WO2017136769A1

WO2017136769A1 - Peptide drug conjugates

Info

Publication number: WO2017136769A1
Application number: PCT/US2017/016575
Authority: WO
Inventors: Sharon MCGONIGLE; Utpal MAJUMDER; Daniel W. CUSTAR; Jiayi Wu; Maarten H.D. POSTEMA
Original assignee: Eisai R&D Management Co., Ltd.
Priority date: 2016-02-04
Filing date: 2017-02-03
Publication date: 2017-08-10

Abstract

Embodiments provide protein-drug conjugates for treatment of cancer. Protein-drug conjugates may include protein-drug conjugates of Formula I: Ctx-L-Cp (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ctx is chlorotoxin or a chlorotoxin analog; Cp is a cryptophycin amide, and L is a linker wherein the linker is bound to Ctx at an X₂ moiety of the linker and at one of a lysine residue and the N-terminus of the chlorotoxin or chlorotoxin analog.

Description

PEPTIDE DRUG CONJUGATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 62/291,270, filed on February 4, 2016. That application is incorporated by reference herein.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] None.

BACKGROUND

[0003] Anti-Cancer Therapeutics

[0004] The need for effective anti-cancer therapeutics is well known. Unfortunately, effective therapeutics require a combination of both potency and selectivity. Although many therapeutic compounds and combinations of compounds have been proposed, none exhibit an ideal combination of potency and selectivity.

[0005] Cryptophycin

[0006] Cryptophycins are antitubulin antitumor agents that were first isolated from the cyanobacterium Nostoc as Cryptophycin- 1. Boinpally, et al. "Pharmacokinetics and Tissue Distribution of Cryptophycin 52 (C-52) Epoxide and Cryptophycin 55 (C-55) Chlorohydrin in Mice with Subcutaneous Tumors," Cancer Chemother. Pharmacol. (2003) 52: 25-33. The structure of Cryptophycin- 1 is below:

[0007] Cryptophycin- 1, a desipeptide, was found to have anticancer activity against murine solid tumor models and human tumor xenografts. Based on these findings, a number of analogs of cryptophycin were synthesized and tested. For example, Cryptophycin-52, an epoxide of Cryptophycin- 1, was clinically evaluated for use as an antitumor agent. See Boinpally at 32. The structure of Cryptophycin-52 is shown below:

[0008] Phase II clinical trials of Cryptophycin-52 were reportedly discontinued due to toxic side effects. See "Bioactive Compounds from Natural Sources, Natural Products as Lead Compounds in Drug Discovery," Second ed., Chapter 2, written by J. Orjala, and edited by C. Tringali, CRC Press, 2012. Hence there is a need for improved chemotherapeutic agents with an improved therapeutic window.

BRIEF SUMMARY

[0009] Embodiments relate generally to compounds that have anti-cancer activity. Particular embodiments relate to compounds that are conjugates of chlorotoxin and cryptophycin derivatives as described herein. Embodiments further relate to methods of use of these conjugates for treatment of cancer in a mammal.

[0010] One embodiment provides a protein-drug conjugate of Formula I: Ctx-L-Cp (I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ctx is chlorotoxin or a chlorotoxin analog;

Cp is a cryptophycin amide having the following formula:

and L is a linker having the following formula:

[0011] wherein the linker is bound to Ctx at the X₂ moiety of the linker and at one of a lysine group and the N-terminus of the chlorotoxin or chlorotoxin analog;

[0012] q is an integer from 0-8 and m is an integer from 0-8;

[0013] Xi is selected from the group consisting of -NH-, -N(CH₃) -, -CH₂ -, -(OR₄NX₃) and -(0-CH₂CH₂-)_p, wherein when XI is -(0-CH₂CH₂-)_p, m is 1 and p is an integer from 1- 8, and wherein R₄ is C[-C₆ alkyl and X₃ is selected from the group consisting of Ci-C₆ alkyl and aryl;

[0014] X₂ is selected from the group consisting of a bond, C]-C₆ alkyl, and -(OCH₂CH₂-)_r, wherein r is an integer from 1-8;

[0015] Ri and R₂ are selected from the group consisting of -H, lower alkyl, lower alkyl and -CH₂CH₂-(OCH₂CH₂)_n-0-R5, wherein n is an integer from 1-6 and R₅ is lower alkyl, or Ri and R₂ together may form a ring selected from the group consisting of a 3 to 6-membered alkyl ring with optional N-Methyl substituent, morpholinyl, and furanyl, wherein Ri and R₂ may not simultaneously be -H; and

[0016] R₃ is at least one substituent independently selected from the group consisting of -H, lower alkyl, aryl, heteroaryl, hydroxy-(lower alkyl), amino-(lower alkyl), N-(lower alkyl)amino-(lower alkyl), N,N-di(lower alkyl)amino-(lower alkyl), N-arylamino-(lower alkyl), N,N-diarylamino-(lower alkyl), N-(heteroaryl)amino-(lower alkyl), N,N- di(heteroaryl)amino-(lower alkyl), hydroxylamino, 0-(lower alkoxy)amino, O-aryloxyamino, O-heteroaryloxyamino, fluoro, chloro, bromo, nitro, hydroxyl, lower alkoxy, aryloxy, hydroxycarbonyl, lower alkanoyl, lower alkanoyloxy, amino, N-(lower alkyl)amino, N,N- di(lower alkyl)amino, formylamino, N-acylamino, Ν,Ν-diacylamino, hydrazido, N-(lower alkyl)hydrazido, N,N-di(lower alkyl)hydrazido, N-arylhydrazido, Ν,Ν-diarylhydrazido, N- (heteroaryl)hydrozido, N,N-di(heteroaryl)hydrazido, carbamoyl, N-(lower alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl, Ν,Ν-diarylcarbamoyl, N- heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl, hydroxysulfonyl, (lower alkoxy)sulfonyl, aryoxysulfonyl, heteroaryloxysulfonyl, hydroxysulfonyl-(lower alkyl), (lower

alkoxy)sulfonyl-(lower alkyl), aryoxysulfonyl-(lower alkyl), heteroaryloxysulfonyl-(lower alkyl), (lower alkyl)sulfonyl, arenesulfonyl, and heteroarenesulfonyl;

[0017] wherein R₅ is selected from the group consisting of -H and -CH₃; and

[0018] wherein the disulfide bond is ortho, meta, or para to the substituent at a.

[0019] In a further embodiment Ctx consists of chlorotoxin having the amino acid sequence of SEQ ID NO: 1. In a further embodiment Ctx consists of a chlorotoxin analog having the amino acid sequence of SEQ ID NO: 2.

[0020] In a still further embodiment Rj is selected from the group consisting of -H and - CH₃, wherein R₂ is selected from the group consisting of -H and -CH₃, and wherein Ri and R₂ may not both be -H. In a still further embodiment Ri and R₂ are each -CH .

[0021] In a further embodiment Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1 ; q is 1; m is 1 ; XI is -CH₂-; X₂ is a bond; wherein one of Ri and R₂ is -CH₃ and the other is -H; Ri is -CH₃; R₂ is -H; R₃ is -H; the linker is bound to chlorotoxin at an L₂₇ lysine residue and the disulfide bond of the linker is ortho to the substituent at position a.

[0022] In a further embodiment, Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1 ; q is 1 ; m is 1 ; Xi is -CH₂-; X₂ is a bond; Ri is -CH₃; R₂ is -CH₃; R₃ is H; the linker is bound to chlorotoxin at an L₂₇ lysine residue; and the disulfide bond of the linker is ortho to the substituent at position a.

[0023] Further embodiments provide methods for treating cancer in a subject, including administering to the subject the protein-drug conjugate as shown herein in an amount sufficient to treat the subject for cancer. The subject may be, for example, a human. The cancer may be, for example, pancreatic cancer, prostate cancer, breast cancer, glioblastoma, or colon cancer.

[0024] Methods, pharmaceutical products, and uses for compositions reported herein are also provided. For example, an embodiment may provide a method of treating cancer comprising administering to a subject any of the previously described compounds. The previously described compounds may be used for treating cancers including but not limited to pancreatic cancer, colon cancer, breast cancer, prostate cancer, and glioblastoma. Pharmaceutical compositions including those compounds are also provided, as are pharmaceutically acceptable salts of those compounds.

DESCRIPTION OF THE FIGURES

[0025] FIG. 1 shows one embodiment, which is referred to herein as Compound 1 and which includes chlorotoxin having SEQ ID NO: 1, a linker, and cryptophycin amide.

[0026] FIG. 2 shows one embodiment, which is referred to herein as Compound 2, and which includes chlorotoxin having SEQ ID NO: 1, a linker, and a cryptophycin amide.

[0027] FIG. 3 shows one embodiment, which is referred to herein as Compound 4, and which includes chlorotoxin analog having SEQ ID NO: 2, a linker, and a cryptophycin amide.

[0028] FIG. 4 shows Antitumor activity of Compounds 2 and 4 in a subcutaneous human prostate cancer PC-3 xenograft model.

[0029] FIG. 5 shows antitumor activity of Compounds 1, 2 and 4 in a subcutaneous human pancreatic cancer MIA-PaCa2 xenograft model.

[0030] FIG. 6 shows antitumor activity of Compound 2 in a subcutaneous human breast cancer MDA-MB-231 xenograft model.

[0031] FIG. 7 shows antitumor activity of Compound 2 in a subcutaneous primary human pancreatic cancer (PDx) xenograft model. [0032] FIG. 8 shows antitumor activity of Compound 2 in a subcutaneous human

glioblastoma U-87 MG xenograft model.

[0033] FIG. 9 shows the reaction scheme of Example 27.

[0034] FIG. 10 shows the reaction scheme of Example 30.

[0035] FIG. 11 shows the reaction scheme of Example 32.

