17.3Hz, 1H), 5.12(dd, J=1.2, 9.2Hz, 1H), 5.04(d, J=17.2Hz, 1H), 4.39(d, J=11.6Hz, 1H), 4.33(d, J=1 1.6Hz, 1H), 4.08(dd, J=6.6, 9.4Hz, 1H), 3.94(dd, J=7.4, 9.4Hz, 1H), 3.93-3.89(m, 1H), 3.79(s, 3H), 3.35(t , J=6.4Hz, 2H), 2.46-2.42(m, 1H), 2.42(s, 3H), 1.74-1.57(m, 2H), 0.78(s, 9H), -0.01(s, 3H), -0.05(s, 3H); 13 C NMR (100MHz, CDCl3) δ 159.30, 144.80, 133.44, 133.15, 130.53, 129.91, 129.39, 128.14, 119.79, 113.92, 72.73, 7 0.54, 68.83, 66.36, 55.43, 48.61, 34.64, 25.91, 21.77, 18.11, -4.25, -4.69;HRMS(ES) calculated value C 28 H 42 O6SSi[M+Na] 557.2364, measured value 557.2370.

[0189] tert-Butyl(((3S,4R)-1-((4-methoxybenzyl)oxy)-4-methylhex-5-en-3-yl)oxy)dimethylsilane (6). 5 To a solution of 5 (3.72 g, 6.96 mmol, 1 equiv) in dry THF (70 mL, 0.1 M) cooled to 0 °C was added lithium triethylborohydride (1 M in THF, 17.39 mL, 17.39 mmol, 2.5 equiv) over 10 min. The reaction was stirred at 0 °C, warmed to room temperature, and stirred for an additional 2 h. The reaction was cooled to 0 °C and quenched by slow addition of water (15 mL), 3 N NaOH (15 mL), 30% HO (15 mL) and stirred for 30 min. The organics were removed under reduced pressure, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organics were washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (10% EtOAc / hexanes) to give 6 (1.87 g, 74% yield) as a clear, colorless oil: R f =0.5 (10% EtOAc / hexane); 1 H NMR (600MHz, CDCl3) δ 7.25(d, J=8.3Hz, 2H), 6.87(d, J=8.2Hz, 2H), 5.76(ddd, J=17.4, 10.1, 7.3Hz, 1H), 4.9 9(d, J=9.8Hz, 1H), 4.98(d, J=18.1Hz, 1H), 4.43(d, J=11.5Hz, 1H), 4.38(d, J=11.5Hz, 1 H), 3.80(s, 3H), 3.76(dt, J=8.0, 4.2Hz, 1H), 3.52-3.42(m, 2H), 2.29(td, J=7.1, 3.9Hz , 1H), 1.74-1.60(m, 2H), 0.99(d, J=6.9Hz, 3H), 0.88(s, 9H), 0.05(s, 3H), 0.03(s, 3H). 13 C NMR (100MHz, CDCl3) δ HRMS(ES) calculated value C21 H 36 O3Si[M+H] 364.2434, found 364.2439.

[0190] (tert-Butyl(((3S,4R,E)-1-((4-methoxybenzyl)oxy)-4-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hex-5-en-3-yl)oxy)dimethylsilane (7). 6 A solution of Hoveyda Grubbs II (0.10 g, 0.16 mmol, 0.05 equiv.) in dry CHCl (15 mL, 0.2 M) was added to a two-neck flask equipped with a reflux condenser under N. To this was added olefin 6 (1.15 g, 3.15 mmol, 1 equiv.), followed by vinylboronic acid pinacol ester (1.07 mL, 6.31 mmol, 3 equiv., passed through a plug of SiO using 10% EtOAc / hexanes as eluent, with the stabilizer removed immediately before use), and the reaction was heated at reflux for 18 h. The residue was cooled, concentrated, and directly purified by flash chromatography (5% EtOAc / hexanes) to give 7 (1.10 g, 74% yield) as a pale yellow oil: R f =0.35 (5% EtOAc / hexane); 1 H NMR (400MHz, CDCl3) δ 7.25(d, J=8.1Hz, 2H), 6.87(d, J=8.0Hz, 2H), 6.56(dd, J=18.1, 6.6Hz, 1H), 5.43(d, J =18.1Hz, 1H), 4.41(d, J=18.0Hz, 1H), 4.38(d, J=17.9Hz, 1H), 3.87-3.75(m, 1H), 3.8 0(d, J=0.8Hz, 3H), 3.55-3.38(m, 2H), 2.40(q, J=6.2Hz, 1H), 1.66(q, J=6.7Hz, 2H), 1 .26(s, 12H), 1.00(d, J=6.9Hz, 3H), 0.87(d, J=0.8Hz, 9H), 0.03(s, 3H), 0.02(s, 3H); 13C NMR (100MHz, CDCl3) δ 159.31, 156.21, 130.88, 129.52, 113.96, 83.26, 72.77, 72.45, 67.42, 55.50, 45.53, 32.93, 26.12, 25.06, 24.96, 18.32, 13.32, -4.20, -4.42; HRMS (ES) calculated value C 27 H 47 BO5Si[M+H] 491.3359, measured value 491.3350.

[0191] (3S,4R,E)-3-((tert-butyldimethylsilyl)oxy)-4-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hex-5-en-1-ol (S4). To a solution of 7 (1.90 g, 3.87 mmol, 1 equiv) in CHCl (25 mL, 0.15 M) and water (1.49 mL) at room temperature, DDQ (1.32 g, 5.81 mmol, 1.5 equiv) was added and the mixture was stirred for 1 h. The reaction was quenched with saturated NaHCO, stirred for 10 min, and diluted with water. The organic layer was separated, the aqueous layer was extracted with CHCl (2 × 50 mL), and the organics were combined, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (12% EtOAc / hexanes) to give S4 (1.21 g, 83% yield) as a colorless oil: R f =0.15 (10% EtOAc / hexane); 1 H NMR (600MHz, CDCl3) δ 6.54(dd, J=18.1, 6.6Hz, 1H), 5.46(dd, J=18.1, 1.5Hz, 1H), 3.87(ddd, J=7.9, 5.0, 3.8Hz, 1H), 3.77-3.67(m, 2H), 2.49(pdd, J=6.8, 5.0, 1 .5Hz, 1H), 2.03(t, J=5.4Hz, 1H), 1.71-1.63(m, 2H), 1.26(d, J=1.7Hz, 12H), 1.01(d, J=6.8Hz, 3H), 0.89(s, 9H), 0.08(s, 3H), 0.07(s, 3H); 13C NMR (150MHz, CDCl3) δ 155.72, 83.07, 74.24, 60.63, 44.92, 34.15, 25.84, 24.80, 24.71, 17.98, 12.81, -4.41, -4.69; HRMS (ES) calculated value C 19 H 39 BO4Si[M+H] 371.2783, found 371.2778.

[0192] (3S,4R,E)-3-((tert-butyldimethylsilyl)oxy)-4-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hex-5-enal (8). To an open round-bottom flask was added alcohol S4 (1.00 g, 4.09 mmol, 1 equiv) in CHCl (41 mL, 0.1 M) and treated with NaHCO (1.72 g, 20.45 mmol, 5 equiv) and Dess-Martin periodinane (2.08 g, 4.91 mmol, 1.2 equiv). The reaction was stirred at room temperature for 1 h, quenched with 10% NaSO solution (50 mL), and stirred until both layers were clear. The organics were separated, and the aqueous layer was extracted with CHCl (2 × 50 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography (10% EtOAc / hexanes) to give 8 (0.768 g, 77% yield) as a colorless oil: R f =0.5 (10% EtOAc / hexane); 1 H NMR (400MHz, CDCl3) δ 9.75 (dd, J=2.1, 1.1Hz, 1H), 6.50 (dd, J=18.1, 6.6Hz, 1H), 5.46 (d, J=18.0Hz, 1H), 4.22 (dt, J=8.1, 4.2Hz, 1H), 2.54-2.4 2(m, 2H), 2.41-2.33(m, 1H), 1.25(s, 12H), 1.01(dd, J=6.9, 0.9Hz, 3H), 0.85(d, J=0.8Hz, 9H), 0.05(s, 3H), 0.02(s, 3H); 13C NMR (100MHz, CDCl3) δ 202.47, 154.68, 83.36, 70.89, 47.17, 45.54, 29.86, 25.92, 24.98, 24.88, 18.16, 13.03, -4.33, -4.56; HRMS (ES) calculated value C 19 H 37 BO4Si[M+H] 368.2554, found 368.2550.

[0193] 2. Synthesis of Unit B S5 was synthesized in three steps from commercially available 9 as shown in Figure 4 and as previously reported. 7

[0194] Methyl (R)-3-(3-chloro-4-methoxyphenyl)-2-(2-(diethoxyphosphoryl)acetamido)propanoate (12). To an open flask was added S5 (1.10 g, 3.20 mmol, 1 equiv.) and 4 M HCl / dioxane (20 mL). The mixture was stirred vigorously for 30 min and concentrated under reduced pressure. The resulting white solid 10 was used directly.

[0195] Compound 10 was suspended in DMF (32 mL, 0.1 M) and treated with 2-(diethoxyphosphoryl)acetic acid 11 (0.692 g, 3.52 mmol, 1.1 equiv.), EDC·HCl (0.731 g, 3.84 mmol, 1.2 equiv.), HOBt hydrate (0.591 g, 3.84 mmol, 1.2 equiv.), and DIPEA (1.03 g, 1.40 mL, 8.00 mmol, 2.5 equiv.) and stirred at room temperature for 18 h. The reaction was quenched with half-saturated aqueous NH4Cl (30 mL), and the aqueous layer was extracted with DCM (3 × 30 mL). The organics were combined, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography system (1-7% methanol / DCM) to give 12 (0.90 mg, 67% yield) as a clear, colorless oil: R f = 0.2 (2.5% methanol / DCM); 11H NMR (600 MHz, CDCl3) δ 7.16 (d, J = 2.2 Hz, 1H), 7.09 (bd, J = 7.5 Hz, 1H), 7.04 (dd, J = 8.4, 2.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 4.78 (td, J = 7.1, 5.4 Hz, 1H), 4.13 (dt, J = 14.9, 7.2 Hz, 1H), 4.06 (dq, J = 8.2, 7.1 Hz, 2H), 3.85 (s, 3H), 3.70 (s, 3H), 3.08 (dd, J = 14.2, 5.4 Hz, 1H), 2.97 (dd, J = 14.2, 6.8 Hz, 1H), 2.85 (d, J = 10.7 Hz, 1H), 2.81 (d, J = 10.5 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H); 13 13C NMR (150 MHz, CDCl3) δ 171.45, 163.95, 163.93, 154.24, 131.09, 129.15, 128.72, 122.48, 112.22, 77.37, 77.16, 76.95, 62.95 (d, J = 15.3 Hz), 62.91 (d, J = 15.3), 56.25, 53.94, 52.52, 36.88, 35.70, 34.83, 16.48, 16.43, 16.39; HRMS (ES) calculated value for C 17 H 25 ClNO7P [M + H] 421.1057, found 421.1053.

[0196] 3. Synthesis and Characterization of Unit A / B Analogs Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)octa-2,7-dienamide)-3-(3-chloro-4-methoxyphenyl)propanoate (13). A suspension of 12 (0.63 g, 1.49 mmol, 1 equiv) in dry THF (15 mL, 0.1 M) was cooled to 0 °C and treated with NaH (60% suspension in oil, 0.055 g, 1.64 mmol, 1.1 equiv). The reaction was stirred for 30 min before the dropwise addition of aldehyde 8 (0.55 g, 1.49 mmol, 1 equiv) in THF (5 mL). The reaction was allowed to stir at 0 °C for 1 h and quenched with half-saturated NH4Cl (10 mL). The organics were removed under reduced pressure, and the remaining aqueous layer was extracted with DCM (3 × 50 mL). The organics were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash chromatography (6–45% EtOAc / hexanes) to afford 13 (0.545 g, 57.4% yield) as a clear, colorless oil: R f =0.25 (25% EtOAc / hexane); 1 H NMR:(400MHz, CD3OD) δ 7.21(d, J=2.2Hz, 1H), 7.10(dd, J=8.5, 2.2Hz, 1H), 6.97(d, J=8.4Hz, 1H), 6.75(dt, J=15.2, 7.5Hz, 1H) , 6.57(dd, J=18.0, 7.7Hz, 1H), 5.96(d, J=15.3Hz, 1H), 5.38(d, J=18.3Hz, 1H), 4.68(dd, J=8.9, 5.5Hz, 1 H), 3.84(s, 3H), 3.73(q, J=5.4Hz, 1H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.90(dd, J=14.0, 9.0 Hz, 1H), 2.41-2.25(m, 3H), 1.26(s, 12H), 1.01(d, J=6.8Hz, 3H), 0.89(s, 9H), 0.06(s, 3H), 0.03(s, 3H); 13 C NMR:(100MHz, CD3OD) δ 173.28, 167.98, 157.33, 155.42, 143.28, 131.82, 131.33, 129.70, 126.29, 123.20, 113.34, 84.41, 76.09, 5 6.55, 55.19, 52.72, 46.48, 38.38, 37.30, 26.47, 25.14, 25.10, 19.00, 15.81, -3.99, -4.30; HRMS (ESI) calculated value C 32 H51 BClNO7Si[M+H] 635.3216, found 635.3215.

[0197] 4. General Suzuki Coupling Procedure To a long tube, 13 (1 equiv.), KPO (2.5 equiv.), aryl iodide (2 equiv.), and Pd(dba) (0.05 equiv.) were suspended in a mixture of 1,2-dichloroethane and water (4:1, 0.1 M) and stirred vigorously until complete, as assessed by TLC (2–12 h). The reaction was diluted with 0.5 M HCl and EtOAc, and the aqueous layer was extracted with EtOAc (3 × 20 mL). The organics were combined, dried over NaSO, filtered, and purified by flash chromatography as described herein.

[0198] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-phenylocta-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14a). The reaction was carried out according to the general Suzuki procedure and purified using a flash chromatography system (SiO, 20-50% EtOAc / hexanes) to give 14a (0.086 g, 93% yield) as a pale yellow oil: R f =0.35 (25% EtOAc / hexane); 1H NMR(400MHz, CD3OD) δ 7.33(d, J=7.5Hz, 2H), 7.27(t, J=7.6Hz, 2H), 7.21(d, J=2.2Hz, 1H), 7.18(t, J=7.2Hz, 1H), 7.10(dd, J=8.4, 2.2Hz, 1H), 6 .96(d, J=8.4Hz, 1H), 6.78(dt, J=15.2, 7.5Hz, 1H), 6.38(d, J=16.0Hz, 1H), 6.19(dd, J=16.0, 8.2Hz, 1H), 5.96(d, J=15.4H) z, 1H), 4.69(dd, J=8.9, 5.6Hz, 1H), 3.83(s, 3H), 3.80(q, 1H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.90(dd, J=14. 0, 9.0Hz, 1H), 2.44(q, J=6.5Hz, 1H), 2.37(t, J=6.9Hz, 2H), 1.11(d, J=6.9Hz, 3H), 0.90(s, 9H), 0.06(s, 3H), 0.05(s, 3H); 13 C NMR (100MHz, CD3OD) 173.28, 168.03, 155.41, 143.50, 139.03, 133.10, 131.82, 131.73, 131.33, 129.67, 129.50, 128.04, 127.06, 126.17, HRMS(ES) calculated value C 32 H 44 ClNO5Si[M+H] 586.2750, found 586.2754.