[0036] FIG. 12 shows a proton NMR of Compound 2. Results for this and other NMR data presented in the figure was obtained using an Avance™ 600Mhz NMR (Bruker,

Switzerland), with a 5 mm-dual-probe, in a solvent of H20/D20=5/95 (3.4mg/ml), at 309K. H20/D20 used 1H ID (P3919GP). For gHSQCAD, average value for 1JCH was 145 Hz; size of FID was 2048 x 512; NMR solvent was Methanol-d4; Probe Temperature was 25 °C; and Chemical shift reference (Methanol-d4) was 3.31 (1H) ; 49.15 (13C).

[0037] FIG. 13 shows a two-dimensional proton ¹³C HSQC two-dimensional NMR for Compound 2.

[0038] FIG. 14 shows a ^I3C NMR for Compound 2.

[0039] FIG. 15 shows ntitumor activity of Compounds 1, 2, 3 and 4 in a subcutaneous human colon cancer COLO 320DM xenograft model.

DETAILED DESCRIPTION

Definitions and Abbreviations

[0040] Unless otherwise stated, terms used in this disclosure have the same meaning as that which they are commonly understood to have by those of ordinary skill in the art. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The embodiments described herein having now been described by way of written description, those of skill in the art will recognize that the embodiments described herein may be practiced in a variety of embodiments and that the description and examples provided herein are for purposes of illustration and not limitation of the claims.

[0041] As used herein, the singular terms "a," "an," and "the" include "at least one" and "one or more" unless stated otherwise. For example, reference to "a pharmaceutically acceptable earner" includes mixtures of two or more carriers as well as a single carrier. [0042] As used herein, the term "effective dosage" or "effective dose" is an amount sufficient to achieve a desired effect. The term "desired effect" refers generally to any result that is anticipated by the skilled artisan, with the benefit of this disclosure, when a compound or composition as taught herein is administered to a subject. In some instances the desired effect may be a complete remission of a cancer. In other instances the desired effect may be partial remission. In still other embodiments the desired effect may be shrinkage of a solid tumor. In still further embodiments the desired effect may be elimination of a solid tumor.

[0043] As used herein, the term "therapeutically effective dose" is an amount sufficient to cure or at least partially arrest a disease and its complications in a patient already suffering from the disease.

[0044] As used herein, the terms "subject" or "patient" refer to any animal, such as mammals, including but not limited to humans, sheep, cows, primates, goats, pigs, horses, cats, dogs, rats, mice, rabbits, guinea pigs, lemurs, or other species. In some embodiments the subject or patient is a human.

[0045] As used herein, the term "chlorotoxin" encompasses chlorotoxin that is isolated from venom of the scorpion Leiurius quinquestriatus or from other organisms in which chlorotoxin may be found. For example, "chlorotoxin" may be a peptide of 36 amino acids in length, having an amino acid sequence as set forth in SEQ ID NO: 1. The term "chlorotoxin" further encompasses recombinant and synthetic chlorotoxin. Chlorotoxin is reported, for example, in PCT International Application Publication No. WO2011/097533, which is incorporated by reference herein.

[0046] As used herein, the term "lower alkyl" refers to straight or branched C[-C₆ alkyl. As used herein, "alkyl", "d, C₂, C₃, C₄, C₅ or C₆ alkyl" or "Ci-C₆ alkyl" is intended to include Ci, C₂, C₃, C₄₎ C₅ or C₆ straight chain (linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbon groups. For example, Ci-C alkyl is intended to include Q, C₂, C₃, C₄, C₅ and C₆ alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n- propyl, /-propyl, n-butyl, sec-butyl, i-butyl, rc-pentyl, seopentyl or n-hexyl.

[0047] In certain embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., Ci-C₆ for straight chain, C₃-C₆ for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. [0048] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated nonaromatic hydrocarbon ring having 3 to 7 carbon atoms (e.g., C₃-C₇). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0049] The term "heterocycloalkyl" refers to a saturated or unsaturated nonaromatic 3-8 membered monocyclic, 7-10 membered fused bicyclic having one or more heteroatoms (such as O, N, or S), unless specified otherwise. Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, tetrahydrothiophenyl, dihydropyranyl, pyranyl, morpholinyl, 1,4- diazepanyl, 1,4-oxazepanyl, and the like.

[0050] Additional examples of heterocycloalkyl groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl,

benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydiOfuro[2,3-b]tetrahydrofuranyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,

octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, l,2,4-oxadiazol-5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,

pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyixolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4- triazolyl and xanthenyl. [0051] The term "optionally substituted alkyl" refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,

aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,

aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl, phosphinyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino,

arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,

trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0052] An "arylalkyl" or an "aralkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). An "alkylaryl" moiety is an aryl substituted with an alkyl (e.g., methylphenyl).

[0053] "Alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term "alkenyl" includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl), and branched alkenyl groups. In certain embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C3-C6 for branched chain). The term "C₂-C₆" includes alkenyl groups containing two to six carbon atoms. The term "C₃-C₆" includes alkenyl groups containing three to six carbon atoms.

[0054] The term "optionally substituted alkenyl" refers to unsubstituted alkenyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,

trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0055] "Alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, "alkynyl" includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl), and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). The term "C₂-C₆" includes alkynyl groups containing two to six carbon atoms. The term "C₃-C₆" includes alkynyl groups containing three to six carbon atoms.

[0056] The term "optionally substituted alkynyl" refers to unsubstituted alkynyl or alkynyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen,- hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,

aminocarbonyl, alkylamin^'ocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl, phosphinyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino,

[0057] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both the unsubstituted moieties and the moieties having one or more of the designated substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl- piperidinyl and 2,2,6,6-tetramethyl-l,2,3,6-tetrahydiOpyridinyl.

[0058] "Aryl" includes groups with aromaticity, including "conjugated," or multicyclic systems with at least one aromatic ring and do not contain any heteroatom in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, etc.

[0059] "Heteroaryl" groups are aryl groups, as defined above, except having from one to four heteroatoms in the ring structure, and may also be referred to as "aryl heterocycles" or "heteroaromatics." As used herein, the term "heteroaryl" is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR' wherein R' is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→0 and S(0)_p, where p = 1 or 2). It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0060] Furthermore, the terms "aryl" and "heteroaryl" include multicyclic aryl and heteroaryl groups, e.g., bicyclic. Non-limiting example of such aryl groups include, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,

methylenedioxyphenyl, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0061] In the case of multicyclic aromatic rings, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline).

[0062] The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can be substituted at one or more ring positions (e.g., the ring-forming carbon or heteroatom such as N) with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,

alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonyl, phosphinyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can also be fused with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl). [0063] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring (as shown by the examples below with substituent R), then such substituent may be bonded to

any atom in the r

[0064] When any variable (e.g., R.sub.l) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 Ri moieties, then the group may optionally be substituted with up to two Ri moieties and Rj at each occurrence is selected independently from the definition of Ri.

[0065] The term "hydroxy" or "hydroxyl" includes groups with an -OH or -O^".

[0066] As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo and iodo. The term "perhalogenated" generally refers to a moiety wherein all hydrogen atoms are replaced by halogen atoms. The term "haloalkyl" or "haloalkoxyl" refers to an alkyl or alkoxyl substituted with one or more halogen atoms.

[0067] "Alkoxyalkyl," "alkylaminoalkyl," and "thioalkoxyalkyl" include alkyl groups, as described above, wherein oxygen, nitrogen, or sulfur atoms replace one or more hydrocarbon backbone carbon atoms.

[0068] The term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,

aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,

aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl, phosphinyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino,

arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonyl, sulfamoyl, sulfonamido, nitro,

trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

[0069] "Isomerism" means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

Stereoisomers that are not mirror images of one another are termed "diastereoisomers," and stereoisomers that are non-superimposable mirror images of each other are termed

"enantiomers" or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a "racemic mixture."

[0070] A carbon atom bonded to four nonidentical substituents is termed a "chiral center."

[0071] "Chiral isomer" means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Calm et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al, Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Calm et al, Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

[0072] As used herein, the term "chlorotoxin analog" refers to chlorotoxin in which one or more lysine residues at positions 15 and 23 have been replaced with alanine.

[0073] The term "crystal polymorphs," "polymorphs" or "crystal forms" means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions. It is understood that the compounds of the present disclosure may exist in crystalline form, crystal form mixture, or anhydride or hydrate thereof. [0074] The compounds disclosed herein include the compounds themselves, as well as their salts and solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on an aryl- or heteroaryl-substituted benzene compound. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on an aryl- or heteroaryl-substituted benzene compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. The aryl- or heteroaryl-substituted benzene compounds also include those salts containing quaternary nitrogen atoms.

[0075] Additionally, compounds of the present disclosure, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0076] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds as reported herein, wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

[0077] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In the salt form, it is understood that the ratio of the compound to the cation or anion of the salt can be 1: 1, or any ratio other than 1 : 1, e.g., 3:1, 2: 1, 1:2, or 1:3.

[0078] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

[0079] "Solvate" means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H₂0.

[0080] Chemicals as named or depicted are intended to include all naturally occurring isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of 1 H hydrogen include tritium and deuterium, and isotopes of 12 C carbon include ¹³C and ^I4C.

[0081] It will be understood that some compounds, and isomers, salts, solvates and polymorphs thereof, of the present disclosure may exhibit greater in vivo or in vitro activity than others. It will also be appreciated that some diseases or conditions may be treated more effectively than others using the compounds, and isomers, salts, solvates and polymorphs thereof, of the present disclosure. [0082] As used herein, "treating" means administering to a subject a pharmaceutical composition to ameliorate, reduce or lessen the symptoms of a disease. As used herein, "treating" or "treat" describes the management and care of a subject for the pmpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term "treat" can also include treatment of a cell in vitro or an animal model.

[0083] Treating cancer may result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as "tumor regression". Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0084] Treating cancer may result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Tumor volume may be measured by any reproducible means of measurement.

[0085] Treating cancer may result in a decrease in number of tumors. Preferably, after treatment, tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x. [0086] Treating cancer may result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified

magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0087] Treating cancer may result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0088] Treating cancer may result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0089] Treating cancer may result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharmaceutically acceptable salt thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0090] Treating cancer may result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[0091] Treating cancer may result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0092] Treating cancer may result in a decrease in tumor regrowth, for example, following attempts to remove it surgically. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.