[0199] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(pyridin-2-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14b). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography (Si-NH, 20-50% EtOAc / hexanes) to give 14b (0.078 g, 45% yield) as a pale yellow oil: R f= 0.15 (50% EtOAc / hexane); 1 H NMR(400MHz, CD3OD) δ 8.44(d, J=4.0Hz, 1H), 7.76(td, J=7.8, 1.8Hz, 1H), 7.46(d, J=8.0Hz, 1H), 7.24(dd, J=7.8, 5.2Hz, 1H), 7.21(d, J=2.1Hz, 1H), 7.10( dd. 98(d, J=15.4Hz, 1H), 4.70(dd, J=9.0, 5.5Hz, 1H), 3.87-3.84(m, 1H), 3.83(m, 3H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.5Hz, 1H), 2.90( dd. 13 C NMR (100MHz, CD3OD) δ 173.27, 167.95, 157.17, 155.41, 149.74, 143.24, 139.05, 138.66, 131.83, 131.34, 131.12, 129.70, 126.38, 123.38, 123 .17, 122.34, 113.34, 76.26, 56.53, 55.15, 52.73, 44.06, 38.62, 37.31, 26.43, 18.97, 16.47, -4.04, -4.34.HRMS(ES) calculated value C 31 H 43 ClN2O5Si[M+H] 587.2703, found 587.2705.

[0200] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(pyridin-3-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14c). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography system (Si-NH, 16-60% EtOAc / hexane) to give 14c (0.049 g, 72% yield) as a pale yellow oil: R f = 0.15 (50% EtOAc / hexane); 1 H NMR (600MHz, CD3OD) δ 8.49(d, J=2.1Hz, 1H), 8.35(dd, J=4.9, 1.5Hz, 1H), 7.85(d, J=8.0Hz, 1H), 7.37(dd, J=8.0, 4.8Hz, 1H), 7.21(d, J=2.1Hz, 1H), 7.10(dd, J= 8.4, 2.2Hz, 1H), 6.96(d, J=8.5Hz, 1H), 6.78(dt, J=15.2, 7.5Hz, 1H), 6.43(d, J=16.1Hz, 1H), 6.36(dd, J=16.1, 7.9Hz, 1H), 5.97(dt, J=15 .3, 1.4Hz, 1H), 4.70(dd, J=8.9, 5.6Hz, 1H), 3.85-3.79(m, 1H), 3.83(s, 3H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.91(dd, J=14.0, 9.0Hz, 1H), 2.49(td, J=7.2, 4.7Hz, 1H), 2.38(ddd, J=7.4, 5.8, 1.4Hz, 2H), 1.13(d, J=6.9Hz, 3H), 0.90(s, 9H), 0.06(s, 3H), 0.05(s, 3H); 13 C NMR (150MHz, CD3OD) δ 173.27, 167.97, 155.43, 148.28, 148.13, 143.21, 136.88, 135.48, 134.71, 131.82, 131.35, 129.68, 127.66, 126.31, 125 .28, 123.20, 113.39, 76.32, 56.56, 55.15, 52.72, 44.24, 38.69, 37.29, 26.42, 18.96, 16.65, -4.00, -4.34;HRMS(ES) calculated value C 31 H 43 ClN2O5Si[M+H] 587.2703, found 587.2702.

[0201] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(pyridin-4-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14d). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography system (Si-NH, 15-55% EtOAc / hexane) to give 14d (0.062 g, 71% yield) as a pale yellow oil: R f =0.25 (50% EtOAc / hexane); 1 H NMR(600MHz, CD3OD) δ δ 8.42(d, J=5.9Hz, 2H), 7.37(d, J=6.3Hz, 2H), 7.21(d, J=2.1Hz, 1H), 7.10(dd, J=8.5, 2.1Hz, 1H), 6.97(d, J=8.4Hz, 1H) , 6.78(dt, J=15.2, 7.5Hz, 1H), 6.56(dd, J=16.0, 8.2Hz, 1H), 6.41(d, J=16.0Hz, 1H), 5.97(d, J=15.3Hz, 1H), 4.69(dd, J =9.0, 5.5Hz, 1H), 3.86-3.81(m, 1H), 3.83(s, 3H), 3.70(s, 3H), 3.11(dd, J=14.1, 5.6Hz, 1H), 2.91(dd, J=14.1, 9.0Hz, 1H), 2.51(td, J=13.3, 6.5Hz, 1H), 2.38(t, J=6.9Hz, 2H), 1.13(d, J=6.8Hz, 3H), 0.90(s, 9H), 0.06(s, 3H), 0.06(s, 3H). 13 C NMR (150 MHz, CD3OD) 13 C NMR (150MHz, CD3OD) δ 173.28, 167.96, 155.44, 150.23, 147.68, 143.05, 139.96, 131.83, 131.36, 129.67, 129.19, 126.37, 123.20, 122 .32, 113.40, 76.19, 56.56, 55.14, 52.73, 44.24, 38.75, 37.28, 26.41, 18.96, 16.56, -4.02, -4.36; HRMS (ES) calculated value C 31 H 43ClN2O5Si[M+H] 587.2703, found 587.2699.

[0202] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(pyrazin-2-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14e). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography system (Si-NH, 20-55% EtOAc / hexane) to give 14e (0.115 g, 65% yield) as a pale yellow oil: R f = 0.2 (50% EtOAc / hexane); 1 H NMR (600MHz, CD3OD) δ 8.57(d, J=1.5Hz, 1H), 8.51(s, 1H), 8.39(d, J=2.6Hz, 1H), 7.21(d, J=2.2Hz, 1H), 7.10(dd, J=8.4, 2.2Hz, 1H), 6.96(d, J =8.5Hz, 1H), 6.90(dd, J=15.9, 8.3Hz, 1H), 6.78(dt, J=15.2, 7.5Hz, 1H), 6.54(d, J=16.0Hz, 1H), 5.98(d, J=15.4Hz, 1H) , 4.70(dd, J=8.9, 5.5Hz, 1H), 3.86-3.84(m, 1H), 3.83(s, 3H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.91(dd, J=14 .0, 9.0Hz, 1H), 2.55(q, J=6.9Hz, 1H), 2.41-2.33(m, 2H), 1.15(d, J=6.8Hz, 3H), 0.89(s, 9H), 0.06(s, 3H), 0.05(s, 3H); 13C NMR (150MHz, CD3OD) δ 173.27, 167.93, 155.42, 152.99, 145.49, 143.89, 143.49, 143.07, 141.77, 131.82, 131.34, 129.69, 127.88, 126.41, HRMS(ES) calculated value C 30 H 42 ClN3O5Si[M+H] 588.2655, found 588.2659.

[0203] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(1-methyl-1H-pyrozol-5-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14f). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography system (Si-NH, 20-70% EtOAc / hexane) to give 14f (0.044 g, 63% yield) as a pale yellow oil: R f = 0.1 (50% EtOAc / hexane); 1H NMR (600MHz, CD3OD) δ 7.34(d, J=2.0Hz, 1H), 7.20(d, J=2.2Hz, 1H), 7.10(dd, J=8.4, 2.2Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.78(dt, J=15.2, 7.5Hz, 1H), 6.38(d, J=15.9Hz, 1H), 6.33(d, J=2.1Hz, 1H), 6.21(dd, J=15.9, 8.3Hz, 1H), 5.97(dt, J=15.4, 1.3Hz, 1H), 4.69(dd, J =8.9, 5.6Hz, 1H), 3.82(s, 3H), 3.81-3.79(m, 1H), 3.80(s, 3H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.91(dd, J=14. 0, 8.9Hz, 1H), 2.48(h, J=6.9Hz, 1H), 2.38(t, J=6.5Hz, 2H), 1.12(d, J=6.9Hz, 3H), 0.89(s, 9H), 0.06(s, 3H), 0.05(s, 3H). 13 C NMR (150MHz, CD3OD) δ 173.28, 167.98, 155.44, 143.09, 142.83, 139.14, 138.52, 131.81, 131.36, 129.67, 126.33, 123.21, 118.11, 113.41 , 103.42, 76.23, 56.57, 55.17, 52.72, 44.20, 38.79, 37.28, 36.45, 26.42, 18.97, 16.77, -3.99, -4.36; HRMS (ES) calculated value C 30 H 44 ClN3O5Si[M+H] 590.2812, found 590.2811.

[0204] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(1-methyl-1H-pyrozol-3-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14 g). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography (Si-NH, 16-55% EtOAc / hexanes) to give 14g (0.065g, 71% yield) as a pale yellow oil: Rf =0.25 (50% EtOAc / hexane); 1 H NMR (600MHz, CD3OD) δ 7.46(d, J=2.3Hz, 1H), 7.21(d, J=2.2Hz, 1H), 7.10(dd, J=8.5, 2.2Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.75(dt, J=15.2 , 7.5Hz, 1H), 6.30(d, J=2.3Hz, 1H), 6.17(dd, J=16.2, 8.1Hz, 1H), 5.96(d, J=15.4Hz, 1H), 4.69(dd, J=9.0, 5.6Hz, 1 H), 3.84(s, 3H), 3.83(s, 3H), 3.78(q, J=5.6Hz, 1H), 3.70(s, 3H), 3.11(dd, J=14.1, 5.6Hz, 1H), 2.91(dd, J=14.0, 9 .0Hz, 1H), 2.42(p, J=6.7Hz, 1H), 2.38-2.30(m, 2H), 1.09(d, J=6.8Hz, 3H), 0.90(s, 9H), 0.06(s, 3H), 0.04(s, 3H); 13 C NMR (150MHz, CD3OD) δ 173.29, 167.99, 155.43, 152.12, 143.43, 134.95, 133.03, 131.82, 131.37, 129.71, 126.28, 123.29, 123.22, 113.38 HRMS(ES) calculated value C 30 H 44 ClN3O5Si[M+H] 590.2812, found 590.2809.

[0205] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-6-methyl-8-(1-methyl-1H-pyrozol-4-yl)octa-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14h). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography system (Si-NH, 16-60% EtOAc / hexanes) to give 14h (0.085 g, 92% yield) as a pale yellow oil: R f = 0.2 (50% EtOAc / hexane); 1 H NMR(400MHz, CD3OD) δ 7.53(s, 1H), 7.47(s, 1H), 7.21(d, J=2.2Hz, 1H), 7.10(dd, J=8.4, 2.2Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.77(dt, J=15.2, 7.5Hz, 1H), 6.18(d, J=16.0Hz, 1H), 5.95(d, J=15.4Hz, 1H), 5.89(dd, J=16.1, 8.2Hz, 1H), 4.69(dd, J= 8.9, 5.6Hz, 1H), 3.84(s, 3H), 3.83(s, 3H), 3.75(q, J=5.5Hz, 1H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2 .90(dd, J=14.0, 9.0Hz, 1H), 2.41-2.27(m, 3H), 1.06(d, J=6.8Hz, 3H), 0.89(s, 9H), 0.05(s, 3H), 0.03(s, 3H). 13 C NMR (150MHz, CD3OD) δ173.29, 168.03, 155.42, 143.67, 137.61, 131.83, 131.58, 131.35, 129.68, 129.33, 126.10, 123.18, 122.45, 121.24 , 113.36, 76.60, 56.55, 55.15, 52.72, 44.16, 38.72, 38.32, 37.28, 26.44, 18.98, 16.45, -4.05, -4.31;HRMS(ES) calculated value C 30 H 44 ClN3O5Si[M+H] 590.2812, found 590.2813.

[0206] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-8-(1-isopropyl-1H-pyrozol-4-yl)-6-methylocta-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14i). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography (amine column, 20-50% EtOAc / hexanes) to give 14i (0.056 g, 83% yield) as a pale yellow oil: R f =0.35 (50% EtOAc / hexane); 1 H NMR (600 MHz, CD3OD) δ 1 H NMR (400MHz, methanol-d4) δ 7.61(s, 1H), 7.48(s, 1H), 7.21(d, J=2.2Hz, 1H), 7.10(dd, J=8.4, 2.2Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.77(dt, J=15.2, 7.5Hz, 1H), 6.20(d, J=16.1Hz, 1H), 5.95(d, J=15.3Hz, 1H), 5.90(dd, J=16.1, 8.1Hz, 1H), 4.69(dd, J=8.9, 5.6Hz, 1H), 4 .46(hept, J=7.0Hz, 1H), 3.83(s, 3H), 3.75(q, J=5.4Hz, 1H), 3.70(s, 3H), 3.11(dd, J=14.0, 5.6Hz, 1H), 2.91(dd, J=14 .0, 8.9Hz, 1H), 2.46-2.29(m, 3H), 1.46(d, J=6.7Hz, 6H), 1.07(d, J=6.9Hz, 3H), 0.90(s, 9H), 0.06(s, 3H), 0.03(s, 3H). 13 C NMR (150 MHz, CD3OD) δ 13 C NMR (101MHz, cd3od) δ 173.29, 168.04, 155.42, 143.72, 137.18, 131.83, 131.35, 129.68, 126.09, 123.19, 121.88, 121.43, 113.36, 76 HRMS(ES) calculated value C 32 H 48 ClN3O5Si[M+H] 618.3125, found 618.3129.

[0207] Methyl (R)-2-((2E,5S,6R,7E)-5-((tert-butyldimethylsilyl)oxy)-8-(3,5-dimethylisoxazol-4-yl)-6-methylocta-2,7-dienamido)-3-(3-chloro-4-methoxyphenyl)propanoate (14j). The reaction was carried out according to the general Suzuki procedure and purified by flash chromatography (amine column, 10-50% EtOAc / hexanes) to give 14j (0.057 g, 85% yield) as a pale yellow oil: R f =0.35 (33% EtOAc / hexane); 1 H NMR:(400MHz, CD3OD) δ 7.20(d, J=2.2Hz, 1H), 7.10(dd, J=8.3, 2.2Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.76(dt, J= 15.2, 7.6Hz, 1H), 6.11(d, J=16.3Hz, 1H), 5.96(d, J=15.4Hz, 1H), 5.90(dd, J=16.4, 8.5 Hz, 1H), 4.68(dd, J=9.0, 5.5Hz, 1H), 3.84(s, 3H), 3.83-3.77(m, 1H), 3.70(s, 3H), 3.1 2(dd, J=14.0, 5.5Hz, 1H), 2.90(dd, J=14.0, 9.0Hz, 1H), 2.46-2.33(m, 3H), 2.38(s, 3H) 2.26(s, 3H), 1.11(d, J=6.9Hz, 3H), 0.90(s, 9H), 0.07(s, 3H), 0.06(s, 3H). 13 C NMR:(100MHz, CD3OD) δ 173.27, 167.98, 166.46, 159.59, 155.42, 143.21, 135.80, 131.81, 131.33, 129.67, 126.21, 123.17, 119.01, 114.23, 11 HRMS(ES) calculated value C 31 H 45 ClN2O6Si[M+H] 605.2808, found 605.2801.