[0093] Treating or preventing a cell proliferative disorder may result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0094] Treating or preventing a cell proliferative disorder may result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

[0095] Treating or preventing a cell proliferative disorder may result in a decrease in size of an area or zone of cellular proliferation. Preferably, after treatment, size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.

[0096] Treating or preventing a cell proliferative disorder may result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiomorphism.

[0097] As used herein, the term "alleviate" is meant to describe a process by which the severity of a sign or symptom of a disorder is decreased. Importantly, a sign or symptom can be alleviated without being eliminated. In a preferred embodiment, the administration of pharmaceutical compositions of the disclosure leads to the elimination of a sign or symptom, however, elimination is not required. Effective dosages are expected to decrease the severity of a sign or symptom. For instance, a sign or symptom of a disorder such as cancer, which can occur in multiple locations, is alleviated if the severity of the cancer is decreased within at least one of multiple locations.

[0098] As used herein, the term "severity" is meant to describe the potential of cancer to transform from a precancerous, or benign, state into a malignant state. Alternatively, or in addition, severity is meant to describe a cancer stage, for example, according to the TNM system (accepted by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other art-recognized methods. Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (spread of cancer into lymph nodes). Alternatively, or in addition, severity is meant to describe the tumor grade by art- recognized methods (see, National Cancer Institute, www.cancer.gov). Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary with each type of cancer. Severity also describes a histologic grade, also called differentiation, which refers to how much the tumor cells resemble normal cells of the same tissue type (see, National Cancer Institute,

www.cancer.gov). Furthermore, severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).

[0099] In another aspect as reported herein, severity describes the degree to which a tumor has secreted growth factors, degraded the extracellular matrix, become vascularized, lost adhesion to juxtaposed tissues, or metastasized. Moreover, severity describes the number of locations to which a primary tumor has metastasized. Finally, severity includes the difficulty of treating tumors of varying types and locations. For example, inoperable tumors, those cancers which have greater access to multiple body systems (hematological and

immunological tumors), and those which are the most resistant to traditional treatments are considered most severe. In these situations, prolonging the life expectancy of the subject and/or reducing pain, decreasing the proportion of cancerous cells or restricting cells to one system, and improving cancer stage/tumor grade/histological grade/nuclear grade are considered alleviating a sign or symptom of the cancer.

[0100] As used herein the term "symptom" is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by non-health-care professionals.

[0101] A "pharmaceutical composition" is a formulation containing a compound of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a

pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0102] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0103] "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient.

[0104] Embodiments reported herein may provide pharmaceutical compositions comprising any compound disclosed herein in combination with at least one pharmaceutically acceptable excipient or earner.

[0105] A pharmaceutical composition as reported herein is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates Di^¬ phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0106] A compound or pharmaceutical composition can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, for treatment of cancers, a compound as reported herein may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches. The dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects. The state of the disease condition (e.g., cancer, precancer, and the like) and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.

[0107] The term "therapeutically effective amount", as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is cancer. In another aspect, the disease or condition to be treated is a cell proliferative disorder.

[0108] For any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio,

LD5₀ ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0109] Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction

sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0110] The pharmaceutical compositions containing active compounds as reported herein may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

[0111] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0112] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0113] Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel® brand cross-linked and carboxymethylated potato starch, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0114] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0115] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0116] The active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0117] It is typically advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms as reported herein are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.

[0118] In therapeutic applications, the dosages of the pharmaceutical compositions used in accordance embodiments reported herein vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the cancer. Dosages can range from about 0.01 mg/kg per day to about 5000 mg kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day. In an aspect, the dose will be in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adjusted for the patient's weight in kg, body surface area in m , and age in years). An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, regression of a tumor in a patient may be measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Regression is also indicated by failure of tumors to reoccur after treatment has stopped. As used herein, the term "dosage effective manner" refers to amount of an active compound to produce the desired biological effect in a subject or cell.

[0119] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. [0120] Techniques for formulation and administration of the disclosed compounds can be found in Remington: the Science and Practice of Pharmacy, 19.sup.th edition, Mack

Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, can be used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.

[0121] Exemplary cancers that may be treated using one or more compounds of the present disclosure include, but are not limited to, pancreatic cancer, colon cancer, prostate cancer, breast cancer, and glioblastoma.

[0122] A cancer that is to be treated can be staged according to the American Joint

Committee on Cancer (AJCC) TNM classification system, where the tumor (T) has been assigned a stage of TX, Tl, Tlmic, Tla, Tib, Tic, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N) have been assigned a stage of NX, NO, Nl, N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant metastasis (M) can be assigned a stage of MX, M0, or Ml. A cancer that is to be treated can be staged according to an American Joint Committee on Cancer (AJCC) classification as Stage I, Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. A cancer that is to be treated can be assigned a grade according to an AJCC classification as Grade GX (e.g., grade cannot be assessed), Grade 1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can be staged according to an AJCC pathologic classification (pN) of pNX, pNO, PN0 (I-), PN0 (I+), PN0 (mol-), PN0 (mol+), PN1, PNl(mi), PNla, PNlb, PNlc, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

[0123] A cancer that is to be treated can include a tumor that has been determined to be less than or equal to about 2 centimeters in diameter. A cancer that is to be treated can include a tumor that has been determined to be from about 2 to about 5 centimeters in diameter. A cancer that is to be treated can include a tumor that has been determined to be greater than or equal to about 3 centimeters in diameter. A cancer that is to be treated can include a tumor that has been determined to be greater than 5 centimeters in diameter. A cancer that is to be treated can be classified by microscopic appearance as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated. A cancer that is to be treated can be classified by microscopic appearance with respect to mitosis count (e.g., amount of cell division) or nuclear pleiomorphism (e.g., change in cells). A cancer that is to be treated can be classified by microscopic appearance as being associated with areas of necrosis (e.g., areas of dying or degenerating cells). A cancer that is to be treated can be classified as having an abnormal karyotype, having an abnormal number of chromosomes, or having one or more chromosomes that are abnormal in appearance. A cancer that is to be treated can be classified as being aneuploid, triploid, tetraploid, or as having an altered ploidy. A cancer that is to be treated can be classified as having a chromosomal translocation, or a deletion or duplication of an entire chromosome, or a region of deletion, duplication or amplification of a portion of a chromosome.

[0124] The compounds, or pharmaceutically acceptable salts thereof are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of certain routes of administration.

[0125] The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

[0126] We have unexpectedly found that protein-drug conjugates including a chlorotoxin or chlorotoxin analog linked to a cryptophycin amide by a disulfide linker provide substantial potency and selectivity that may be useful for the treatment of cancer. In some embodiments the cancer may be, for example, colon cancer, glioblastoma, breast cancer, prostate cancer, or pancreatic cancer. In some embodiments the patient in need of treatment for cancer is a human patient. Without wishing to be bound by theory, it is believed that the chlorotoxin or chlorotoxin analog acts as a targeting moiety that delivers the cytotoxic cryptophycin amide to cancer cells. Delivery to cancer cells or to close proximity to cancer cells is believed to potentially minimize the effect of the cytotoxic on non-cancerous tissue.

[0127] Embodiments of those protein-drag conjugates include protein-drug conjugates of Formula I:

Ctx-L-Cp (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ctx is chlorotoxin or a chlorotoxin analog;

Cp is a cryptophycin amide having the following formula:

and L is a linker having the following formula:

wherein the linker is bound to Ctx at the X₂ moiety of the linker and at one of a lysine residue and the N-terminus of the chlorotoxin or chlorotoxin analog. One embodiment is shown in FIG. 1 and another embodiment is shown in FIG. 2. In some embodiments the aromatic group in the linker is absent, and the disulfide is bound directly to a methylene group at q.

[0128] In embodiments as reported herein, q is an integer from 0-8 and m is an integer from 0-8;

Xi is selected from the group consisting of -ΝΗ-, -N(CH₃) -, -CH₂ -(OR₄NX₃) and -(O- CH₂CH₂-)_P, wherein when Xj is -(0-CH₂CH₂-)p, m is 1 and p is an integer from 1-8, and wherein R₄ is Ci-C₆ alkyl and X₃ is selected from the group consisting of Ci-C₆ alkyl and aryl;

X₂ is selected from the group consisting of a bond, Ci-C₆ alkyl, and -(OCH CH₂-)_1; wherein r is an integer from 1-8;

Ri and R₂ are selected from the group consisting of -H,; lower alkyl and -CH₂CH₂- (OCH₂CH₂)_n-0-R₅, wherein n is an integer from 1-6 and R₅ is lower alkyl; wherein Ri and R₂ may not simultaneously be -H; or Rj and R₂ together may form a ring selected from the group consisting of a 3 to 6-membered alkyl ring with optional N-methyl substituent, morpholinyl, and furanyl; and

R₃ is at least one substituent independently selected from the group consisting of -H, lower alkyl, aryl, heteroaryl, hydroxy-(lower alkyl), amino-(lower alkyl), N-(lower alkyl)amino- (lower alkyl), N,N-di(lower alkyl)amino-(lower alkyl), N-arylamino-(lower alkyl), N,N- diarylamino-(lower alkyl), N-(heteroaryl)amino-(lower alkyl), N,N-di(heteroaryl)amino- (lower alkyl), hydroxylamino, 0-(lower alkoxy)amino, O-aryloxyamino, O- heteroaryloxyamino, fluoro, chloro, bromo, nitro, hydroxyl, lower alkoxy, aryloxy, hydroxycarbonyl, lower alkanoyl, lower alkanoyloxy, amino, N-(lower alkyl)amino, N,N- di(lower alkyl)amino, formylamino, N-acylamino, /V,N-diacylamino, hydrazido, N-(lower alkyl)hydrazido, N,N-di(lower alkyl)hydrazido, N-arylhydrazido, NN-diarylhydrazido, N- (heteroaryl)hydrozido, N,N-di(heteroaryl)hydrazido, carbamoyl, N-(lower alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl, N,7V-diarylcarbamoyl, N-heteroarylcarbamoyl, N/V-di(heteroaryl)carbamoyl, hydroxysulfonyl, (lower alkoxy)sulfonyl, aryoxysulfonyl, heteroaryloxysulfonyl, hydroxysulfonyl-(lower alkyl), (lower alkoxy)sulfonyl-(lower alkyl), aryoxysulfonyl-(lower alkyl), heteroaryloxysulfonyl-(lower alkyl), (lower alkyl)sulfonyl, arenesulfonyl, and heteroarenesulfonyl; wherein the disulfide bond of the linker is ortho, meta, or para to the substituent at position a; and wherein R₅ is -CH₃ or -H.

[0129] In a further embodiment, Ctx consists of chlorotoxin having the amino acid sequence of SEQ ID NO: 1. In another embodiment, Ctx consists of a chlorotoxin analog having the amino acid sequence of SEQ ID NO: 2. Chlorotoxin and chlorotoxin analogs may be bioproducts or may be synthetic. [0130] In a further embodiment, Rj and R₂ are both -CH₃. In a still further embodiment, one of Ri and R₂ is -CH₃, and the other is -H.

[0131] In a further embodiment, Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1; q is 1 ; m is 1; Xi is -CH₂-; X₂ is a bond; Ri is -CH₃; R₂ is -H; R₃ is H; the linker is bound to chlorotoxin at the L₂₇ lysine residue; and the disulfide bond of the linker is ortho to the substituent at position a. This embodiment is shown in FIG. 1.

[0132] In a still further embodiment, Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1; q is 1; m is 1; Xi is -CH₂-; X₂ is a bond; R, is -CH₃; R₂ is -CH₃; R₃ is H; the linker is bound to chlorotoxin at the L₂₇ lysine residue; and the disulfide bond of the linker is ortho to the substituent at position a. This embodiment is shown in FIG. 2.

[0133] Targeting Moieties

[0134] In a typical embodiment, the targeting moiety is chlorotoxin having SEQ ID NO: 1. This may be a synthetic version of a 36 amino acid chlorotoxin peptide (SEQ ID NO: 1) that occurs in the venom of the scorpion Leiurus quinquestriatus. Chlorotoxin having SEQ ID NO: 1 includes three lysine residues at positions 15, 23, and 27. All three of these lysine residues, as well as the nitrogen terminus of chlorotoxin having SEQ ID NO: 1, provide opportunities for conjugation to a linker. In typical embodiments conjugation to Lys₂₃, Lys₂₇, and the N-terminus is more likely than conjugation to Lys₁₅.

[0135] Another useful targeting moiety is a chlorotoxin analog having SEQ ID NO: 2.

Chlorotoxin analog having SEQ ID NO: 2 is a chlorotoxin analog that replaces Lysi₅ and Lys₂₃ with alanine. The replacement of those lysine residues with alanine ensures that all lysine conjugation occurs at the Lys₂ residue, which may be useful. This may particularly be useful during synthesis.

[0136] In various embodiments the targeting moiety has one or more cleaved cysteine linkages. In further embodiments cysteine linkage is prevented by, for example, capping the cysteine moiety with an acetamide or other protecting group. In a still further embodiment cysteine linkages are prevented by replacement of cysteine with serine.

[0137] Linkers

[0138] Protein-drug conjugates as taught and suggested herein may incorporate linkers between the chlorotoxin targeting moiety and the cytotoxic cryptophycin amide. Linkers may serve to simplify the connection of the targeting moiety and the cytotoxic. Cleavable linkers may also allow release of the cytotoxic after it has reached a location of interest in a patient.

[0139] Embodiments as presented herein utilize a disulfide-based cleavable linker. Release of the cryptophycin amide is triggered by cleavage of the disulfide bond upon reduction by glutathione or other reducing agents. Intracellular glutathione concentration is high relative to the concentration in blood. In addition, the concentration of glutathione in and around tumor cells is typically high relative to normal cells and tissue.

[0140] Synthesis

[0141] Although embodiments are not limited by their means of synthesis, a useful general method of synthesis is set forth below.

[0142] Example 1

[0143] Compound 101: 3-(bromotriphenylphosphoranyl)propanoic acid (Compound 100) (20.74 g, 49.9 mmol) was sonicated with THF (120 mL) for 45 min and then 4-(((4- methoxybenzyl)oxy)methyl)-benzaldehyde (8.0 g, 31.2 mmol) in THF (34 mL) was added. See, e.g. , Bhuniya,R., Tetetrahedron: Asymmetry, 2011, 22, 1125-1132.

[0144] The mixture was cooled to -20°C and potassium tert-butoxide in THF (1M, 100 mL, 100 mmol) was added over 2 h at -20→-15°C. The reaction mixture was slowly warmed to room temperature over 3 h, cooled back down to 0°C and then iodomethane (17.1 mL, 273 mmol) was added and the reaction stirred for 2 days at room temperature. Ether (200 mL) was added followed by water (200 mL ) to quench the reaction and the aqueous layer was extracted with ether (2 x 200 mL). The combined ethereal extracts were washed with saturated NaHC0₃ (1 x 100 mL), 5% KHS0₄ solution (1 x 100 mL), dried and concentrated in vacuo to give a crude residue. The crude material was purified on silica gel using 15% to 35% EtOAc-heptane to furnish the desired product Compound 101 (7.2 g, 71%) as a colorless oil; Ή NMR (400 MHz, CDC1₃) δ 7.37 (d, J = 8.4 Hz, 2H), 7.32-7.28 (m, AH), 6.90 (dd, J = 2.0, 6.8 Hz, 2H), 6.50 (d, / = 16.0 Hz, 1H), 6.30 (ddd, / = 7.2, 7.2, 15.6 Hz, 1H), 4.51 (s, 2H), 4.48 (s, 2H), 3.82 (s, 3H), 3.73 (s, 3H), 3.27 (dd, J = 1.4, 7.0 Hz, 2H).

[0145] Example 2

ER-898 (101) ER-898; (102)

[0146] Compound 102: Potassium osmate dihydrate (88 mg, 0.25 mmol), potassium ferrocyanide (24.38 g, 66.2 mmol) and potassium carbonate (9.15 g, 66.2 mmol) were dissolved in water (116 ml). 7¾rf-butanol (90 mL) was added, followed by (DHQD)₂-PHAL (189 mg, 0.243 mmol) and methanesulfonamide (2.09 g, 21.9 mmol). The solution was cooled to 0° C and (E)-methyl 4-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)butan-3-enoate (7.2 g, 22.1 mmol) in teri-butanol (26 mL) was added. After stirring for 48 h at 0°C, solid sodium sulfite (33.4 g, 264.7 mmol) and water (45 mL) were added. The cooling bath was removed and the reaction stirred for additional 2h. The organic layer was separated and aqueous layer was extracted with ether (3 x 150 mL). The combined organic layers were washed with water (1 x 100 mL), brine (1 x 100 mL), dried and concentrated in vacuo. The crude material was purified on silica gel using 20% to 60% EtO Ac/heptane to deliver the desired product Compound 102 (6.02 g, 83%); Ή NMR (400 MHz, CDC1₃) δ 7.46 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 6.91 (dd, J = 2.0, 6.4 Hz, 2H), 5.39 (d, J = 3.2 Hz, 1H), 4.62 (br s, 1H), 4.55 (s, 2H), 4.54 (s, 2H), 3.82 (s, 3H), 2.91 (dd, J = 5.2, 17.6 Hz, lH), 2.76 (d, / = 16.8 Hz, lH), 1.39 (s, 1H).

[0147] Example 3

ER-898: (102) ER-898 (103)

[0148] Compound 103: n-BuLi in hexanes (1.6 M, 7.2 ml, 11.5 mmol) was added to a solution of dusopropylamine (1.65 ml, 11.6 mmol) in THF (30 ml) at -78°. The solution was stirred for 20 min and the cooling bath was removed and stirring continued for 20 min. The reaction was cooled back down to 0°C, stirred for 20 min and then cooled back to -78°C. (4R,5R)-4-Hydroxy-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)dihydrofuran-2(3H)-one (1.54 g, 4.69 mmol) in THF (20 ml) was added to the LDA solution over 80 min. The resulting solution was stirred for additional 45 min at -78°C, cooled to -90°C and then methyl iodide (0.08 ml, 12.8 mmol) in THF (15 ml) was added over 120 min. After the addition was completed, the reaction mixture was stirred for 2 days at -78°C. The reaction was quenched by the addition of AcOH (1.5 mL) in THF (4 mL) and warmed up to 23°C. The solvent was removed under vacuum and the resulting pink residue was partitioned between water (100 mL) and ether (100 mL). The organic layer was seperated and aqueous layer was extracted with ether (2 x 75 mL). The combined organic layers were washed with 5% KHS0₄, brine, dried over Na₂S0₄ and concentrated in vacuo. The material was purified on silica gel using 10% to 35% EtO Ac-heptane to isolate the product Compound 103 (1.4 g, 87%); 1H NMR (400 MHz, CDC1₃) δ 7.45 (d, / = 8.4 Hz, 2H), 7.35 (d, 7 = 8.4 Hz, 2H), 7.30 (d, / = 8.4 Hz, 2H), 6.90 (d, 7 = 8.4 Hz, 2H), 5.62 (d, / = 3.6 Hz, 1H), 4.55 (s, 2H), 4.53 (app s, 2H), 4.28 (dd, 7 = 3.6, 6.4 Hz, 1H), 3.82 (s, 3H), 2.76 (dq, 7 = 2.8, 7.6 Hz, 1H), 1.41 (app d, J = 7.6 Hz, 3H), 1.36 (d, 7 = 3.6 Hz, 1H).