[0208] 5. Characterization of Unit C / D

[0209] [ka]

[0210] As shown in Figure 6B, 19 and 20 were synthesized from commercially available 15. All spectra were in accordance with published literature. 8~11

[0211] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((tert-butoxycarbonyl)amino)-2-methylpropanoate (21). To a solution of 19 in DMF (0.1 M) was added EDC HCl (1.5 equiv.) and HOBt (1.2 equiv.), and the reaction was stirred for 30 min. NAc was added and stirred for 10 min before adding catalytic DMAP (0.05 equiv.). The reaction was stirred for 12 h, diluted with water and EtOAc, the aqueous layer extracted (2 × 40 mL), the organics combined, washed with saturated NH4Cl (2 × 100 mL), and dried over sodium sulfate. The organics were removed under reduced pressure, and the remaining residue was purified by flash chromatography (1–10% methanol / DCM) to give 21 as a white solid. Spectroscopy was in accordance with published literature. 12

[0212] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl 3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropanoate (22). To a solution of 20 in DMF (0.1 M) was added EDC HCl (1.5 equiv.) and HOBt (1.2 equiv.), and the reaction was stirred for 30 min. NAc was added and stirred for 10 min before adding catalytic DMAP (0.05 equiv.). The reaction was stirred for 12 h, diluted with water and EtOAc, the aqueous layer extracted (2 × 40 mL), the organics combined, washed with saturated NH4Cl (2 × 100 mL), and dried over sodium sulfate. The organics were removed under reduced pressure, and the remaining residue was purified using the flash chromatography system described herein (1–10% methanol / DCM) to give 22 as a white solid. Spectroscopy was in accordance with published literature. 12

[0213] 6. Secocryptophycin Synthesis and Characterization of Analogues General Peptide Coupling Procedure 14a-m (1 equiv.) was suspended in 1,2 dichloroethane (0.2 M), treated with trimethyltin hydroxide (4 equiv.), and heated at 80° C. for 4 h. The reaction was cooled, diluted with DCM, and washed with 1 N HCl (2×). The crude acid was used directly.

[0214] Concurrently, 21 or 22 (1.1 equiv) was suspended in 4M HCl / dioxane (5 mL), stirred at room temperature for 1 h, concentrated, and used directly.

[0215] The acids 14a-m were suspended in DCM, cooled to 0 °C, and treated with HATU (1.1 equiv.). The amine salts of 21 or 22 were suspended in DCM, treated with DIPEA (2.5 equiv.), cooled to 0 °C, and added to the mixture. The reaction was allowed to stir overnight and warm to room temperature. The mixture was diluted with half-saturated sodium bicarbonate, the aqueous layer was extracted with DCM (3 × 10 mL), and the organics were combined, washed with brine, dried over Na2SO4, filtered, concentrated, and purified as specified herein. When utilized for analytical analysis, further HPLC purification was performed to remove any diastereomers generated during the coupling procedure using a 20–80% water / acetonitrile gradient at a flow rate of 3 mL / min before deprotection on a HydroRP C18 (250 × 10.0 mm, 4 micron). Semi-preparative reactions were carried out using intermediates immediately after purification on the flash chromatography system disclosed herein.

[0216] General deprotection procedure. The crude coupling product was suspended in acetonitrile (0.1 M) in an open polypropylene vial. It was treated with 33% aqueous HF (2 equivalents) and allowed to stir until the reaction was complete, as monitored by TLC. The reaction was diluted with DCM and quenched by slowly adding saturated NaHCO3 until the aqueous layer was basic. The aqueous layer was then extracted with DCM (3 x 10 mL), and the organics were combined, washed with brine, dried over Na2SO4, filtered, concentrated, and purified as specified herein. The diastereomeric ratio (dr) is reported as seen by NMR.

[0217] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-phenylocta-2,7-dienamido)propanamido)-2-methylpropanoate (23a). The reaction was carried out according to the general coupling procedure and purified by flash chromatography (1–10% methanol / DCM).。 R f = 0.5 (5% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2–14% methanol / DCM) to give 23a (0.051 g, 42% yield over three steps, 10:1 dr) as a clear, colorless oil: R f = 0.35 (10% methanol / DCM). 1 H NMR:(600MHz, CD3OD) δ 7.37(d, J=7.0Hz, 2H), 7.27(t, J=7.9Hz, 2H), 7.25(d, J=2.2Hz, 1H), 7.18(t, J=7.3Hz, 1 H), 7.14(dd, J=8.4, 2.2Hz, 1H), 6.97(d, J=8.4Hz, 1H), 6.81(dt, J=15.1, 7.3Hz, 1H), 6. 41(d, J=15.9Hz, 1H), 6.23(dd, J=15.9, 8.5Hz, 1H), 6.01(d, J=15.4Hz, 1H), 5.21(dd, J= 9.6, 4.0Hz, 1H), 4.58(dd, J=8.1, 7.0Hz, 1H), 3.83(s, 3H), 3.65(dt, J=8.7, 4.6Hz, 1H), 3.48(dd, J=13.5, 6.6Hz, 1H), 3.34-3.27(m, 2H), 3.19(dd, J=13.5, 7.0Hz, 1H), 3.06-2. 97(m, 3H), 2.85(dd, J=13.7, 8.1Hz, 1H), 2.70(h, J=7.0Hz, 1H), 2.44-2.35(m, 2H), 2.35 -2.28(m, 1H), 1.91(s, 3H), 1.80-1.74(m, 1H), 1.74-1.69(m, 1H), 1.66-1.59(m, 1H), 1. 15(d, J=6.9Hz, 3H), 1.10(d, J=7.1Hz, 3H), 0.94(d, J=6.5Hz, 3H), 0.91(d, J=6.5Hz, 3H). 13C NMR:(150MHz, CD3OD) 200.34, 175.06, 173.49, 173.42, 168.07, 155.40, 143.51, 139.05, 132.50, 132.09, 131.91, 131.50, 129.84, 129.48, 128.05, 127.14, 126.08, 123.20, 113.35, 78.63, 75.34, 56.57, 56.24, 44.21, 42.73, 41.95, 40.57, 39.85, 38.82, 38.07, 28.58, 25.74, 23.48, 22.54, 21.97, 17.53, 14.87; HRMS (ES) calculated C 39 H 52 ClN3O8S[M+H] 758.3236, found 758.3238.

[0218] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyridin-2-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23b). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give 23b (0.025 g, 38% yield over three steps, 7:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR: (600 MHz, CD3OD) δ 8.44 (d, J = 4.1 Hz, 1H), 7.76 (td, J = 7.7, 1.9 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.25 - 7.22 (m, 1H), 7.14 (dd, J = 8.4, 2.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.81 (dt, J = 15.0, 7.3 Hz, 1H), 6.67 (dd, J = 16.0, 8.4 Hz, 1H), 6.52 (d, J = 16.0 Hz, 1H), 6.02 (d, J = 15.4 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.58 (dd, J = 8.1, 6.9 Hz, 1H), 3.84 (s, 3H), 3.68 (dt, J = 8.7, 4.7 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (s, 2H), 3.21 (dd, J = 13.5, 7.0 Hz, 1H), 3.05 - 2.99 (m, 3H), 2.85 (dd, J = 13.8, 8.1 Hz, 1H), 2.71 (h, J = 7.0 Hz, 1H), 2.51 - 2.44 (m, 1H), 2.44 - 2.37 (m, 1H), 2.37 - 2.29 (m, 1H), 1.91 (s, 3H), 1.81 - 1.69 (m, 2H), 1.67 - 1.59 (m, 1H), 1.18 (d, J = 6.9 Hz, 3H), 1.11 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.34, 175.09, 173.49, 173.42, 168.05, 157.30, 155.43, 149.71, 143.29, 138.74, 138.61, 131.92, 131.56, 131.35, 129.85, 126.27, 123.36, 122.32, 113.42, 111.43, 78.67, 75.12, 56.61, 56.25, 44.08, 42.75, 41.97, 40.60, 39.87, 38.75, 38.09, 28.61, 25.76, 23.46, 22.54, 21.99, 17.10, 14.86; HRMS (ES) calculated for C 38 H 51 ClN4O8S [M + H] 759.3189, found 759.3184.

[0219] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyridin-3-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23c). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23c (0.037 g, 51% yield over three steps, 7:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 8.52 (d, J = 2.2 Hz, 1H), 8.36 (dd, J = 4.9, 1.6 Hz, 1H), 7.89 (dt, J = 8.1, 1.9 Hz, 1H), 7.37 (ddd, J = 8.0, 4.9, 0.9 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.4, 2.3 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.81 (dt, J = 15.2, 7.3 Hz, 1H), 6.46 (d, J = 16.1 Hz, 1H), 6.41 (dd, J = 16.0, 7.9 Hz, 1H), 6.02 (d, J = 15.4 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.59 (dd, J = 8.0, 6.8 Hz, 1H), 3.84 (s, 3H), 3.67 (dt, J = 8.0, 4.6 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (m, 2H) 3.20 (dd, J = 13.5, 6.9 Hz, 1H), 3.04 - 3.00 (m, 3H) 2.85 (dd, J = 13.7, 8.1 Hz, 1H) 2.70 (h, J = 7.0 Hz, 1H), 2.51 - 2.36 (m, 2H), 2.36 - 2.28 (m, 1H), 1.91 (s, 3H), 1.83 - 1.68 (m, 2H), 1.65 - 1.61 (m, 1H), 1.17 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H); 13 13C NMR (150 MHz, CD3OD) δ 200.34, 175.08, 173.48, 173.42, 168.04, 155.42, 148.27, 148.22, 143.30, 136.34, 135.52, 134.79, 131.91, 131.53, 129.85, 127.95, 126.19, 125.24, 123.23, 113.41, 78.66, 75.16, 56.60, 56.24, 44.33, 42.75, 41.97, 40.59, 39.86, 38.87, 38.08, 28.60, 25.76, 23.47, 22.54, 21.99, 17.34, 14.86; HRMS (ES) calculated value C 38 H 51 ClN4O8S [M + H] 759.3189, found 759.3192.

[0220] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyridin-4-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23e). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23e (0.048 g, 49% yield over three steps, 11:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 8.42 (d, J = 6.3 Hz, 1H), 7.40 (d, J = 6.3 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.5, 2.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.80 (dt, J = 15.0, 7.3 Hz, 1H), 6.61 (dd, J = 16.0, 8.5 Hz, 1H), 6.44 (d, J = 15.9 Hz, 1H), 6.01 (d, J = 15.4 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.58 (dd, J = 8.1, 6.9 Hz, 1H), 3.84 (s, 3H), 3.67 (dt, J = 7.9, 4.7 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (m, 2H) 3.20 (dd, J = 13.5, 7.0 Hz, 1H), 3.04 - 3.00 (m, 3H), 2.85 (dd, J = 13.8, 8.1 Hz, 1H), 2.70 (h, J = 6.9 Hz, 1H), 2.51 - 2.42 (m, 1H), 2.42 - 2.25 (m, 2H), 1.91 (s, 3H), 1.82 - 1.68 (m, 2H), 1.63 (ddd, J = 13.1, 8.1, 4.0 Hz, 1H), 1.17 (d, J = 6.8 Hz, 3H), 1.10 (d, J = 7.1 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.34, 175.08, 173.48, 173.42, 168.02, 155.43, 150.20, 147.73, 143.18, 139.50, 131.92, 131.53, 129.84, 129.42, 126.25, 123.24, 122.39, 113.42, 78.67, 75.03, 56.61, 56.23, 44.30, 42.75, 41.97, 40.60, 39.87, 38.90, 38.09, 28.61, 25.76, 23.46, 22.54, 21.99, 17.19, 14.86.; HRMS (ES) calculated value C 38 H 51 ClN4O8S [M + H] 759.3189, found 759.3187.

[0221] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyrazin-2-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23 g). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23g (0.021g, 25% yield over three steps, 9:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (400 MHz, CD3OD) δ 8.62 (s, 1H), 8.51 (s, 1H), 8.39 (d, J = 2.6 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.5, 2.1 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.93 (dd, J = 15.9, 8.5 Hz, 1H), 6.81 (dt, J = 14.9, 7.3 Hz, 1H), 6.57 (d, J = 16.0 Hz, 1H), 6.02 (d, J = 15.4 Hz, 1H), 5.21 (dd, J = 9.4, 3.9 Hz, 1H), 4.58 (t, J = 7.5 Hz, 1H), 3.69 (dt, J = 9.0, 4.7 Hz, 1H), 3.48 (dd, J = 13.4, 6.6 Hz, 1H), 3.37 - 3.24 (m, 2H), 3.20 (dd, J = 13.5, 7.1 Hz, 1H), 3.07 - 2.97 (m, 1H), 3.03 (t, J = 6.8 Hz, 2H), 2.85 (dd, J = 13.7, 8.2 Hz, 1H), 2.70 (h, J = 6.9 Hz, 1H), 2.56 - 2.42 (m, 1H), 2.41 - 2.25 (m, 2H) 1.91 (s, 3H), 1.82 - 1.68 (m, 2H), 1.62 (ddd, J = 13.1, 8.1, 3.9 Hz, 1H), 1.19 (d, J = 6.8 Hz, 3H), 1.10 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H). 13 13C NMR (151 MHz, CD3OD) δ 200.34, 175.08, 173.47, 173.42, 168.03, 155.42, 153.09, 145.39, 143.89, 143.46, 143.20, 141.38, 131.91, 131.54, 129.86, 128.22, 126.28, 123.24, 113.42, 78.67, 75.01, 56.62, 56.24, 44.21, 42.75, 41.97, 40.60, 39.86, 38.80, 38.09, 28.61, 25.76, 23.46, 22.54, 21.99, 16.99, 14.86. HRMS (ES) calculated value C 37 H 50 ClN5O8S [M + H] 760.3141, found 760.3143.

[0222] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(1-methyl-1H-pyrozol-5-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23h). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23h (0.022, 29% yield over three steps, 9:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 7.34 (d, J = 2.2 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.4, 2.2 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.81 (dt, J = 15.0, 7.3 Hz, 1H), 6.42 (d, J = 15.9 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H), 6.25 (dd, J = 15.9, 8.6 Hz, 1H), 6.01 (dd, J = 15.4, 1.5 Hz, 1H), 5.21 (dd, J = 9.5, 3.9 Hz, 1H), 4.58 (dd, J = 8.1, 7.0 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.65 (dt, J = 8.0, 4.6 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (m, 2H) 3.20 (dd, J = 13.5, 7.0 Hz, 1H), 3.03 (td, J = 6.6, 1.8 Hz, 2H), 3.03 - 3.00 (m, 1H) 2.85 (dd, J = 13.8, 8.1 Hz, 1H), 2.70 (h, J = 6.9 Hz, 1H), 2.50 - 2.36 (m, 2H), 2.36 - 2.28 (m, 1H), 1.91 (s, 3H), 1.82 - 1.68 (m, 2H), 1.63 (ddd, J = 13.2, 8.1, 3.9 Hz, 1H), 1.15 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). 13 13C NMR: (150 MHz, CD3OD) δ 200.34, 175.07, 173.47, 173.41, 168.03, 155.41, 143.25, 142.84, 139.10, 137.95, 131.90, 131.51, 129.84, 126.20, 123.21, 118.41, 113.40, 103.56, 78.66, 75.00, 56.60, 56.24, 44.34, 42.74, 41.96, 40.59, 39.86, 38.86, 38.09, 28.60, 25.75, 23.47, 22.54, 21.99, 17.31, 14.87; HRMS (ES) calculated value C 37 H 52 ClN5O8S [M + H] 762.3298, found 762.3295.

[0223] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(1-methyl-1H-pyrozol-3-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23i). The reaction was carried out according to the general coupling procedure and purified by flash chromatography (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography (2-15% methanol / DCM) to give the final product 23i (0.019, 34% yield over three steps, 11:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 7.46 (d, J = 2.3 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H), 7.14 (dd, J = 8.5, 2.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.79 (dt, J = 15.1, 7.3 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 16.6 Hz, 1H), 6.21 (dd, J = 16.1, 8.4 Hz, 1H), 6.01 (d, J = 15.4 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.58 (dd, J = 8.1, 7.0 Hz, 1H), 3.84 (s, 6H), 3.63 (dt, J = 8.7, 4.6 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.29 (m, 2H), 3.20 (dd, J = 13.5, 7.0 Hz, 1H), 3.05 - 2.99 (m, 3H), 2.85 (dd, J = 13.7, 8.1 Hz, 1H), 2.70 (h, J = 7.0 Hz, 1H), 2.41 - 2.33 (m, 2H), 2.30 (dt, J = 15.4, 7.8 Hz, 1H), 1.91 (s, 3H), 1.82 - 1.68 (m, 2H), 1.63 (ddd, J = 13.2, 8.1, 3.9 Hz, 1H), 1.13 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 7.2 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H). 13 13C NMR: (150 MHz, CD3OD) δ 200.35, 175.08, 173.50, 173.43, 168.07, 155.42, 152.17, 143.44, 134.45, 133.02, 131.91, 131.54, 129.87, 126.17, 123.62, 123.23, 113.39, 103.27, 78.65, 75.19, 56.59, 56.27, 44.02, 42.75, 41.97, 40.59, 39.86, 38.72, 38.60, 38.09, 28.59, 25.76, 23.47, 22.54, 21.98, 17.24, 14.86. HRMS (ES) calculated value for C 37 H 52 ClN5O8S [M + H] 762.3298, found 762.3295.

[0224] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(1-methyl-1H-pyrozol-4-yl)octa-2,7-dienamido)propanamido)-2-methylpropanoate (23j). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23j (0.065 g, 48% yield over three steps 11:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 7.54 (s, 1H), 7.50 (s, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 8.2, 2.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.80 (dt, J = 15.0, 7.3 Hz, 1H), 6.22 (d, J = 16.0 Hz, 1H), 6.00 (d, J = 16.1, 1H), 5.94 (dd, J = 16.0, 8.4 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.58 (t, J = 7.5 Hz, 1H), 3.84 (s, 3H), 3.84 (s, 3H), 3.60 (dt, J = 8.5, 4.5 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (m, 2H), 3.20 (dd, J = 13.5, 6.9 Hz, 1H), 3.03 (t, J = 5.8 Hz, 2H), 3.03 - 3.00 (m, 1H), 2.85 (dd, J = 13.7, 8.2 Hz, 1H), 2.70 (h, J = 7.0 Hz, 1H), 2.40 - 2.23 (m, 3H), 1.91 (s, 3H), 1.81 - 1.68 (m, 2H), 1.63 (td, J = 9.2, 8.6, 4.4 Hz, 1H), 1.10 (d, J = 7.0 Hz, 6H), 0.95 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.35, 175.08, 173.49, 173.42, 168.10, 155.43, 143.57, 137.72, 131.92, 131.54, 130.89, 129.84, 129.39, 126.05, 123.24, 122.47, 121.60, 113.42, 78.67, 75.39, 56.61, 56.24, 44.14, 42.75, 41.97, 40.60, 39.86, 38.71, 38.65, 38.08, 28.60, 25.76, 23.46, 22.54, 21.99, 17.38, 14.86. HRMS (ES) calculated value for C 37 H 52 18H15ClN5O8S [M + H]+ 762.3298, found 762.3294.

[0225] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-8-(1-isopropyl-1H-pyrozol-4-yl)-6-methylocta-2,7-dienamido)propanamido)-2-methylpropanoate (231). The reaction was carried out according to the general coupling procedure and purified by flash chromatography (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography (2-15% methanol / DCM) to give the final product 23l (0.025, 38% yield over three steps, 9:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 7.64 (s, 1H), 7.51 (s, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.4, 2.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.80 (dt, J = 15.0, 7.3 Hz, 1H), 6.23 (d, J = 16.0 Hz, 1H), 6.00 (d, J = 15.4 Hz, 1H), 5.94 (dd, J = 16.0, 8.5 Hz, 1H), 5.21 (dd, J = 9.5, 4.0 Hz, 1H), 4.58 (t, J = 7.5 Hz, 1H), 4.46 (hept, J = 6.6 Hz, 1H), 3.84 (s, 3H), 3.61 (dt, J = 8.6, 4.5 Hz, 1H), 3.48 (dd, J = 13.5, 6.6 Hz, 1H), 3.33 - 3.30 (m, 2H), 3.20 (dd, J = 13.5, 7.0 Hz, 1H), 3.03 (t, J = 6.6 Hz, 2H), 3.03 - 3.00 (m, 1H), 2.85 (dd, J = 13.8, 8.1 Hz, 1H), 2.70 (h, J = 6.9 Hz, 1H), 2.43 - 2.23 (m, 3H), 1.91 (s, 3H), 1.82 - 1.69 (m, 2H), 1.63 (ddd, J = 13.2, 8.1, 3.9 Hz, 1H), 1.47 (d, J = 6.7 Hz, 6H), 1.11 (d, J = 6.9 Hz, 3H), 1.10 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.36, 175.08, 173.49, 173.44, 168.11, 155.42, 143.59, 137.30, 131.92, 131.52, 130.64, 129.85, 126.04(2), 123.22, 121.90, 121.82, 113.39, 78.65, 75.41, 56.59, 56.24, 54.99, 44.15, 42.74, 41.97, 40.59, 39.86, 38.67, 38.07, 28.59, 25.76, 23.48, 23.08, 22.54, 21.97, 17.44, 14.86. HRMS (ESI) calculated value for C 39 H 56 ClN5O8S [M + H] 790.3611, found 790.3608.

[0226] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl (R)-3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-8-(3,5-dimethylisoxazol-4-yl)-5-hydroxy-6-methylocta-2,7-dienamido)propanamido)-2-methylpropanoate (23m). The reaction was carried out according to the general coupling procedure and purified by flash chromatography (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography (2-15% methanol / DCM) to give the final product 23m (0.015 g, 43% yield over three steps, 8:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 1H NMR (600MHz, CD3OD) δ 7.25(d, J=2.1Hz, 1H), 7.14(dd, J=8.4, 2.2Hz, 1H), 6.97(d, J=8.4Hz, 1H), 6.81( dt, J=15.0, 7.3Hz, 1H), 6.14 (d, J=16.3Hz, 1H), 6.01 (d, J=15.4Hz, 1H), 5.93 (dd, J=16.3, 8.5Hz, 1H), 5.21 (dd, J=9.5, 4.0Hz, 1H), 4.57 (t, J=7.5Hz, 1H), 3.84 (s, 3H), 3.63(dt, J=8.5, 4.7Hz, 1H), 3.48(dd, J=13.5, 6.6Hz, 1H), 3.33-3.30(m, 2H) 3.20(dd, J=13.5, 7.0Hz, 1H), 3.03(t, J=6.8Hz, 2H), 3.02-2.99(m, 1H) 2.85(dd, J=13.7, 8.1Hz, 1H), 2.70(h, J=7.0Hz, 1H), 2.39(s, 3H), 2.38-2.30(m, 3H), 2.28(s, 3H), 1.91(s, 3H), 1.82-1.67(m, 2H ), 1.63 (ddd, J=12.6, 8.2, 3.9Hz, 1H), 1.14 (d, J=6.8Hz, 3H), 1.10 (d, J=7.1Hz, 3H), 0.95 (d, J=6.4Hz, 3H), 0.92 (d, J=6.4Hz, 3H). 13 C NMR (150MHz, CD3OD) δ 200.36, 175.08, 173.49, 173.44, 168.04, 166.54, 159.73, 155.42, 143.35, 1 35.53, 131.90, 131.50, 129.85, 126.13, 123.22, 119.18, 114.25, 113.38, 78. 65, 75.09, 56.59, 56.26, 44.85, 42.74, 41.97, 40.59, 39.86, 38.94, 38.09, 2 8.59, 25.76, 23.48, 22.53, 21.97, 17.69, 14.86, 11.47, 11.29.HRMS(ES) calculation value C 38 H 53 ClN4O9S[M+H]777.3295, measured value 777.3293.

[0227] 7.ジェムジメチルセコクリプトフィシン (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl 3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyridin-3-yl)octa-2,7-dienamido)propanamido)-2,2-dimethylpropanoate (23d). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23d (0.019 g, 51% yield over three steps, 10:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 8.52 (d, J = 2.3 Hz, 1H), 8.35 (dd, J = 4.8, 1.6 Hz, 1H), 7.89 (dt, J = 8.0, 2.0 Hz, 1H), 7.37 (dd, J = 8.0, 4.9 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.15 (dd, J = 8.4, 2.3 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 6.80 (dt, J = 15.0, 7.3 Hz, 1H), 6.46 (d, J = 16.1 Hz, 1H), 6.40 (dd, J = 16.0, 7.9 Hz, 1H), 6.01 (d, J = 15.4 Hz, 1H), 5.20 (dd, J = 9.5, 3.8 Hz, 1H), 4.65 (dd, J = 8.5, 6.5 Hz, 1H), 3.92 (p, J = 6.2 Hz, 2H), 3.83 (s, 3H), 3.66 (dt, J = 8.7, 4.6 Hz, 1H), 3.44 - 3.36 (m, 2H), 3.33 - 3.29 (m, 2H), 3.11 - 3.00 (m, 3H), 2.85 (dd, J = 13.9, 8.5 Hz, 1H), 2.44 (td, J = 7.2, 4.3 Hz, 1H), 2.39 (dt, J = 13.0, 6.4 Hz, 1H), 2.32 (dt, J = 14.9, 7.6 Hz, 1H), 1.91 (s, 3H), 1.83 - 1.69 (m, 2H), 1.64 (ddd, J = 13.4, 8.4, 3.9 Hz, 1H), 1.14 (s, 3H) 1.14 (d, 3H), 1.13 (d, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.53, 176.86, 173.76, 173.42, 168.17, 155.38, 148.26, 148.21, 143.36, 136.35, 135.52, 134.80, 131.89, 131.69, 129.82, 127.96, 126.16, 125.25, 123.23, 113.41, 78.81, 75.15, 64.74, 56.61, 56.39, 47.89, 44.69, 44.31, 41.94, 39.86, 38.88, 37.86, 28.73, 25.87, 25.26, 23.49, 23.26, 23.18, 22.59, 21.95, 17.35. HRMS (ES) calculated value C 39 H 53ClN4O8S[M+H] 773.3345, found 773.3353.

[0228] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl 3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(pyridin-4-yl)octa-2,7-dienamido)propanamido)-2,2-dimethylpropanoate (23f). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23f (0.039 g, 45% yield over three steps, 12:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 11H NMR (600 MHz, CD3OD) δ 8.41 (d, J = 6.1 Hz, 1H), 7.40 (d, J = 6.1 Hz, 1H), 7.27 (d, J = 1.9 Hz, 1H), 7.15 (dd, J = 8.5, 2.2 Hz, 1H), 6.96 (dd, J = 8.5, 1.0 Hz, 1H), 6.79 (dt, J = 14.5, 7.1 Hz, 1H), 6.61 (dd, J = 16.0, 8.5 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 6.00 (d, J = 15.4 Hz, 1H), 5.20 (dd, J = 9.5, 3.8 Hz, 1H), 4.65 (dd, J = 8.4, 6.6 Hz, 1H), 3.83 (s, 3H), 3.66 (dt, J = 8.5, 4.4 Hz, 1H), 3.40 (d, J = 13.6 Hz, 1H), 3.36 (d, J = 12.9 Hz, 1H), 3.32 - 3.29 (m, 2H), 3.12 - 2.99 (m, 3H), 2.85 (dd, J = 14.0, 8.5 Hz, 1H), 2.51 - 2.42 (m, 1H), 2.41 - 2.34 (m, 1H), 2.35 - 2.27 (m, 1H), 1.90 (s, 3H), 1.81 - 1.68 (m, 2H), 1.68 - 1.60 (m, 1H), 1.16 (s, 6H), 1.15 (d, J = 5.1 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 200.53, 176.86, 173.67, 173.42, 168.14, 155.38, 150.15, 147.78, 143.23, 139.54, 131.90, 131.68, 129.81, 129.42, 126.21, 123.24, 122.41, 113.43, 78.81, 75.01, 56.61, 56.33, 47.78, 44.69, 44.28, 41.94, 39.86, 38.92, 37.86, 28.73, 25.87, 23.49, 23.26, 23.18, 22.59, 21.95, 17.20.; HRMS (ES) calculated value for C 39 H 53 ClN4O8S [M + H] 773.3345, found 773.3353.