[0149] Example 4

ER-898 (103) ER-898 (104)

[0150] Compound 104: (3i?,4R,5tf)-4-hydroxy-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-3-methyldihydrofuran-2(3H)-one (1.51 g, 4.41 mmol) was dissolved in methanol (5 mL) and 2,2-dimethoxypropane (18 ml). Dry Amberlyst-15 (280 mg) was added and the slurry was stirred for 5 days at room temperature. The formed solid was filtered off and washed with a mixture of heptane and ethyl acetate (5: 1, 250 mL). The organic layer was washed with brine (1 x 100 mL), dried and concentrated in vacuo. The crude material was purified on silica gel using 20% to 60% EtO Ac-heptane to give the desired product Compound 104 (1.41 g, 77%); Ή NMR (400 MHz, CDC1₃) δ 7.36 (s, 4H), 7.28 (d, 7 = 9.2 Hz, 2H), 6.90 (app d, 7 = 2.0, 6.8 Hz, 2H), 4.76 (d, 7 = 8.4 Hz, 1H), 4.52 (s, 2H), 4.47 (s, 2H), 4.11 (dd, 7 = 6.0, 8.4 Hz, 1H), 3.82 (s, 3H), 3.45 (s, 3H), 2.69 (dq, 7 = 7.2, 12.4 Hz, 1H), 1.57 (s, 3H), 1.48 (s, 3H), 1.28 (d, 7 = 6.8 Hz, 3H).

[0151] Example 5

ER-898J (104) ER-89: (105)

[0152] Compound 105: DIBAL-H (1M, 27.7 ml, 27.7 mmol) was added to a solution of (R)-methyl 2-((4R,5/?)-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl- 1 ,3- dioxolan-4-yl)propanoate (4.6 g, 11.1 mmol) in CH₂C1₂ (100 mL) at -70°C over 20 minutes, warmed up to 0°C over 1.5 h and stirred for 1 h at 0°C. The solution was cooled back to - 70°C and methanol (2.7 mL) was slowly added to quench the reaction and then the reaction was permitted to warm up to room temperature. The reaction mixture was diluted with a mixture of AcOH (200 mL), heptane (200 mL) and EtOAc (200 mL). The organic layer was separated and aqueous layer was extracted with heptane/ether (1:1, 2 x 200 mL). The combined organic extracts were washed with AcOH (1M, 50 mL), brine, dried and passed through a pad of celite (rinsing with heptane/ether (1: 1, 100 mL). The filtrate concentrated in vacuo and the crude material was purified on silica gel using 20% to 60% EtOAc-heptane to afford the product Compound 105 (3.72 g, 87%); 1H NMR (400 MHz, CDC1₃) δ 7.37 (s, 4 H), 7.29 (dd, J = 2.2, 6.8 Hz, 2H), 6.90 (dd, / = 2.0, 6.8 Hz, 2H), 4.80 (d, J = 8.4 Hz, 1H), 4.53 (s, 2H), 4.49 (s, 2H), 3.95 (dd, / = 3.2, 8.8 Hz, 1H), 3.82 (s, 3H), 3.61 (dd, J = 4.6, 4.6 Hz, 2H), 1.89-1.84 (m, 2H), 1.57 (s, 3H), 1.50 (s, 3H), 1.08 (d, J = 7.2 Hz, 3H).

[0153] Example 6

[0154] Compound 106: (5)-2-((4R,5R)-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)-2,2- dimethyl-l,3-dioxolan-4-yl)propan-l-ol (3.72 g, 9.63 mmol) was dissolved in CH₂C1 (100 mL) cooled to -15°C, Hiinig's base (10.1 mL, 57.8 mmol) was added, followed by pyridine sulfur trioxide (4.60 g, 28.9 mmol) and then DMSO (7.12 mL, 96.3 mmol) was added dropwise over 30 min. The reaction mixture was stirred for 1.5 h at -10→ -5° C. Saturated NaHC0₃ (50 mL) was added to quench the reaction and the crude mixture was extracted with EtOAc/heptane (1:1; 2 x 250 mL), washed with saturated NaHC0₃ (3 x 60 mL) dried and concentrated in vacuo to obtain the crude aldehyde (3.70 g. 100%) which was used directly in the next step without any further purification. Ή NMR (400 MHz, CDC1₃) δ 9.52 (d, / = 0.8 Hz, 1H), 7.27-7.26 (m, 4H), 7.18 (d, / = 8.8 Hz, 2H), 6.79 (dd, J = 2.0, 6.4 Hz, 2H), 4.67 (d, J = 8.8 Hz, 1H), 4.43 (s, 2H), 4.38 (s, 2H), 4.13 (dd, / = 3.2, 8.8 Hz, 1H), 3.71 (s, 3H), 2.43 (ddq, / = 2.4, 7.2, 14.0 Hz, 1H), 1.48 (s, 3H), 1.38 (s, 3H), 1.13 (d, 7 = 6.8 Hz, 3H). Crude (R)-2-((4R,5R)-5-(4-(((4-methoxybenzyl)oxy) methyl)phenyl)-2,2-dimethyl-l,3-dioxolan-4- yl)propanal (3.7 g, 9.6 mmol) in CH₂C1₂ (30 mL) was added to a suspension of magnesium bromide diethyl etherate (5.96 g, 23.1 mmol) in CH₂C1₂ (30 mL) at -78°C, stiiTed for 40 min. A solution of allyl tri-n-butyltin (7.16 mL, 23.097 mmol) in CH₂C1₂ (45 mL) was added over 20 minutes at -78°C. The resulting solution was stirred for additional 18 h at -78°C, saturated NaHC0₃ (120 mL) was added and the mixture warmed to room temperature. A mixture of ether (500 mL) and water (100 mL) was added and the mixture was stitrred for 20 minutes. The organic layer was separated and aqueous layer was extracted with ether (2 x 150 mL). The combined ethereal layers were washed with brine, dried and concentrated in vacuo. The crude residue was purified on silica gel using 20% to 60% EtO Ac-heptane to afford homoallylic alcohol Compound 106 (3.72 g, 91 %) as a colorless oil; [<X]D = - 15.0^° (c = 1, CH₂C1₂); FT-rR (CH₂C1₂ cast) 3504 (br), 2933, 1613, 1514, 1370, 1247, 1037 cm^{- 1} ; Ή NMR (400 MHz, CDC1₃) δ 7.36 (app s, 4 x ArH), 7.35-7.28 (m, 2 Η, 2 x ArH), 6.92-6.88 (m, 2 Η, 2 x ArH), 5.67 (dddd, J = 7.2, 7.2, 10.4, 17.2 Hz, 1H), 5.06-4.89 (m, 2H), 4.79 (d, J = 8.8 Hz, 1H), 4.53 (s, 2H, CH₂), 4.48 (s, 2 Η, CH_2, OH), 4.11 (dd, / = 2.0, 8.8 Hz, 1H), 3.82 (s, OCHj), 3.80 (dddd, J = 2.0, 2.0, 2.0, 10 Hz, 1H), 2.31 (d, J = 6.0 Hz, 1H), 2.36-2.22 (m, 1H), 2.14 (ddd, J = 8.8, 8.8, 15.6 Hz, 1H), 1.77 (ddd, J = 2.4, 7.2, 7.2 Hz, 1H), 1.56 (s, G¾), 1.50 (s, CHj), 1.08 (d, / = 7.2 Hz, CHj); ¹³C NMR (100 MHz, CDC1₃) δ 159.5, 138.8, 137.2, 135.1, 130.5, 129.6, 128.2, 127.0, 1 18.0, 114.0, 109.0, 82.8, 80.0, 73.8, 72.0, 71.7, 55.5, 39.7, 36.5, 27.4, 27.3, 10.9; MS (m/z) calcd for C₂₆H₃₆N0₆ (M+N¾): 444.2750; found: 444.2748.

[0155] Example 7

[0156] Compound 107: DIEA (15.7 ml, 89.9 mmol) was added to a 0°C THF (191 mL) solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanoic acid (9.96 g, 28.2 mmol). This was followed by the addition of 2,4,6-tnchlorobenzoyl chloride (5.1 mL, 32.6 mmol) and the resulting mixture was stirred for 0.5 h at 0°C, allowed to warm to room temperature over 30 min and then stirred for additional 1.5 h at room temperature. At this point, the reaction mixture was cooled to 0°C, a solution of Compound 106, (25,3S)-2- ((4R,5R)-5-(4-(((4-methoxybenzyl)oxy)methyl) phenyl)-2,2-dimefhyl-l,3-dioxolan-4-yl)hex- 5-en-3-ol (10.45 g, 24.5 mmol) and DMAP (4.49 g, 36.7 mmol) in THF (135 mL) was added at 0°C over 15 min. The mixture was stirred overnight under an atmosphere of nitrogen at 0°C at which point TLC (silica, 10% EtOAc-heptane) and uPLC-MS showed the reaction was complete. The reaction was quenched by the addition of saturated NH4CI (50 mL) and the resulting solution was diluted with ether:EtOAc (1 : 1 ; 300 mL) and water (20 mL). The organic layer was separated and aqueous layer was extracted with EtOAc:Ether (1 : 1 , 2 x 200 mL). The combined extracts were washed with saturated NaHC0₃ (2 x 100 mL), brine (1 x 50 mL) dried and concentrated in vacuo. Chromatography of the residue on silica gel using 0% to 20% EtOAc-heptane gave Compound 107 (16.28 g, 87%) as a white solid; [a]_D = - 2.9 ^° (c = 0.1, CDC1₃); FT-IR (CH₂C1₂ cast) 3337, 2956, 1724, 1612, 1514, 1451 , 1247, 1043 cm^"1; Ή NMR (400 MHz, CDC1₃) δ 7.78 (app d, J = 7.4 Hz, 2 x ArH), 7.58 (app d, = 7.4 Hz, 2 x ArH), 7.39 (app dd, / = 7.4 Hz, 2 x ArH), 7.34 (app s, 4 x ArH), 7.33-7.24 (m, 4 x ArH), 6.92-6.88 (m, 2 Η, 2 x ArH), 5.46 (dddd, / = 7.2, 7.2, 10.4, 17.2 Hz, 1H), 5.20 (d, / = 9.4 Hz, 1H), 4.92-4.78 (m, 3H), 4.69 (d, J = 8.6 Hz, 1H), 4.51 (s, 2 Η, CH₂), 4.45 (s, 2 Η, CH₂), 4.48-4.26 (m, 2H), 4.21 (dd, / = 7.0, 7.0 Hz, 1H), 3.86 (dd, J = 2.7, 8.6 Hz, 1H), 3.80 (s, 3 Η, OCHj), 2.20 (app dd, / = 6.6, 6.6 Hz, 2H), 1.91 (ddd, / = 2.4, 7.2, 7.2 Hz, 1H), 1.69- 1.45 (m, 4H), 1.56 (s, CH₃), 1.55 (s, CH , 1.45 (s, CH₃), 1.08 (d, / = 7.2 Hz, CH₃), 0.91 (app dd, J = 5.9, 5.9 Hz, 2 x CH₃) ¹³C NMR (100 MHz, CDC1₃) δ 172.2, 159.2, 155.8, 143.8, 141.3, 138.8, 136.9, 133.0, 130.2, 129.4, 128.1, 127.7, 127.0, 126.9, 125.1, 125.0, 120.0, 119.9, 118.1, 113.8, 108.9, 82.0, 80.4, 75.8, 71.7, 71.4, 66.8, 55.3, 52.8, 47.2, 42.1, 35.6, 35.4, 27.2, 27.0, 24.7, 23.0, 21.8, 9.9; TOF-MS (m/z) calcd for C₄₇H₅₉N₂0₈ (M+NH₄):