[0229] (S)-1-((2-acetamidoethyl)thio)-4-methyl-1-oxopentan-2-yl 3-((R)-3-(3-chloro-4-methoxyphenyl)-2-((2E,5S,6R,7E)-5-hydroxy-6-methyl-8-(1-methyl-1H-pyrozol-4-yl)octa-2,7-dienamido)propanamido)-2,2-dimethylpropanoate (23k). The reaction was carried out according to the general coupling procedure and purified by flash chromatography system (1–10% methanol / DCM). 。 R f = 0.65 (10% methanol / DCM). This was then deprotected according to the general deprotection procedure and purified by flash chromatography system (2-15% methanol / DCM) to give the final product 23k (0.060, 41% yield over three steps, 10:1 dr) as a clear, colorless oil: R f =0.25(10% methanol / DCM); 1 H NMR (600 MHz, CD3OD) δ 7.55(s, 1H), 7.50(s, 1H), 7.28(d, J=2.1Hz, 1H), 7.15(dd, J=8.5, 2.2Hz, 1 H), 6.96(d, J=8.5Hz, 1H), 6.79(dt, J=14.9, 7.3Hz, 1H), 6.21(d, J=16.1Hz, 1H), 5.99(d, J=15.4Hz, 1H), 5.93(dd, J=16.0, 8.4Hz, 1H), 5.20(dd, J=9.6 , 3.9Hz, 1H), 4.65(dd, J=8.6, 6.5Hz, 1H), 3.84(s, 3H), 3.83(s, 3H), 3.60(d t, J=8.8, 4.6Hz, 1H), 3.40(d, J=13.5Hz, 1H), 3.35(d, J=13.9Hz, 1H), 3.33 -3.29(m, 2H), 3.12-2.98(m, 3H), 2.85(dd, J=13.9, 8.6Hz, 1H), 2.32(m, 3H) , 1.91(s, 3H), 1.83-1.68(m, 2H), 1.64(ddd, J=12.8, 8.4, 3.8Hz, 1H), 1.16( s, 6H), 1.10(d, J=6.9Hz, 3H), 0.96(d, J=6.5Hz, 3H), 0.92(d, J=6.5Hz, 3H).13 C NMR (150MHz, CD3OD) δ 200.54, 176.85, 173.68, 173.44, 168.22, 155.38, 143.62, 137.71, 131.90, 1 31.68, 130.86, 129.81, 129.39, 126.00, 123.23, 122.47, 121.60, 113.40, 78. 80, 75.37, 56.60, 56.35, 47.77, 44.68, 44.12, 41.94, 39.86, 38.72, 38.67, 37.85, 28.73, 25.87, 23.49, 23.25, 23.17, 22.59, 21.94, 17.40. HRMS (ES) calculated value C 38 H 54 ClN5O8S[M+H] 776.3454, found 776.3457.

[0230] 8. Biocatalytic Cryptophycin Synthesis and Characterization of Analogues General procedure analysis scale response. Phosphate buffer (pH = 7.2, 100 mM, 300 μL), DMSO (5%), and seco substrate (50 μM) suspended in DMSO were added to a 1.5 mL Eppendorf tube. This was then treated with CrpTE enzyme (in phosphate buffer, pH = 7.2, 100 mM, 0.5 μM) and shaken at 30 °C for 12 hours. Upon completion of the reaction, the aqueous layer was extracted with 0.300 mL of ethyl acetate, and 150 μL was removed, dried directly into an HPLC vial, and resuspended in 70 μL of methanol. This was used for analysis by TOF-MS.

[0231] General Procedure for Semi-Preparative Scale Reactions. DMSO (5%) was added to a 250 mL Erlenmeyer flask, and the substrate (75 μM) was suspended in DMSO. This was diluted with phosphate buffer (pH = 7.2, 100 mM) and warmed to 30 °C for 20 min before treatment with CrpTE enzyme (0.5 μM). The reaction was shaken at 100 RPM at 30 °C for 12 h before diluting 1:1 v / v with acetone and cooling to -20 °C in a freezer. The precipitated protein was filtered through Celite, washed with acetone, the organics were removed under reduced pressure, and the remaining aqueous layer was extracted with 3 × 20 mL of DCM. The organics were combined, dried over sodium sulfate, filtered, and concentrated. The reaction was purified using a HydroRP C18 (250 × 10.0 mm, 4 micron) column using a 20–80% water / acetonitrile gradient at a flow rate of 3 mL / min.

[0232] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(pyridin-2-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25b). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1H NMR(600MHz、CD3OD) δ 8.46(dd、J=5.0、0.9Hz、1H)、7.78(td、J=7.7、1.8Hz、1H)、7.48(d、J=7.9Hz、1H)7. 28(d、J=2.2Hz、1H)、7.26(ddd、J=7.5、5.0、1.1Hz、1H)、7.17(dd、J=8.5、2.2Hz、1H) 6.98(d、J=8.5Hz、1H)、6.71(ddd、J=15.1、11.1、3.9Hz、1H)、6.61-6.57(m、2H)5.9 3(dd、J=15.1、1.9Hz、1H)、5.09(ddd、J=11.3、6.9、1.9Hz、1H)、4.93(dd、J=9.9、3.6H z、1H)、4.52(dd、J=11.3、3.9Hz、1H)、3.84(s、3H)、3.58(dd、J=13.8、3.3Hz、1H)3. 27(dd、J=13.8、3.0Hz、1H)、3.18(dd、J=14.5、3.9Hz、1H)、2.80-2.73(m、2H)、2.73-2 .65(m、2H)、2.39(dt、J=14.5、11.2Hz、1H)、1.69-1.52(m、2H)、1.34(ddd、J=14.1、8 .8、3.6Hz、1H)、1.18(d、J=7.1Hz、6H)、0.74(d、J=6.5Hz、3H)、0.71(d、J=6.6Hz、3H). 13 C NMR (151MHz, CD3OD) δ 177.53, 174.04, 172.22, 168.33, 156.60, 155.36, 149.99, 143.41, 8.74, 137.58, 132.23, 132.10, 131.49, 129.28, 125.62, 123.71, 123.2 7, 122.82, 113.50, 78.48, 72.86, 57.35, 56.60, 43.46, 41.19, 40.88, 38.98, 37.99, 36.36, 5.62, 23.20, 21.66, 17.53, 15.06.HRMS(ES) Introduction C.C 34 H 42 ClN3O7[M+H]640.2784;

[0233] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(pyridin-3-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25c). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600MHz, CD3OD) δ 8.53(d, J=2.2Hz, 1H), 8.38(dd, J=4.8, 1.6Hz, 1H), 7.92(d, J=8.0Hz, 1H), 7.40(dd, J= 8.0, 4.9Hz, 1H), 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.4, 2.2Hz, 1H), 6.98(d, J=8.4Hz, 1H), 6.71(ddd, J=15.1, 11.2, 3.9Hz, 1H), 6.53(d, J=15.9Hz, 1H), 6.30(dd, J=15.9, 8. 9Hz, 1H), 5.93(dd, J=15.2, 1.9Hz, 1H), 5.08(ddd, J=11.2, 7.2, 1.9Hz, 1H), 4.93(dd, J= 9.9, 3.7Hz, 1H), 4.52(dd, J=11.2, 3.9Hz, 1H), 3.84(s, 3H), 3.58(dd, J=13.8, 3.3Hz, 1 H), 3.27(dd, J=13.8, 3.0Hz, 1H), 3.18(dd, J=14.5, 3.9Hz, 1H), 2.80-2.63(m, 4H), 2.3 7(dt, J=14.4, 11.1Hz, 1H), 1.69-1.55(m, 2H), 1.31(ddd, J=14.1, 8.9, 3.7Hz, 1H), 1.1 9(d, J=3.5Hz, 3H), 1.18(d, J=2.9Hz, 3H), 0.74(d, J=6.5Hz, 3H), 0.70(d, J=6.5Hz, 3H). 13C NMR (150 MHz, CD3OD) δ 180.30, 177.53, 174.03, 172.21, 168.33, 155.36, 148.71, 148.40, 143.42, 135.55, 134.96, 134.86, 132.21, 131.49, 129.28, 128.77, 125.62, 125.40, 113.49, 78.47, 72.80, 57.36, 56.60, 43.75, 41.18, 40.94, 38.97, 37.88, 36.35, 25.60, 23.18, 21.69, 17.50, 15.06. HRMS (ES) Calculated C 34 H 42 ClN3O7[M+H] 640.2784, found 640.2789

[0234] (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-((R,E)-4-(pyridin-3-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25d). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 11H NMR (600 MHz, CD3OD) δ 8.53 (s, 1H), 8.38 (d, J = 4.7 Hz, 1H), 7.92 (dt, J = 8.0, 1.9 Hz, 1H), 7.40 (dd, J = 8.0, 4.8 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.4, 2.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.72 (ddd, J = 15.2, 11.2, 3.9 Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H), 6.30 (dd, J = 16.0, 8.9 Hz, 1H), 5.92 (dd, J = 15.1, 1.9 Hz, 1H), 5.07 (ddd, J = 11.1, 7.2, 1.8 Hz, 2H), 4.96 (dd, J = 9.9, 3.4 Hz, 1H), 4.51 (dd, J = 11.3, 3.8 Hz, 1H), 3.84 (s, 3H), 3.46 (d, J = 13.6 Hz, 1H), 3.18 (dd, J = 14.5, 3.8 Hz, 1H), 3.08 (d, J = 13.7 Hz, 1H), 2.74 (dd, J = 14.5, 11.4 Hz, 1H), 2.74 - 2.62 (m, 2H), 2.36 (dt, J = 14.4, 11.2 Hz, 1H), 1.66 - 1.55 (m, 2H), 1.37 - 1.26 (m, 3H), 1.20 (s, 3H), 1.18 (d, J = 6.9 Hz, 3H), 1.16 (s, 3H), 0.74 (d, J = 6.4 Hz, 3H), 0.70 (d, J = 6.4 Hz, 3H). 13 13C NMR (150 MHz, CD3OD) δ 180.03, 178.94, 173.68, 172.01, 168.22, 155.37, 148.71, 148.40, 143.63, 135.55, 134.85, 132.17, 131.46, 129.26, 128.78, 125.43, 123.28, 113.50, 78.43, 72.58, 57.48, 56.60, 47.39, 44.02, 43.75, 40.92, 37.92, 36.48, 31.64, 25.85, 23.32, 23.29, 23.22, 21.65, 17.49. HRMS (ES) calculated value for C 35 H 44 ClN3O7 [M + H] 654.2941, found 654.2940.

[0235] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(pyridin-4-yl)butan-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25e). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600 MHz, CD3OD) δ 8.43(d, J=6.2Hz, 2H), 7.42(d, J=6.3Hz, 2H), 7.27(d, J=2.1Hz, 1H), 7.15( dd, J=8.5, 2.1Hz, 1H), 6.96(d, J=8.4Hz, 1H), 6.69(ddd, J=15.1, 11.1, 3.9 Hz, 1H), 6.50-6.47(m, 2H), 5.91(dd, J=15.2, 1.8Hz, 1H), 5.08(dd, J=10.7, 8.3Hz, 1H), 4.90(dd, J=9.9, 3.6Hz, 1H), 4.51(dd, J=11.2, 3.9Hz, 1H), 3. 82(s, 3H), 3.56(dd, J=13.8, 3.3Hz, 1H), 3.25(dd, J=13.8, 2.9Hz, 1H), 3.1 6(dd, J=14.5, 3.9Hz, 1H), 2.77-2.62(m, 4H), 2.34(dt, J=14.7, 11.2Hz, 1H ), 1.67-1.51(m, 2H), 1.28(ddd, J=12.9, 8.7, 3.5Hz, 1H), 1.17(d, J=2.6Hz , 3H), 1.16(d, J=2.0Hz, 3H), 0.72(d, J=6.4Hz, 3H), 0.68(d, J=6.5Hz, 3H). 13 C NMR (150MHz, CD3OD) δ 177.55, 174.02, 172.17, 168.31, 155.36, 150.42, 147.12, 143.33, 138.63, 132.21, 131.49, 130.23, 129.28, 125.66, 123.27, 122.4 9, 113.49, 78.35, 72.78, 57.36, 56.60, 43.67, 41.17, 40.91, 38.96, 37.88, 36.35, 25.60, 23.15, 21.66, 17.35, 15.06.HRMS(ES) calculated value C 34 H42 ClN3O7[M+H] 640.2784, found 640.2787.

[0236] (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-((R,E)-4-(pyridin-4-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25f). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600 MHz, CD3OD) δ 8.45(d, J=5.7Hz, 2H), 7.44(d, J=6.3Hz, 2H), 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.4, 2.2Hz, 1H), 6.98(d, J=8.5Hz, 1H), 6.72(ddd, J=15.1, 11.2, 3.9Hz, 1H) , 6.51(s, 1H), 6.50(d, J=4.6Hz, 1H), 5.92(dd, J=15.1, 1.9Hz, 1H), 5.09(ddd, J=11.3, 7.2, 1.9Hz, 1H), 4.95(dd, J=9.9, 3.3Hz, 1H), 4.51(dd, J=11.3, 3.7Hz , 1H), 3.84(s, 3H), 3.46(d, J=13.6Hz, 1H), 3.18(dd, J=14.6, 3.8Hz, 1H), 3.08 (d, J=13.6Hz, 1H), 2.74(dd, J=14.5, 11.3Hz, 1H), 2.74-2.66(m, 2H), 2.35(dt , J=14.5, 11.2Hz, 1H), 1.67-1.54(m, 2H), 1.37-1.24(m, 1H), 1.19(s, 3H), 1.1 8(d, J=6.9Hz, 3H), 1.16(s, 3H), 0.74(d, J=6.3Hz, 3H), 0.71(d, J=6.4Hz, 3H). 13C NMR (150MHz, CD3OD) δ 177.59, 174.05, 172.32, 168.36, 155.34, 143.63, 137.86, 132.21, 131.48, 130.26, 129.63, 129.28, 125.50, 123.24, 122.46, 121.98, 1 13.46, 78.68, 72.91, 57.37, 56.58, 43.68, 41.18, 40.87, 38.95, 38.75, 37.84, 36.36, 25.64, 23.18, 21.67, 17.75, 15.07.HRMS(ES) calculated value C 35 H 44 ClN3O7[M+H] 654.2941, found 654.2947.

[0237] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(pyrazin-2-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25 g). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1H NMR(600MHz、CD3OD) δ 8.61(d、J=1.5Hz、1H)、8.54(dd、J=2.6、1.5Hz、1H)、8.42(d、J=2.6Hz). 1H)、7.28(d、J=2.2Hz、1H)、7.17(dd、J=8.4、2.2Hz、1H)、6.98(d、J=8.5Hz、1H) 6.84(dd、J=15.7、8.9Hz、1H)、6.71(ddd、J=15.2、11.2、3.9Hz、1H)、6.64(d、J= 15.7Hz、1H)、5.93(dd、J=15.1、1.9Hz、1H)、5.11(ddd、J=11.3、6.8、2.0Hz、1H) 4.94(dd、J=9.7、3.7Hz、1H)、4.53(dd、J=11.2、3.9Hz、1H)、3.84(s、3H)、3.58(d d、J=13.8、3.4Hz、1H)、3.27(dd、J=13.7、3.0Hz、1H)、3.18(dd、J=14.5、3.9Hz); 1H)、2.81-2.72(m、3H)、2.69(ddt、J=14.2、3.7、1.9Hz、1H)、2.38(dt、J=14.6、1 1.2Hz、1H)、1.70-1.57(m、2H)、1.36(ddd、J=14.1、8.7、3.7Hz、1H)、1.19(d、J=6 .9Hz、3H)、1.19(d、J=7.5Hz、3H)、0.76(d、J=6.5Hz、3H)、0.73(d、J=6.5Hz、3H). 13 C NMR(150MHz、CD3OD) δ 177.53, 174.03, 172.18, 168.32, 155.36, 152.52, 145.69, 144.15, 143.92 143.33, 140.15, 132.22, 131.48, 129.28, 128.83, 125.65, 123.27, 113.50 78.45, 72.83, 57.35, 56.60, 43.37, 41.19, 40.91, 38.98, 38.03, 36.35, 65, 23.23, 21.74, 17.37, 15.07.