779.4271 ; found: 779.4265.

[0157] Example 8

(107) (108)

[0158] Compound 108: (S)-(2S,3S)-2-((4R,5R)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl- 1 ,3-dioxolan-4-yl)hex-5-en-3-yl 2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanoate (7.75 g, 10.17 mmol) was dissolved in acetonitrile (743 mL) and diethylamine (372 mL, 3.55 mol) was added to the solution at room temperature. The mixture was stirred for 1 h at which point uPLC-MS indicated that the reaction was complete. The solvent and volatile organics were removed in vacuo to afford the crude free amine (5.60 g. 102%) which was azeotroped with toluene (3 x 50 mL) before the next step. 3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,2- dimethylpropanoic acid (3.80 g, 11.21 mmol) was dissolved in CH₂C1₂ (25 mL), cooled to 0°C and DIPEA (5.73 mL, 32.8 mmol) was then added to the mixture, followed by 3H- [l,2,3]triazolo[4,5-b]pyridin-3-ol (1.94 g, 14.3 mmol) (ΗΟΑΤ) and Nl- ((ethylimino)methylene)-N,N-dimethylpropane-l,3-diamine hydrochloride (3.13 g, 16.3 mmol). The mixture was stirred at 0°C for 15 min. The cmde product, described above, was dissolved in CH₂C1₂ (93 mL) and added to the reaction mixture which was stirred overnight under an atmosphere of nitrogen at room temperature in the dark. At this point, uPLC-MS showed the reaction was complete. The mixture was diluted with ether EtOAc (1: 1, 200 mL,) and washed with 20 mL of water (1 x 20 mL), 5% KHS0₄ (1 x20 mL) and saturated NaHC0₃ (1 x 20 mL). The individual aqueous layers were extracted EtOAc Et₂0 (1: 1, 1 x 100 mL) and the combined organic layers were washed with brine (1 x 20 mL), dried, filtered and concentrated in vacuo. The crude residue was purified on silica gel using 0% to 40% EtOAc-heptane to afford Compound 108 (S)-(2S,3,S)-2-((4R,5R)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl- 1 ,3-dioxolan-4-yl)hex-5-en-3-yl 2-(3- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethylpropanamido)-4- methylpentanoate (7.70 g, 8.94 mmol, 88 % yield) as a colorless foam: [ajo = +2.6^° (c = 1, CH₂C1₂); FT-IR (cast) 3440, 3353, 2958, 1724,1653, 1514, 1451, 1246, 1039 cm^"1; 1H NMR (400 MHz, CDC1₃) δ 7.76 (app d, J = 7.8 Hz, 2 x ArH), 7.60 (app dd, / = 7.4 Hz, 2 x ArH), 7.42-7.24 (m, 10 x ArH), 6.90-6.88 (m, 2 Η, 2 x ArH), 5.94 (d, J = 7.8 Hz, 1H), 5.66 (dd, J = 5.9, 5.9 Hz, 1H), 5.56-5.44 (m, 1H), 4.96-4.82 (m, 3H), 4.69 (d, / = 9.0 Hz, 1H), 4.48 (app d, 7 = 16 Hz, 6H, O-CH2), 4.38 (app d, = 7.0 Hz, 2H) 4.20 (dd, / = 7.4, 7.4 Hz, lH), 3.85 (dd, J = 2.7, 9.0 Hz, 1H), 3.80 (s, 3H, OCHj), 3.27 (app d, / = 7.1 Hz, 2H), 2.90-2.18 (m, 2H), 1.92 (ddd, / = 2.3, 6.6, 6.6 Hz, 1H), 1.66 (m, 1H), 1.50 (s, C¾), 1.43 (s, G¾), 1.38- 1.19 (m, 1H), 1.18 (app d, / = 5.0 Hz, 2 x C¾), 1.07 (d, J = 7.2 Hz, CHj), 0.91 (app d, J = 5.0 Hz, 2 x C¾); ¹³C NMR (100 MHz, CDC1₃) δ 176.7, 172.6, 159.2, 156.9, 144.0, 141.3,

138.7, 137.0, 133.0, 130.2, 129.4, 128.1, 127.6, 127.0, 127.0, 126.9, 125.2, 119.9, 118.1,

113.8, 108.9,82.0, 80.3, 76.1 , 71.8, 71.4, 66.7, 55.3, 51.0, 49.5, 47.3, 43.3, 41.2, 35.5, 35.3, 27.2, 27.1 , 25.1, 23.5, 23.0, 23.0, 21.8, 9.7; TOF-MS (m/z) calcd for C₅₂H₆₅N₂0₉ (M+H): 861.4690; found: 861.4677.

[0159] Example 9

[0160] Compound 109 : (S)-(2S,3S)-2-((4R,5R)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl-l ,3-dioxolan-4-yl)hex-5-en-3-yl 2-(3- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethylpropanamido)-4- methylpentanoate (5.8 g, 6.74 mmol) was dissolved in acetonitrile (500 mL) and

diethylamine (246 mL, 2.36 mol) was added to the solution at room temperature and the mixture was stirred at room temperature for 1 h. uPLC-MS indicated that the reaction was completed so the solvent and volatile organics were removed in vacuo to afford the crude free amine (4.41 g. 102%) which was azeotroped with toluene (3 x 50 mL) before used for the next step. (R)-2-acrylamido-3-(3-chloro-4-methoxyphenyl)propanoic acid (2.67 g, 9.42 mmol) was dissolved in CH₂C1₂ (18 ml), cooled to 0°C and DIPEA (3.79 mL, 21.7 mmol) was added followed by 3H [l,2,3]triazolo[4,5-b]pyridin-3-ol (1.28 g, 9.42 mmol) (ΗΟΑΤ) and Nl-((ethylimino)methyl-ene)-N/Y-dimethylpropane-l,3-diamine hydrochloride (2.07 g, 10.8 mmol). The mixture was stin-ed at 0°C for 15 min. The crude amine (4.41g) dissolved in CH₂C1₂ (60 mL) was added to the reaction mixture and the resultant mixture was stirred overnight under an atmosphere of nitrogen at room temperature and in the dark. uPLC-MS then showed the reaction was complete so the mixture was diluted with ether /EtOAc (1 : 1 ; 200 mL) and washed with water (1 x 20 mL), 5% KHS0₄ (1 x 20 mL) and saturated NaHC0₃ (1 x 20 mL). The crude residue was purified on silica gel using 0% to 20% to 80% EtO Ac- heptane to give Compound 109, (S)-(2S,35)-2-((4R,5R)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl-l,3-dioxolan-4-yl)hex-5-en-3-yl 2-(3-((R 2-acrylamido-3-(3-chloro-4-methoxyphenyl) propanamido)-2,2-dimefhylpropanamido)-4- methylpentanoate (4.19 g, 4.63 mmol, 68.8 % yield) as a colorless solid; [a]_D = +4. Γ (c = 1.0, CH₂C1₂); FT-IR (CH₂C1₂ cast) 3290, 2959, 1736,1652, 1514, 1248, 1067 cm^{" 1} ; Ή NMR (400 MHz, CDC1₃) δ 7.38 (app s, 4 x ArH), 7.26 (d, 7 = 6.6 Hz, 2 x ArH), 7.04 (dd, 7 = 2.3, 8.6 Hz, 1 x ArH), 6.88 (app d, 7 = 6.6 Hz, 2 x ArH), 6.89-6.84 (m, 1H), 6.81 (app d, / = 8.6 Hz, 2 x ArH), 6.28-6.19 (m, 2H), 6.04 (dd, 7 = 10.1 , 16.8 Hz, 1H), 5.90 (d, 7 = 8.2 Hz, 1H), 5.62 (dd, / = 1.6, 10.6 Hz, 1H), 5.51 (dddd, / = 7.0, 7.0, 10.2, 17.2 Hz, 1H), 4.98-4.82 (m, , 3H), 4.70 (d, 7 = 9.0 Hz, 1H), 4.63 (dd, 7 = 6.4, 14.4 Hz, 1H), 4.49 (d, 7 = 16.4 Hz, 1H), 4.38 (ddd, 7 = 4.3, 4.3, 9.8 Hz, 1H), 3.87 (dd, 7 = 1.6, 9.0, 1H), 3.84 (s, 3H, OCH₅), 3.81 (s, 3H, OCH , 3.33 (dd, J = 6.6, 13.3 Hz, 1H), 3.23 (dd, 7 = 5.5, 13.3 Hz, 1H), 3.07-2.96 (m, 2H), 2.30-2.19 (m, 2H), 1.90 (ddd, 7 = 2.3, 6.6, 6.6 Hz, 1H), 1.60-1.54 (m, 2H), 1.51 (s, CH₃), 1.45 (s, CHj), 1.12 (s, CHj), 1.08 (d, 7 = 7.0 Hz, CH₃), 1.06 (s, CH_?), 0.90 (d, 7 = 6 Hz, CH₃), 0.86 (d, 7 = 5.9 Hz, CH₃); ¹³C NMR (100 MHz, CDC1₃) δ 176.6, 172.9, 170.9, 165.2, 159.2, 153.9, 138.7, 137.0, 132.9, 131.1, 130.5, 130.2, 129.7, 129.4, 128.6, 128.1, 126.9, 126.9, 122.2, 118.1 , 1 13.8, 112.1 , 108.9, 81.9, 80.3, 76.4, 71.8, 71.4, 56.1 , 55.3, 54.7, 51.2, 47.8, 42.8, 40.5, 37.2 ; MS (m/z) calcd for C₅oH6₇ClN₃0₁₀ (M+H): 904.4515; found: 904.4526.