[0238] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(1-methyl-1H-pyrozol-5-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25h). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600MHz, CD3OD) δ 7.36(d, J=2.1Hz, 1H), 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.5, 2.2Hz, 1H), 6.9 8(d, J=8.4Hz, 1H), 6.71(ddd, J=15.1, 11.2, 3.9Hz, 1H), 6.52(d, J=15.8Hz, 1H) , 6.42(d, J=2.1Hz, 1H), 6.14(dd, J=15.8, 9.0Hz, 1H), 5.93(dd, J=15.1, 1.9Hz, 1H), 5.07(ddd, J=11.3, 6.9, 1.9Hz, 1H), 4.93(dd, J=9.8, 3.6Hz, 1H), 4.52(dd, J=11.3, 3.9Hz, 1H), 3.84(s, 6H), 3.59(dd, J=13.8, 3.3Hz, 1H), 3.27(dd, J=13. 8, 3.0Hz, 1H), 3.18(dd, J=14.5, 3.9Hz, 1H), 2.79-2.73(m, 2H), 2.73-2.62(m, 2 H), 2.35(dt, J=14.6, 11.2Hz, 1H), 1.71-1.57(m, 2H), 1.35(ddd, J=14.0, 8.6, 3 .5Hz, 1H), 1.19(d, J=7.4Hz, 3H), 1.17(d, J=6.9Hz, 3H), 0.77(d, J=6.4Hz, 6H). 13C NMR:(150MHz, CD3OD) δ 177.58, 174.04, 172.22, 168.32, 155.35, 143.40, 142.29, 139.27, 136.81, 132.20, 131.48, 129.28, 125.61, 123.24, 119.18, 113.46, 1 03.68, 78.39, 72.84, 57.39, 56.58, 43.68, 41.16, 40.96, 38.96, 37.96, 36.51, 36.36, 25.69, 23.25, 21.76, 17.53, 15.08.HRMS(ES) calculated value C 33 H 43 ClN4O7[M+H] 643.2893, found 643.2890.

[0239] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(1-methyl-1H-pyrozol-3-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25i). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1H NMR(600MHz、CD3OD) δ 7.47(d、J=2.3Hz、1H)、7.26(d、J=2.2Hz、1H)、7.15(dd、J=8.4、2.2Hz、1H)、6.96(d、J=8.5 Hz、1H)、6.68(ddd、J=15.1、11.1、3.9Hz、1H)、6.36(d、J=16.0Hz、1H)、6.33(d、J=2.3Hz、1H )、6.05(dd、J=16.0、9.0Hz、1H)、5.90(dd、J=15.2、1.9Hz、1H)、5.00(ddd、J=11.3、7.1、2. 0Hz、1H)、4.90(dd、J=10.0、3.6Hz、1H)、4.50(dd、J=11.2、3.9Hz、1H)、3.56(dd、J=13.8、3. 3Hz、1H)、3.25(dd、J=13.8、3.0Hz、1H)、3.16(dd、J=14.5、3.9Hz、1H)、2.77-2.71(m、2H) 2.65(ddt、J=14.5、4.1、2.1Hz、1H)、2.61-2.51(m、1H)、2.34(dt、J=14.6、11.2Hz、1H)、1.6 8-1.61(m、1H)、1.57(ddd、J=14.8、10.0、5.0Hz、1H)、1.35(ddd、J=14.2、9.0、3.6Hz、1H)1 .17(d、J=7.4Hz、3H)、1.12(d、J=6.8Hz、3H)、0.76(d、J=6.5Hz、3H)、0.74(d、J=6.6Hz、3H). 13 C NMR(150MHz、CD3OD) δ 177.56, 174.05, 172.28, 168.35, 155.35, 151.52, 143.52, 133.58 133.15, 132.23, 131.49, 129.28, 125.57, 124.52, 123.26, 113.49,1 03.64, 78.52, 72.93, 57.36, 56.60, 43.57, 41.19, 40.83, 38.97, 65.37.89.36.36.25.68.23.16.21.62.17.61.15.06.HRMS(ES)Designation C.C 35 H 47 ClN4O7[M+H]671.3206;

[0240] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-((R,E)-4-(1-methyl-1H-pyrozol-4-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25j). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600MHz, CD3OD) δ 7.61(s, 1H), 7.51(s, 1H), 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.4, 2.2Hz, 1H), 6.98 (d, J=8.4Hz, 1H), 6.70(ddd, J=15.2, 11.2, 3.9Hz, 1H), 6.27(d, J=15.9Hz, 1H), 5.92 (dd, J=15.2, 1.9Hz, 1H), 5.80(dd, J=15.9, 8.9Hz, 1H), 4.99(ddd, J=11.3, 7.1, 1.9H z, 1H), 4.92(dd, J=10.0, 3.6Hz, 1H), 4.52(dd, J=11.3, 3.8Hz, 1H), 3.84(d, J=1.8Hz , 6H), 3.58(dd, J=13.8, 3.3Hz, 1H), 3.27(dd, J=13.8, 2.9Hz, 1H), 3.18(dd, J=14.5 , 3.8Hz, 1H), 2.81-2.70(m, 2H), 2.69-2.60(m, 1H), 2.50(p, J=7.1Hz, 1H), 2.33(dt, J=14.5, 11.2Hz, 1H), 1.71-1.56(m, 2H), 1.38(ddd, J=13.2, 9.0, 3.6Hz, 1H), 1.18(d , J=7.5Hz, 3H), 1.11(d, J=6.8Hz, 3H), 0.78(d, J=6.5Hz, 3H), 0.76(d, J=6.6Hz, 3H). 13C NMR (150MHz, CD3OD) δ 177.59, 174.05, 172.32, 168.36, 155.34, 143.63, 137.86, 132.21, 131.48, 130.26, 129.63, 129.28, 125.50, 123.24, 122.46, 121.98, 1 13.46, 78.68, 72.91, 57.37, 56.58, 43.68, 41.18, 40.87, 38.95, 38.75, 37.84, 36.36, 25.64, 23.18, 21.67, 17.75, 15.07.HRMS(ES) calculated value C 33 H 43 ClN4O7[M+H] 643.2893, found 643.2890.

[0241] (3S,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-((R,E)-4-(1-methyl-1H-pyrozol-4-yl)but-3-en-2-yl)-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone. The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 11H NMR (600 MHz, CD3OD) δ 7.60 (s, 1H), 7.51 (s, 1H), 7.27 (d, J = 2.1 Hz, 1H), 7.16 (dd, J = 8.5, 2.2 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.70 (ddd, J = 15.2, 11.2, 3.9 Hz, 1H), 6.27 (d, J = 15.9 Hz, 1H), 5.90 (dd, J = 15.1, 1.9 Hz, 1H), 5.79 (dd, J = 15.9, 8.9 Hz, 1H), 4.97 (ddd, J = 11.3, 7.1, 1.6 Hz, 1H), 4.94 (dd, J = 9.9, 3.4 Hz, 1H), 4.50 (dd, J = 11.4, 3.7 Hz, 1H), 3.83 (s, 3H), 3.83 (s, 3H), 3.45 (d, J = 13.6 Hz, 1H), 3.17 (dd, J = 14.5, 3.7 Hz, 1H), 3.07 (d, J = 13.6 Hz, 1H), 2.73 (dd, J = 14.5, 11.4 Hz, 1H), 2.65 (dt, J = 14.7, 2.4 Hz, 1H), 2.50 (h, J = 7.2 Hz, 1H), 2.32 (dt, J = 14.4, 11.2 Hz, 1H), 1.68 - 1.57 (m, 2H), 1.43 - 1.36 (m, 1H), 1.19 (s, 3H), 1.15 (s, 3H), 1.11 (d, J = 6.9 Hz, 3H), 0.77 (t, J = 6.7 Hz, 6H). 13 13C NMR (150 MHz, CD3OD) δ 178.97, 173.70, 172.12, 168.25, 155.37, 143.83, 137.86, 132.17, 131.46, 130.26, 129.63, 129.26, 125.33, 123.27, 122.48, 121.98, 113.49, 78.65, 72.69, 57.47, 56.59, 47.40, 44.01, 43.68, 40.85, 38.75, 37.88, 36.49, 25.90, 23.32, 23.28, 23.20, 21.64, 17.74. HRMS (ES) Calcd for C 34 H 45 ClN4O7 [M + H] 656.2977, found 656.2973.

[0242] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-16-((R,E)-4-(1-isopropyl-1H-pyrozol-4-yl)but-3-en-2-yl)-6-methyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (251). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1 H NMR (600MHz, CD3OD) δ 7.70(s, 1H), 7.53(s, 1H), 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.4, 2.2Hz, 1H), 6.98(d, J =8.5Hz, 1H), 6.70(ddd, J=15.1, 11.2, 3.9Hz, 1H), 6.28(d, J=15.8Hz, 1H), 5.92(dd, J=15 .2, 1.9Hz, 1H), 5.79(dd, J=15.9, 8.9Hz, 1H), 4.98(ddd, J=11.2, 7.5, 2.0Hz, 1H), 4.91( dd, J=10.0, 3.5Hz, 1H), 4.52(dd, J=11.2, 3.8Hz, 1H), 4.47(p, J=6.7Hz, 1H), 3.84(s, 3H) , 3.58(dd, J=13.8, 3.3Hz, 1H), 3.27(dd, J=13.7, 3.0Hz, 1H), 3.18(dd, J=14.5, 3.8Hz, 1 H), 2.79-2.71(m, 2H), 2.71-2.63(m, 1H), 2.50(h, J=7.2Hz, 1H), 2.33(dt, J=14.5, 11.2H z, 1H), 1.72-1.54(m, 2H), 1.46(d, J=6.7Hz, 6H), 1.37(ddd, J=14.2, 9.0, 3.6Hz, 1H), 1. 18(d, J=7.4Hz, 3H), 1.12(d, J=6.9Hz, 3H), 0.77(d, J=6.5Hz, 3H), 0.74(d, J=6.6Hz, 3H). 13C NMR (150MHz, CD3OD) δ 177.60, 174.05, 172.33, 168.37, 155.35, 143.64, 137.40, 132.21, 13 1.48, 130.12, 129.28, 126.34, 125.51, 123.25, 122.67, 121.38, 113. 46, 78.67, 72.93, 57.38, 56.59, 55.07, 43.80, 41.18, 40.87, 38.96, 37.85, 36.37, 25.64, 23.31, 23.09, 21.70, 17.79, 15.07.HRMS(ES) calculated value C 35 H 47 ClN4O7[M+H] 671.3206, found 671.3025.

[0243] (3S,6R,10R,16S,E)-10-(3-chloro-4-methoxybenzyl)-16-((R,E)-4-(3,5-dimethylisoxazol-4-yl)but-3-en-2-yl)-3-isobutyl-6-methyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone (25m). The reaction was carried out and purified according to the general procedure for semi-preparative scale reactions. 1H NMR (600MHz, CD3OD) δ 7.28(d, J=2.2Hz, 1H), 7.17(dd, J=8.4, 2.2Hz, 1H), 6.98(d, J=8.4Hz, 1H), 6.69(ddd, J=15.1, 11.1, 3.9Hz, 1H), 6.23 (d, J=16.2Hz, 1H), 5.93 (dd, J=15.1, 1.9Hz, 1H), 5.80 (dd, J=16.2, 8.9Hz, 1H), 5.06 (ddd, J=11.3, 6.7, 1.9Hz, 1H), 4.93 (dd, J=9.7, 3.8Hz, 1H), 4.51(dd, J=11.2, 3.9Hz, 1H), 3.84(s, 3H), 3.57(dd, J=13.7, 3.3Hz, 1H), 3.27(d d, J=13.8, 3.0Hz, 1H), 3.17 (dd, J=14.5, 3.9Hz, 1H), 2.80-2.71 (m, 2H), 2.67 (ddt, J= 14.6, 4.0, 2.0Hz, 1H), 2.58 (dt, J=8.8, 6.6Hz, 1H), 2.40 (s, 3H), 2.35 (dt, J=14.5, 11 .4Hz, 1H), 2.28(s, 3H), 1.70-1.53(m, 2H), 1.33(ddd, J=14.1, 8.7, 3.8Hz, 1H), 1.18( d, J=7.4Hz, 3H), 1.15 (d, J=6.9Hz, 3H), 0.79 (d, J=3.7Hz, 3H), 0.78 (d, J=3.8Hz, 3H). 13 C NMR (150MHz, CD3OD) δ 177.53, 174.04, 172.23, 168.34, 166.95, 159.53, 155.35, 143.40, 13 4.36, 132.19, 131.48, 129.29, 125.65, 123.24, 120.19, 113.78, 113. 46, 78.53, 72.77, 57.40, 56.58, 44.22, 41.16, 41.02, 38.95, 37.87, 36.35, 25.62, 23.20, 21.77, 17.83, 15.07, 11.55, 11.45.HRMS (ES) calculation value C 34 H 44 ClN3O8[M+H]658.2890, measured value 658.2893.

[0244] Ordinary Nanako high-quality handmade handcrafts The protein was grown and purified using a modified protocol. 12 The pET-28 b(+)-CrpTE construct was transformed into BL21(DE3) and grown overnight at 37°C on LB agar plates supplemented with 50 μg / mL kanamycin. Colonies were picked and grown overnight at 37°C in 25 mL LB broth supplemented with 50 μg / mL kanamycin. Six liters of TB expression medium containing 50 μg / mL kanamycin were each inoculated with 4 mL of the overnight culture, and the OD 600 The mixture was incubated (37°C, 200 rpm) for approximately 5 hours until the RI reached 1.2. The flask was removed from the shaker and cooled in an ice bath for 15 minutes (temperature approximately 20°C). The flask was returned to the shaker, and protein expression was initiated by the addition of 100 μM IPTG and allowed to proceed for 18 hours at 20°C. The cells were harvested by centrifugation at 6,000 x g for 30 minutes.

[0245] The cell pellet was then resuspended in lysis buffer (10 mL / g cell pellet, 100 mM sodium phosphate, 20 mM imidazole, 300 mM NaCl, pH 8) and treated with 1 mg Dnase (Sigma). The solution was placed on ice and subjected to sonication (12 × 10 s with a 50 s pause). The suspension was then centrifuged at 50,000 × g for 30 min at 4 °C. The supernatant was collected and passed through a 0.45 μm filter before being loaded onto a 10 mL NiNTA resin column. The column was washed with 10 column volumes of wash buffer (100 mM sodium phosphate buffer, 50 mM imidazole, 300 mM NaCl, pH 8), and the protein was eluted using 15 mL of elution buffer (100 mM sodium phosphate, 300 mM imidazole, 300 mM NaCl, pH 8). The eluate was then concentrated to 2.5 mL and subjected to a PD-10 buffer exchange / desalting column (pre-equilibrated with storage buffer, 100 mM sodium phosphate, 150 mM NaCl, pH 7.2), flash-frozen in liquid nitrogen, and placed at -80 °C for storage. Samples were run on a NuPAGE 4-12% Bis-Tris protein gel to confirm purity. See Example 2 1.Shankaraiah,G.;Kumar,T.V.;Reddy,G.V.;Rao,J.M.;Babu,K.S.Stereoselective Synthesis of(-)-Pinidinone.Helvetica Chimica Acta 2013,96,990-996. 2.Gao,X.;Han,H.;Krische,M.J.Direct Generation of Acyclic Polypropionate Stereopolyads via Double Diastereo- and Enantioselective Iridium-Catalyzed Crotylation of 1,3-Diols:Beyond Stepwise Carbonyl Addition in Polyketide Construction.Journal of the American Chemical Society 2011,133,12795-12800. 3.Phukan,P.;Bauer,M.;Maier,M.E.Facile generation of alkenes and dienes from tosylates.Synthesis-Stuttgart 2003,1324-1328. 4.Kotoku,N.;Kato,T.;Narumi,F.;Ohtani,E.;Kamada,S.;Aoki,S.;Okada,N.;Nakagawa,S.;Kobayashi,M.Synthesis of 15,20-triamide analogue with polar substituent on the phenyl ring of arenastatin A,an extremely potent cytotoxic spongean depsipeptide.Bioorganic&Medicinal Chemistry 2006,14,7446-7457. 5.Krishnamurthy,S.Rapid Reduction of Alkyl Tosylates with Lithium Triethylborohydride-Convenient and Advantageous Procedure for Deoxygenation of Simple and Hindered Alcohols-Comparison of Various Hydride Reagents.Journal of Organometallic Chemistry 1978,156,171-181. 6.Morrill,C.;Grubbs,R.H.Synthesis of functionalized vinyl boronates via ruthenium-catalyzed olefin cross-metathesis and subsequent conversion to vinyl halides.Journal of Organic Chemistry 2003,68,6031-6034. 7.McCubbin,J.A.;Maddess,M.L.;Lautens,M.Total synthesis of cryptophycin analogues via a scaffold approach.Org Lett 2006,8,2993-6. 8.Ghosh,A.K.;Bischoff,A.Asymmetric syntheses of potent antitumor macrolides cryptophycin B and arenastatin A. European Journal of Organic Chemistry 2004,2131-2141. 9.Ghosh,A.K.;Swanson,L.Enantioselective synthesis of(+)-cryptophycin 52(LY355703),a potent antimitotic antitumor agent.J Org Chem 2003,68,9823-6. 10.Buck,SB;Huff,JK;Himes,RH;Georg,GITotal synthesis and anti-tubulin activity of epi-c3 analogues of cryptophycin-24.J Med Chem 2004,47,3697-9. 11.Mast, CA; Eissler, S.; Stoncius, A.; Stammler, HG; Neumann, B.; Sewald, N. Efficient and versatile stereoselective synthesis of cryptophycins. Chemistry 2005,11,4667-77. 12.Beck,ZQ;Aldrich,CC;Magarvey,NA;Georg,GI;Sherman,DHChemoenzymatic synthesis of cryptophycin / arenastatin natural products.Biochemistry 2005,44,13457-13466.

[0246] Example 3 Synthesis of secocryptophycin chain elongation intermediates. To test the substrate scope of CrpTE and generate novel cryptophycin macrolactones, we developed a scalable synthesis of a NAc-activated seco-chain-elongating intermediate suitable for later diversification of the aryl group of unit A. Analogs were synthesized convergently using two key intermediates, including unit AB and unit CD-NAc. The formulation of unit AB utilized chiral auxiliary chemistry, similar to the previously reported Suzuki coupling strategy. 38 A final Horner-Wadsworth-Emmons olefination (HWE) was used to form the key junction between units A and B. Unit CD, containing the NAc recognition element, was generated by peptide coupling of commercially available and easily engineered amino acid derivatives.

[0247] To that end, unit A was synthesized using an Evans asymmetric aldol containing N-crotonyl oxazolidinone 1 and aldehyde 2 (Figure 3), which afforded the desired (2R,3S) adduct in excellent yield with a high dr (>20:1). Subsequent silylation with TBS trifluoromethanesulfonate gave 3, which was then reductively cleaved with a chiral auxiliary to afford alcohol 4. 39 Tosylation produced 5, which upon subsequent reductive deoxygenation with lithium triethylborohydride gave the desired intermediate 6. 40 At this stage, a vinylboronic acid pinacol ester was introduced via Hoveyda-Grubbs cross-metathesis to afford 7 with the desired Suzuki handle for future diversification. 38 Removal of the p -methoxybenzyl (PMB) protecting group with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) followed by Dess-Martin periodinane (DMP) oxidation to 8 provided the unit A aldehyde fragment required for HWE olefination. 41

[0248] The phosphonate partner 12 (Figure 5) was synthesized starting with commercially available D-tyrosine methyl ester hydrochloride 9, which was converted to 10 in four steps as previously described. 42 Subsequent peptide coupling with diethylphosphonoacetic acid (11) afforded the desired phosphonate 12. From here, HWE olefination conditions were explored, with optimal conditions obtained using sodium hydride in THF, which afforded the diversifiable unit AB fragment 10 in 57% yield of the correct isomer (70% overall yield, approximately 5:1 E:Z product ratio).

[0249] The unit CD-NAc was readily synthesized from commercially available leucine acid 15 (Figure 6). Initial benzyl protection of the acid functionality produced 16, which was subsequently coupled with β-amino acids 17 or 18 to generate the desired esters. These esters were readily deprotected via H / Pd hydrogenolysis to give acids 19 or 20. 43These acids were coupled with N-acetylcysteamine to give the desired unit CD fragment 21 or 22.

[0250] With the diversifiable substrate 13 in hand, coupling with 21 or 22 and sequential Suzuki diversification were explored. Due to the labile functional group and racemization, Suzuki diversification was not possible prior to the final peptide coupling. Screening Suzuki conditions for substrate 13 yielded a procedure utilizing Pd(dba) and KPO to generate a series of novel unit AB cryptophycin analogs 14a-m (Figure 5) in good to excellent yields without detectable racemization.

[0251] Elaboration of the final secocryptophycin chain-elongation intermediate was achieved via coupling of units AB and CD (Figure 7). Saponification of methyl esters 14a–m proved susceptible to racemization, and screening of various hydrolysis procedures afforded trimethyltin hydroxide, which provided the corresponding acid in good yield without detectable racemization (Figure 7). 44 Simultaneous Boc deprotection of 21 or 22 with 4 M HCl / dioxane, followed by peptide coupling with 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) at 0 °C, produced the TBS-protected intermediates in good yields with minimal racemization (7:1 to 12:1 dR, Figure 7). The diastereomers were resolved using reverse-phase HPLC before removal of the TBS group. Finally, deprotection of the silyl groups using aqueous HF in acetonitrile afforded the desired secocryptophycin NAc analogs (Figure 7, 23a-m).

[0252] Example 4 Analytical substrate conversion assay for CrpTEs with unit A heterocycles. Armed with a versatile synthesis, we began investigating the flexibility of CrpTE toward newly generated Unit A chain elongation intermediates (Figure 2, 23a-m). The initial set of CrpTE analogs contained a six-membered heterocycle in place of the native benzene ring (Figure 2, 23b-g). Initial analytical-scale reactions revealed significant turnover to product compared to the native benzyl substrate. Here, the native benzyl substrate (23a) showed 68% overall conversion at a 9:1 cyclization:hydrolysis ratio. In contrast, the 2-, 3-, 4-, and pyrazine substrates showed complete turnover of starting material and almost undetectable levels of hydrolysis byproducts (Figure 2, 23b, 23c, 23e, 23g). This is reflected in the % conversions and cyclization to hydrolysis ratios seen in Figure 8 (24b, 24c, 24e, and 24g), all of which were significantly greater (91–96% conversion, >10:1 cyclization:hydrolysis ratio) when compared to the native substrate 24a.

[0253] The set of unnatural unit A analogs was expanded to include five-membered aromatic heterocycles with various alkyl chains. 2-, 3-, and 4-methylpyrazole derivatives (23h-j) were synthesized and tested in the same analytical assay, utilizing the benzyl substrate 23a as a control. The 2-methylpyrazole and 3-methylpyrazole groups showed slightly lower conversions than the previous six-membered heterocycles, at 85% and 84% (Figure 8, 24h and 24i). Despite these substrates being processed less efficiently than the six-membered analogs, they retain higher conversions to the corresponding depsipeptide cryptophycin analogs than the native substrates, catalyzed by wild-type CrpTE. Interestingly, incorporation of the 4-methylpyrazole ring (23j) showed nearly complete conversion to the product, with no measurable starting material or hydrolysis by-products.

[0254] Incorporating a larger alkyl chain, the 4-isopropylpyrazole group (Figure 2, 23i), provided important insight into the potential size limitations of the CrpTE binding pocket. While no hydrolysis products were observed, incomplete consumption of the starting material after reaction completion resulted in a lower conversion (83%) compared to its methyl counterpart, which showed near-quantitative conversion to product. This larger alkyl substituent may indicate steric constraints within the enzyme, leading to the reduced overall conversion seen with this analog. Finally, the dimethylisoxazole substrate 23m was examined. This compound showed a similar conversion (Figure 8, 24m) compared to the native substrate, with a significant amount of starting material remaining.

[0255] Example 5 Isolation and characterization of unit A cryptophycin analogues To obtain isolated yields, complete structural characterization, and biological evaluation of the reaction products, all reactions were performed on a 10 mg semi-preparative scale using the same conditions as the analytical reactions. These results closely corresponded to the percent conversions observed in the analytical reactions. Six-membered heterocycles 24b, c, e, and f were isolated in good yields of 62–66% (Figure 8). Five-membered rings 24h–24j, 24i, and 24m were also performed on a 10 mg scale and isolated in yields varying from 55% to 69% (Figure 8). All novel cryptophycin analogs produced from these chemoenzymatic reactions were characterized by HRMS, 1 H NMR, and 13 The compounds were confirmed by CNMR and then tested for biological activity.

[0256] Example 6 Biological evaluation of novel styrene cryptophycin analogues. Each of the cryptophycin analogs was evaluated using a zone assay for relative comparison of potency across various cell lines (see Table 1), and the IC for each analog was 50 was determined in the HCT-116 human colon cancer cell line. The potency of the initial monomethyl unit C analogs showed significant variation in activity depending on the heterocyclic ring present (Figure 9). For six-membered heterocycles, the IC 50Values ​​also vary widely within the pyridyl set of analogs, with the 2-pyridyl analog having an IC of 102 nM, three orders of magnitude less than the 3-pyridyl-(24c) and 4-pyridyl-(24e)-containing analogs (0.860 nM and 0.51 nM, Figure 9 ). 50 The five-membered ring exhibited IC values ​​over almost six orders of magnitude. 50 The inclusion of an isoxazole ring (24m) significantly reduced the activity, resulting in an IC 50 The value was 1.4 μM. However, the introduction of the 4-methylpyrazole ring (24j) provided a low-picomolar analog, making it one of the most potent cryptophycin analogs observed to date.

[0257] [Table 1]

[0258] [Table 2]

[0259] Example 7 Formulation and biological evaluation of Gem-dimethyl analogues. Utilizing the data disclosed herein to guide the present design of analogs for testing with the CrpTE biocatalyst, geminal dimethyl unit C analogs were synthesized using the chemistry described herein for unit A with the top three analogs: terminal 3-pyridyl-, 4-pyridyl-, or 4-methyl-pyrazole. The ester bond between units C and D is known to be metabolically labile, and the addition of a second methyl group adjacent to this labile position is known to improve the half-life of the drug. 45~46 It was shown that the inclusion of gem-dimethyl (unit C) in the benzyl-containing unit A resulted in an increase in hydrolysis by-products (10:1 vs. 6:1 cyclization to hydrolysis). 37New analogs 20d, f, and k were first tested on an analytical scale to directly compare the ratio of hydrolysis to cyclization, as well as the % conversion to their monomethyl counterparts. All three showed increased hydrolytic activity (Figures 2 and 8) and a higher percentage of unreacted starting material when incubated with CrpTE, consistent with previous findings. Despite lower overall conversion to the macrocycle, the corresponding chain-elongation intermediates were processed with nearly the same efficiency as the natural substrate, further demonstrating the remarkable flexibility of CrpTE toward substrates containing unnatural functional groups in both the PKS- and NRPS-derived portions of the molecule.

[0260] These analogs were also compared in the zone assay and the IC of HCT-116. 50 Incorporation of the gemdimethyl moiety resulted in a decrease in the potency of all three analogs in this cell line, raising the expectation that alternative methods of blocking metabolism at this site would result in analogs with greater potency.