[0161] Example 10

[0162] Compound 110: (S)-(2S,3S)-2-((4/?,5i?)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl-l,3-dioxolan-4-yl)hex-5-en-3-yl 2-(3-((R)- 2-acrylamido-3-(3-chloiO-4-methoxyphenyl)propanamido)-2,2-dimethylpropanamido)-4- methylpentanoate (4.19 g, 4.632 mmol) was dissolved in CH₂C1₂ (298 mL, 4632.26 mmol), and the catalyst of [1,3-Bis {2,4, 6-tri CH₃ phenyl }-2-imidazoli dinylidene]di CI

{phenylmethyle ne} [tricycloh exylphosphine] Ru (334 mg, 0.394 mmol; Sig ma Al dritch) was added to the mixture at room temperature. [0163] The reaction mixture was heated to reflux (41 °C) for 3h at which it was deemed complete as shown by TLC (silica, 60% EtO Ac-heptane) and uPLC-MS. The mixture was concentrated in vacuo and chromatography of the residue on silica gel using 0% to 40% EtOAc-heptane to obtain Compound 110, (35,10R,165,£)-10-(3-chloro-4-methoxybenzyl)- 3-isobutyl-16-((5)-l-((4R,5/?)-5-(4-(((4-methoxybenzyl)oxy)methyl)-phenyl)-2,2-dimethyl- l,3-dioxolan-4-yl)ethyl)-6,6-dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9, 12- tetraone (3.49 g, 3.98 mmol, 86 % yield) as a light brownish foam; [<X]D = -30.0^° (c = 1 , CH₂C1₂); FT-IR (CH₂C1₂ cast) 3301, 2959, 1743,1652, 1506, 1247, 1174,1066 cm^{" 1} ; Ή NMR (400 MHz, CDC1₃) δ 7.26-7.39 (m, 3 x ArH), 7.16 (app s, 1 x ArH), 7.05 (app dd, / = 8.2 Hz, 1 x ArH), 6.80-6.90 (app dd, 5 x ArH), 6.30 (ddd, / = 3.5, 11.3, 14.5 Hz, 1 x ArH), 5.86 (d, / = 14.9 Hz, lH), 5.62 (d, J = 14.9 Hz, 1H), 5.39 (d, / = 7.0 Hz, 1H), 5.10 (m, 1 Η, CH), 4.69 (d, J = 8.6 Hz, 1 H), 4.51 (app s, 2 H, CH₂), 4.47 (app s, 2 Η, CH₂), 4.39 (ddd, J = 7.0, 9.0, 9.0 Hz, 1 H), 3.87 (s, 3 H, OC¾), 3.81 (s, 3 H, OCHj), 3.78 (d, J = 9.0 Hz, 1 H), 3.45 (dd, / = 9.4, 12.9 Hz, 1H), 3.16 (app d, 2 Η), 3.08 (dd, / = 5.1, 14.8 Hz, 1H), 2.96 (dd, / = 7.8, 14.8 Hz, 1H), 2.43 (app d, 1H), 2.12-2.24 (m, 1H), 1.76-1.85 (m, 1H), 1.48 (s, CH₃), 1.49 (s, C¾), 1.32-1.56 (m, 3H), 1.17 (s, CHj), 1.12 (s, CH₃), 0.89 (d, J = 6.3 Hz, G¾), 0.84 (d, / = 6.3 Hz, CHj),; ¹³C NMR (100 MHz, CDC1₃) δ 177.6, 172.7, 170.6, 164.8, 159.2, 154.1, 142.4, 138.8, 136.8, 130.8, 130.2, 129.4, 129.4, 128.1 , 126.1, 124.4, 122.5, 113.8, 112.4, 109.0. 82.2, 79.9, 74.9, 71.8, 71.3, 56.1 , 55.3, 54.7, 50.4, 46.8, 43.2, 41.6, 36.6, 35.8, 35.5, 27.2, 27.0, 24.9, 24.9, 22.8, 22.4, 21.7, 9.4; MS (m/z) calcd for C₄8H₆₃Cl ₃Oio (M+H):

876.4202; found: 876.4200.

[0164] Example 11

[0165] Compound 111: (3₁S,107?,165,E)-10-(3-chloiO-4-methoxybenzyl)-3-isobutyl-16-((_l )- l-((4/?,5 ?)-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl-l,3-dioxolan-4- yl)ethyl)-6,6-dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9,12-tetraone (18.92 g, 21.6 mmol) was dissolved in acetonitrile (500 mL) and water (250 mL). The reaction mixture was cooled to 0°C and then TFA (41.6 ml, 0.540 mol) was added dropwise at 0°C. The reaction mixture was slowly warmed up to room temperature and stirred for 2 days (until SM was consumed as shown by TLC). After the reaction was completed, reaction mixture was cooled to -70°C and diluted with pre-cooled CH₂C1₂ (500 mL). Solid NaHC0₃ (98 g, 1.16 mol) was carefully added at -70°C and slowly warmed to 0°C while the mixture was vigorously stirred and then an additional water (15 mL) was added and the reaction was stirred for 15 min. The organic layer was seperated and the aqueous layer was extracted with CH₂C1₂ (2 x 300 mL). The combined layers were washed with saturated NaHC0₃ (1 x 150 mL), dried, filtered and concentrated in vacuo to obtain the free diol Compound 111

(3^,10R,165,E)-10-(3-chloro-4-methoxybenzyl)-16-((2R,3R,4R)-3,4-dihydroxy-4-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)butan-2-yl)-3-isobutyl-6,6-dimethyl-l-oxa-4,8,l l- triazacyclohexadec-13-ene-2,5,9,12-tetraone (17.7 g, 98 % yield) as a light grey foam: [ct]o = -16.5^° (c = 1, CH₂C1₂); FT-IR (CH₂C1₂ cast) 3414, 3322, 2960, 1741,1646, 1506, 1257, 1 180, 1066 cm^"1 ; 1H NMR (400 MHz, CDC1₃) δ 7.35 (app d, / = 7.8 Hz, 2H), 7.32-7.25 (m, 3 x ArH), 7.16 (d, / = 2.0 Hz 1 x ArH), 7.03 (dd, 7 = 2.0, 8.2 Hz, 1 x ArH), 6.93-6.80 (m, 4 x ArH), 6.69 (ddd, J = 3.5, 10.9, 14.9 Hz, 1 x ArH), 5.95 (d, / = 8.2 Hz, 1H), 5.68 (dd, / = 1.2, 16.0 Hz, 1H), 5.39 (d, J = 7.0 Hz, 1H), 5.15-5.08 (m, 1H), 4.65-4.55 (m, 2H), 4.54-4.46 (m, 4H), 4.42 (ddd, J = 5.1 , 9.0 Hz, 1H), 3.87 (s, 3 Η, OCHj), 3.81 (s, 3 Η, OCHi), 3.73 (d, J = 8.6 Hz, 1H), 3.45 (dd, / = 9.4, 13.3 Hz, 1H), 3.16 (d, / = 10.9 Hz, lH), 3.08 (dd, 7 = 4.3, 14.5 Hz, 1H), 2.90 (dd, / = 8.6, 14.8 Hz, 1H), 2.48 (d, / = 14.5 Hz, 1H), 2.20-2.10 (m, 1H), 1.60-1.40 (m, AH), 1.18 (s, CH₃), 1.12 (s, G¾), 0.99 (d, J = 6.3 Hz, CH₅), 0.90 (d, J = 6.3 Hz, CH₃), 0.78 (d, J = 6.3 Hz, CH₃); ¹³C NMR (100 MHz, CDC1₃) δ 177.7, 173.2, 171.0,

165.2, 159.2, 154.0, 142.9, 140.1, 138.4, 130.7, 130.2, 129.6, 129.4, 128.1, 128.0, 126.9,

124.3, 122.3, 113.8, 112.5, 75.4, 74.8, 72.0, 71.4, 56.1 , 55.3, 54.9, 50.5, 47.0, 43.1, 41.3, 38.0, 36.1 , 35.6, 25.0, 24.8, 22.9, 22.6, 21.8, 9.4; MS (m/z) calcd for C₄₅H₅9ClN₃Oio (M+H): 836.3889; found: 836.3873.