[0261] References 1.Weissman, KJ, Genetic engineering of modular PKSs: from combinatorial biosynthesis to synthetic biology. Nat Prod Rep 2016, 33(2), 203-30. 2.Walsh,CT,Insights into the chemical logic and enzymatic machinery of NRPS assembly lines.Nat Prod Rep 2016,33(2),127-35. 3. Driggers, EM; Hale, SP; Lee, J.; Terrett, NK, The exploration of macrocycles for drug discovery-an underexploited structural class. Nature Reviews Drug Discovery 2008, 7(7), 608-624. 4.Horsman,M.E.;Hari,T.P.;Boddy,C.N.,Polyketide synthase and non-ribosomal peptide synthetase thioesterase selectivity:logic gate or a victim of fate?Nat Prod Rep 2016,33(2),183-202. 5.Kohli,R.M.;Walsh,C.T.,Enzymology of acyl chain macrocyclization in natural product biosynthesis.Chem Commun(Camb)2003,(3),297-307. 6.Clouthier,C.M.;Pelletier,J.N.,Expanding the organic toolbox:a guide to integrating biocatalysis in synthesis.Chem Soc Rev 2012,41(4),1585-1605. 7.Weissman,K.J.;Leadlay,P.F.,Combinatorial biosynthesis of reduced polyketides.Nat Rev Microbiol 2005,3(12),925-36. 8.Mortison,J.D.;Sherman,D.H.,Frontiers and opportunities in chemoenzymatic synthesis.J Org Chem 2010,75(21),7041-51. 9.Kopp,F.;Grunewald,J.;Mahlert,C.;Marahiel,M.A.,Chemoenzymatic design of acidic lipopeptide hybrids: New insights into the structure-activity relationship of daptomycin and A54145.Biochemistry-Us 2006,45(35),10474-10481. 10.Yeh,E.;Lin,H.N.;Clugston,S.L.;Kohli,R.M.;Walsh,C.T.,Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent.Chem Biol 2004,11(11),1573-1582. 11.Trauger,J.W.;Kohli,R.M.;Mootz,H.D.;Marahiel,M.A.;Walsh,C.T.,Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.Nature 2000,407(6801),215-218. 12.Trauger,J.W.;Kohli,R.M.;Walsh,C.T.,Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase.Biochemistry-Us 2001,40(24),7092-7098. 13.Bruner,S.D.;Weber,T.;Kohli,R.M.;Schwarzer,D.;Marahiel,M.A.;Walsh,C.T.;Stubbs,M.T.,Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain SrfTE.Structure 2002,10(3),301-310. 14.Samel,S.A.;Wagner,B.;Marahiel,M.A.;Essen,L.O.,The thioesterase domain of the fengycin biosynthesis cluster:A structural base for the macrocyclization of a non-ribosomal lipopeptide.J Mol Biol 2006,359(4),876-889. 15.Akey,D.L.;Kittendorf,J.D.;Giraldes,J.W.;Fecik,R.A.;Sherman,D.H.;Smith,J.L.,Structural basis for macrolactonization by the pikromycin thioesterase.Nature Chemical Biology 2006,2(10),537-542. 16.Tsai,S.C.;Miercke,L.J.W.;Krucinski,J.;Gokhale,R.;Chen,J.C.H.;Foster,P.G.;Cane,D.E.;Khosla,C.;Stroud,R.M.,Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel.P Natl Acad Sci USA 2001,98(26),14808-14813. 17.Giraldes,J.W.;Akey,D.L.;Kittendorf,J.D.;Sherman,D.H.;Smith,J.L.;Fecik,R.A.,Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels.Nature Chemical Biology 2006,2(10),531-536. 18.Pinto,A.;Wang,M.;Horsman,M.;Boddy,C.N.,6-Deoxyerythronolide B Synthase Thioesterase-Catalyzed Macrocyclization Is Highly Stereoselective.Org Lett 2012,14(9),2278-2281. 19.Hansen,D.A.;Koch,A.A.;Sherman,D.H.,Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.J Am Chem Soc 2017,139(38),13450-13455. 20.Koch,A.A.;Hansen,D.A.;Shende,V.V.;Furan,L.R.;Houk,K.N.;Jimenez-Oses,G.;Sherman,D.H.,A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst.J Am Chem Soc 2017,139(38),13456-13465. 21.Magarvey,N.A.;Beck,Z.Q.;Golakoti,T.;Ding,Y.;Huber,U.;Hemscheidt,T.K.;Abelson,D.;Moore,R.E.;Sherman,D.H.,Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins.Potent anticancer agents from cyanobionts.ACS chemical biology 2006,1(12),766-79. 22.Ding,Y.;Rath,C.M.;Bolduc,K.L.;Hakansson,K.;Sherman,D.H.,Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module.J Am Chem Soc 2011,133(37),14492-5. 23.Schwartz,R.E.;Hirsch,C.F.;Sesin,D.F.;Flor,J.E.;Chartrain,M.;Fromtling,R.E.;Harris,G.H.;Salvatore,M. J.;Liesch,J.M.;Yudin,K.,Pharmaceuticals from Cultured Algae.J Ind Microbiol 1990,5(2-3),113-123. 24.Smith,C.D.;Zhang,X.Q.;Mooberry,S.L.;Patterson,G.M.L.;Moore,R.E.,Cryptophycin-a New Antimicrotubule Agent Active against Drug-Resistant Cells.Cancer Res 1994,54(14),3779-3784. 25.D’Agostino,G.;del Campo,J.;Mellado,B.;Izquierdo,M.A.;Minarik,T.;Cirri,L.;Marini,L.;Perez-Gracia,J.L.;Scambia,G.,A multicenter phase II study of the cryptophycin analog LY355703 in patients with platinum-resistant ovarian cancer.International journal of gynecological cancer:official journal of the International Gynecological Cancer Society 2006,16(1),71-6. 26.Stevenson,J.P.;Sun,W.;Gallagher,M.;Johnson,R.;Vaughn,D.;Schuchter,L.;Algazy,K.;Hahn,S.;Enas,N.;Ellis,D.;Thornton,D.;O’Dwyer,P.J.,Phase I trial of the cryptophycin analogue LY355703 administered as an intravenous infusion on a day 1 and 8 schedule every 21 days.Clinical cancer research:an official journal of the American Association for Cancer Research 2002,8(8),2524-9. 27.Sessa,C.;Weigang-Kohler,K.;Pagani,O.;Greim,G.;Mora,O.;De Pas,T.;Burgess,M.;Weimer,I.;Johnson,R.,Phase I and pharmacological studies of the cryptophycin analogue LY355703 administered on a single intermittent or weekly schedule.European journal of cancer 2002,38(18),2388-2396. 28.Edelman,M.J.;Gandara,D.R.;Hausner,P.;Israel,V.;Thornton,D.;DeSanto,J.;Doyle,L.A.,Phase 2 study of cryptophycin 52(LY355703) in patients previously treated with platinum based chemotherapy for advanced non-small cell lung cancer.Lung cancer 2003,39(2),197-9. 29.Eggen,M.;Georg,G.I.,The cryptophycins:their synthesis and anticancer activity.Medicinal research reviews 2002,22(2),85-101. 30.Nahrwold,M.;Weiss,C.;Bogner,T.;Mertink,F.;Conradi,J.;Sammet,B.;Palmisano,R.;Royo Gracia,S.;Preusse,T.;Sewald,N.,Conjugates of modified cryptophycins and RGD-peptides enter target cells by endocytosis.Journal of medicinal chemistry 2013,56(5),1853-64. 31.Leamon,C.P.;Wang,Y.;Vlahov,I.R.;You,F.;Kleindl,P.J.;Santhapuram,H.K.R.Conjugates containing hydrophilic spacer linkers.WO2009 / 002993A1、2008 / 12 / 31 / ,2008. 32.Su,D.;Kozak,K.R.;Sadowsky,J.;Yu,S.F.;Fourie-O’Donohue,A.;Nelson,C.;Vandlen,R.;Ohri,R.;Liu,L.N.;Ng,C.;He,J.T.;Davis,H.;Lau,J.;Del Rosario,G.;Cosino,E.;dela Cruz-Chuh,J.;Ma,Y.;Zhang,D.L.;Darwish,M.;Cai,W.W.;Chen,C.J.;Zhou,H.X.;Lu,J.W.;Liu,Y.C.;Kaur,S.;Xu,K.Y.;Pillow,T.H.,Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification.Bioconjugate Chem 2018,29(4),1155-1167. 33.Verma,V.A.;Pillow,T.H.;DePalatis,L.;Li,G.;Phillips,G.L.;Polson,A.G.;Raab,H.E.;Spencer,S.;Zheng,B.,The cryptophycins as potent payloads for antibody drug conjugates.Bioorg Med Chem Lett 2015,25(4),864-868. 34.Bigot,A.;Bouchard,H.;Brun,M.-P.;Clerc,F.;Zhang,J.Novel Cryptophycin Compounds and Conjugates,Their Preparation and Their Therapeutic Use.WO2017 / 076998(A1)、2017 / 05 / 11 / ,2017. 35.Steinkuhler,M.C.;Gallinari,M.P.;Osswald,B.;Sewald,N.;Ritzefeld,M.;Frese,M.Cryptophycin-Based Antibody-Drug Conjugates with Novel Self-Immolative Linkers.WO2016 / 146638(A1)、2016 / 09 / 22 / ,2016. 36.Bouchard,H.;Brun,M.-P.;Commercon,A.;Zhang,J.Novel Conjugates,Preparation Thereof,and Therapeutic Use Thereof.WO2011 / 001052(A1)、2011 / 01 / 06 / ,2011. 37.Beck,Z.Q.;Aldrich,C.C.;Magarvey,N.A.;Georg,G.I.;Sherman,D.H.,Chemoenzymatic synthesis of cryptophycin / arenastatin natural products.Biochemistry-Us 2005,44(41),13457-13466. 38.Bolduc,K.L.;Larsen,S.D.;Sherman,D.H.,Efficient,divergent synthesis of cryptophycin unit A analogues.Chem Commun(Camb)2012. 39.Kotoku,N.;Kato,T.;Narumi,F.;Ohtani,E.;Kamada,S.;Aoki,S.;Okada,N.;Nakagawa,S.;Kobayashi,M.,Synthesis of 15,20-triamide analogue with polar substituent on the phenyl ring of arenastatin A,an extremely potent cytotoxic spongean depsipeptide.Bioorgan Med Chem 2006,14(22),7446-7457. 40.Krishnamurthy,S.,Rapid Reduction of Alkyl Tosylates with Lithium Triethylborohydride-Convenient and Advantageous Procedure for Deoxygenation of Simple and Hindered Alcohols-Comparison of Various Hydride Reagents.J Organomet Chem 1978,156(1),171-181. 41.Ghosh,A.K.;Swanson,L.,Enantioselective synthesis of(+)-cryptophycin 52(LY355703),a potent antimitotic antitumor agent.J Org Chem 2003,68(25),9823-6. 42.McCubbin,J.A.;Maddess,M.L.;Lautens,M.,Total synthesis of cryptophycin analogues via a scaffold approach.Org Lett 2006,8(14),2993-6. 43.Mast,C.A.;Eissler,S.;Stoncius,A.;Stammler,H.G.;Neumann,B.;Sewald,N.,Efficient and versatile stereoselective synthesis of cryptophycins.Chemistry 2005,11(16),4667-77. 44.Nicolaou,K.C.;Estrada,A.A.;Zak,M.;Lee,S.H.;Safina,B.S.,A mild and selective method for the hydrolysis of esters with trimethyltin hydroxide.Angew Chem Int Edit 2005,44(9),1378-1382. 45.Wagner,M.M.;Paul,D.C.;Shih,C.;Jordan,M.A.;Wilson,L.;Williams,D.C.,In vitro pharmacology of cryptophycin 52(LY355703)in human tumor cell lines.Cancer chemotherapy and pharmacology 1999,43(2),115-125. 46.Golakoti,T.;Ogino,J.;Heltzel,C.E.;LeHusebo,T.;Jensen,C.M.;Larsen,L.K.;Patterson,G.M.L.;Moore,R.E.;Mooberry,S.L.;Corbett,T.H.;Valeriote,F.A.,Structure determination,conformational analysis,chemical stability studies,and antitumor evaluation of the cryptophycins.Isolation of 18 new analogs from Nostoc sp strain GSV 224(vol 117,pg 12030,1995).J Am Chem Soc 1996,118(13),3323-3323.

[0262] It will be understood from the disclosure herein that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications can be made without departing from the spirit and scope of the disclosure.

Claims

1. A compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 During the ceremony, Ar is a 5- or 6-membered heterocyclic aryl group having 1 to 3 ring N atoms, and optionally 1 to 3 C １－５ is substituted with alkyl, R ３ and R ４ each independently represents H, or C １－６ is alkyl, R ５ But C １－６ A compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof, wherein:

2. 2. The compound or salt of claim 1, wherein Ar comprises pyridyl, pyrazinyl, imidazolyl, or oxazolyl, optionally substituted with 1 to 3 substituents selected from methyl and isopropyl.

3. R ３ is C １－６ 3. The compound or salt of claim 1, wherein the aryl group is alkyl.

4. R ４ The compound or salt of any one of claims 1 to 3, wherein is H.

5. R ４ is C １－６ 3. The compound or salt of claim 1, wherein the aryl group is alkyl.

6. (1) Ar is 【Chemistry 2】 and R ３ But CH ３ and R ４ is H, (2) Ar is 【Transformation 3】 and R ３ But CH ３ and R ４ is H or CH ３ and (3) Ar is 【Chemistry 4】 and R ３ But CH ３ and R ４ is H or CH ３ and (4) Ar is 【Transformation 5】 and R ３ But CH ３ and R ４ is H, (5) Ar is 【Transformation 6】 and R ３ But CH ３ and R ４ is H, (6) Ar is 【Transformation 7】 and R ３ But CH ３ and R ４ is H, (7) Ar is 【Transformation 8】 and R ３ But CH ３ and R ４ is H or CH ３ and (8) Ar is 【Chemistry 9】 and R ３ But CH ３ and R ４ is H or CH ３ and (9) Ar is 【Chemistry 10】 and R ３ But CH ３ and R ４ The compound or salt of claim 1 , wherein is H.

7. (1) Ar is 【Chemistry 11】 and R ３ But CH ３ and R ４ is H or CH ３ and R ５ But CH (CH ３ ) ２ and (2) Ar is 【Chemistry 12】 and R ３ But CH ３ and R ４ is H or CH ３ and R ５ But CH (CH ３ ) ２ and (3) Ar is 【Chemistry 13】 and R ３ But CH ３ and R ４ is H or CH ３ and R ５ But CH (CH ３ ) ２ 7. The compound or salt of claim 6, wherein:

8. 2. The compound or salt of claim 1, wherein the structure is selected from the group consisting of: 【Chemistry 14】

9. 9. A method of producing the compound or salt of any one of claims 1 to 8, comprising contacting a secocryptophycin intermediate with a cryptophycin thioesterase under conditions suitable for macrocyclization to form the compound or salt.

10. 10. The method of claim 9, wherein the macrocyclization is macrolactonization.

11. 11. The method of claim 10, wherein the cryptophycin thioesterase is derived from a polyketide synthase protein complex, a non-ribosomal protein synthetase protein complex, or a hybrid polyketide synthase / non-ribosomal peptide synthetase protein complex.

12. 12. The method of claim 11, wherein the cryptophycin thioesterase is derived from a hybrid polyketide synthase / non-ribosomal peptide synthetase protein complex.

13. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

14. A conjugate comprising a compound or salt according to any one of claims 1 to 8 and a peptide, protein or antibody.

15. 15. The conjugate of claim 14, wherein the antibody and the compound or salt are covalently bonded via the reactive chemical group of the compound or salt and a complementary reactive group on the antibody.

16. 16. The conjugate of claim 15, wherein the complementary reactive group on the antibody comprises an amine.

17. The conjugate of claim 16, wherein the amine is the ε-amine of a lysine on the antibody.

18. The conjugate according to any one of claims 14 to 17, wherein the antibody is a monoclonal antibody or a nanobody.

19. 19. The conjugate of claim 18, wherein the nanobody is a single domain antibody or a camelid antibody.

20. 19. The conjugate of claim 18, wherein the monoclonal antibody is brentuximab, cetuximab, gemtuzumab, panitumumab, ofatumumab, rituximab, or trastuzumab.

21. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 8, or a conjugate according to any one of claims 14 to 20, for the treatment of cancer.

22. 22. The pharmaceutical composition of claim 21, wherein the cancer is colon, breast, leukemia, prostate, ovarian, central nervous system, or non-small cell lung cancer.

Images