[0166] Example 12

(111) (112)

[0167] Compound 112: (3. ,10R,16_lS',E)-10-(3-chloro-4-methoxybenzyl)-16-((2R,3R,4R)- 3,4-dihydroxy-4-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)butan-2-yl)-3-isobutyl-6,6- dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9,12-tetraone (5.72 g, 6.84 mmol) was dissolved in CH₂C1₂ (341 mL), trimethyl orthoformate (113 mL, 1.03 mol) was added to the mixture followed by p-PTS (1.41 g, 5.61 mmol). The reaction mixture was stirred for 3.5 h at room temperature. TLC and uPLCMS showed that there was no SM remaining so the reaction mixture was diluted with dry CH2CI2 (250 mL), filtered through a short pad of silica gel and rinsed with 80% EtOAc/CH₂Cl₂ (1.5 L). The filtrate was concentrated in vacuo and then kept under high vacuum for lh to obtain (3>S,10/?,16S,E)-10-(3-chloiO-4- methoxybenzyl)-3 -isobutyl- 16-(( 15)- 1 -((4R,5R)-2-methoxy-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-l,3-dioxolan-4-yl)ethyl)-6,6-dimethyl-l-oxa-4,8,l l- triazacyclohexadec-13-ene-2,5,9,12-tetraone (6.05 g, 101 % yield). The residue was azeotroped with toluene (3 x 50 mL) before the next step. The crude was dissolved in CH₂C1₂ (175 mL) and a solution of bromotrimethylsilane (1.78 mL, 13.7 mmol) in CH₂C1₂ (12.9 mL) was added to the mixture at room temperature and stirred for 1.5 h until the starting material was consumed (TLC). The reaction mixture was diluted with dry CH₂C1₂ (150 mL), cooled to -50°C and saturated NaHC0₃ (50 mL) and water (30 mL) were added. The resultant mixture was gently warmed to melt the frozen solid. The organic layer was seperated and aqueous layer was extracted with CH₂C1₂ (2 x 100 mL). The combined organic layers were washed with brine (1 x 50 mL), dried and concentrated in vacuo to give

(l/?,2 ?,35)-l-bromo-3-((35,10R,16₁S,E)-10-(3-chloiO-4-methoxybenzyl)-3-isobutyl-6,6- dimethyl-2,5,9,12-tetraoxo-l-oxa-4,8, 11-triazacyclohexadec- 13-en- 16-yl)- 1 -(4-(((4- methoxybenzyl)oxy)methyl)-phenyl)butan-2-yl formate (6.41g, 101%); mlz 927.3. The crude material (6.34 g, 6.84 mmol) was azeotroped with dry toluene (3 x 50 mL), dissolved in mixture of THF (36 mL) and methanol (110 mL), potassium bicarbonate (1.58 g, 34.2 mmol) was added to the mixture at room temperature. The reaction mixture was stirred for 8 h at 42°C, diluted with CH₂C1₂ (300 mL) and saturated NH₄C1 (50 mL) was added followed by water (20 mL). The organic layer was seperated and aqueous layer was extracted with CH₂C1₂ (2 x 300 mL). The combined organic layers were washed with brine (1 x 50 mL), dried and concentrated in vacuo to obtain (3₁S',10_JR,16_lS',E)-10-(3-chloro-4-methoxybenzyl)-3- isobutyl-16-((5')-l-((2/?,3R)-3-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)oxiran-2-yl)ethyl)- 6,6-dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9,12-tetraone (5.65g, 101%), mlz 818.4. The crude material (35,10R,165',E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-16- ((,S')-l-((2R,3R)-3-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)oxiran-2-yl)ethyl)-6,6- dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9,12-tetraone (5.60 g, 6.84 mmol) was dissolved in CH₂C1₂ (280 mL) and water (32.7 mL), DDQ (3.42 g, 15.1 mmol) was added to the mixture and stirred for 2.5 h until the starting material was consumed. The reaction mixture was diluted with CH₂C1₂ (50 mL) and saturated NaHC0₃ (40 mL) was added and the organic layer seperated and the aqueous layer was extracted with CH₂C1₂ (2 x 200 mL). The combined organic layers were washed with NaHC0₃ (3 x 50 mL) and brine (1 x 50 mL), dried, filtered and concentrated in vacuo. The residue was purified by silica column using 65% to 100% EtO Ac-heptane to obtain Compound 112, (3S, 10^,16S,E)-10-(3 -chloro-4- methoxybenzyl)-16-((,S)-l-((2/?,3i?)-3-(4-(hydroxymethyl)phenyl)oxiran-2-yl)ethyl)-3- isobutyl-6,6-dimethyl-l-oxa-4,8,l l-triazacyclohexadec-13-ene-2,5,9,12-tetraone (3.92 g, 5.61 mmol, 82 % yield) as a colorless foam: [ct]_D = +8.5° (c = 1 , CH₂C1₂); FT-IR (CH₂C1₂ cast) 3392, 2960, 1741, 1651, 1504, 1258 cm^{" 1} ; Ή NMR (400 MHz, CDC1₃) δ 7.37 (app d, / = 7.8 Hz, 2H), 7.22 (d, / = 8.2 Hz, 2 x ArH), 7.16 (d, / = 2.0 Hz 1 x ArH), 7.00 (dd, / = 2.0, 8.2 Hz, 1 x ArH), 6.93-6.88 (m, 1 x ArH), 6.83 (d, J = 8.6 Hz, 1 x ArH), 6.72 (ddd, / = 3.5, 10.9, 14.9 Hz, 1 x ArH), 5.82 (d, J = 8.9 Hz, 1H), 5.62 (dd, J = 1.6, 14.8 Hz, 1H), 5.23 (ddd, 7 = 2.0, 4.7, 11.4 Hz, 1H), 4.71 (app d, / = 3.9 Hz, 2H), 4.64 (ddd, J = 5.1, 7.8, 7.8 Hz, 1H), 4.43 (ddd, J = 5.5, 9.7 Hz, 1H), 3.87 (s, 3H, OCHj), 3.68 (d, / = 1.9 Hz, 1H), 3.46 (dd, J = 9.8, 13.3 Hz, 1H), 3.14 (dd, J = 4.7, 14.4 Hz, 1H), 3.08 (dd, J = 4.3, 14.5 Hz, lH), 2.97 (dd, / = 7.8, 14.4 Hz, 1H), 2.90 (d, / = 1.9, 7.4 Hz, lH), 2.58-2.30 (m, 1H), 2.44-2.34 (m, 1H), 1.83-1.70 (m, 2H), 1.59-1.30 (b m, 3H), 1.18 (s, G¾), 0.89 (s, C¾), 0.88 (d, / = 6.3 Hz, CHj), 0.84 (d, / = 6.3 Hz, G¾), 0.78 (d, J = 6.3 Hz, G¾); ^I3C NMR (100 MHz), CDC1₃ δ 177.8, 173.1, 170.6, 164.7, 154.1, 141.5, 136.0, 130.6, 129.5, 128.1, 127.4, 125.8, 124.9, 122.4, 112.4, 75.0, 64.8, 63.2, 58.9, 56.1, 54.8, 50.4, 46.8, 43.2, 41.3, 40.4, 36.7, 35.6, 24.9, 24.8, 22.7, 22.4, 21.6, 13.5; MS (m/z) calcd for C₃₇H₄₉C1N₃0₈ (M+H): 698.3208; found: 698.3199.

[0168] Example 13

(113) OH (114)

[0169] Compound 114: Hunig's base (4.1 mL, 23.4 mmol) was added to a solution of Compound 113 (2.6 g, 10.6 mmol) in DMF (20 mL) followed by addition of tert-butyl piperazine-l-carboxylate (3.96 g, 21.3 mmol). The mixture was stirred for 6 h at 23°C and then heated to 40°C . After 14 h uPLC-MS showed that the reaction was complete. The mixture was concentrated in vacuo and then saturated aqueous NaHC0₃ (30 mL) was added followed by H₂0 (30 mL). The resulting mixture was extracted with CH₂C1₂ (3 x 150 mL). The combined organic layers were washed with brine (1 x 20 mL), dried with Na₂S0₄, filtered and concentrated in vacuo. Chromatography of the residue on silica gel using 0% to 15% MeOH-CH₂Cl₂ gave Compound 114 (2.54 g, 92%) as a clear oil; Ή NMR (400 MHz, CDC1₃) δ 5.74 (br.s, 1 H), 3.98-3.94 (m, 1 H), 3.43-3.38 (m, 4 H), 2.66-2.54 (m, 4 H), 2.34- 2.30 (m, 2 H), 1.68-1.1.45 (m, 2 H), 1.45 (s, 9 H), 1.16 (d, 3 Η, / = 6.4 Hz); MS (m/z) calcd for Ci₃H₂₇N₂0₃ (M+H): 259.20; found: 259.20.

[0170] Example 14

[0171] Compound 115: To a solution of Compound 114 (2.54 g, 9.83 mmol) in pyridine (25 mL) was added TsCl (5.62 g, 29.5 mmol) portion-wise over 15 minutes at 0°C. The reaction was allowed to warm to 23°C over ~1 hour and then stirred for 16 h at which point uPLC-MS showed the reaction was complete. The mixture was concentrated in vacuo and diluted with CH₂C1₂ (200 mL) and washed sequentially with saturated aqueous NaHC0₃ (1 x 30 mL), H₂0 (1 x 30 mL) and brine (1 x 30 mL). The organic layer was dried with Na₂S0₄, filtered and concentrated in vacuo and chromatography of the residue on silica gel using 30% to 100% EtO Ac-heptane then 0% to 15% MeOH-CH₂Cl₂ gave Compound 115 (3.4 g, 84%) as a clear oil: 1H NMR (400 MHz, CDC1₃) δ 7.78 (d, / =8.4 Hz, 2H), 7.32 (d, / = 8.0 Hz, 2H), 4.70 (dq, J = 6.2, 12.8 Hz, 1H), 3.33 (dd, / = 5.2, 5.2 Hz, AH), 2.43 (s, 2.29-2.03 (m, 6H), 1.78 (dddd, / = 7.6, 7.6, 7.6,14.2 Hz, 1H), 1.67-1.60 (m, 1H), 1.44 (s, 9H), 1.27 (d, / = 6.0 Hz, 3H).

[0172] Example 15

[0173] Compound 116: O-Ethyl carbonodithioate (2.5 g, 15.6 mmol) was added portion- wise over 15 minutes to a 23°C EtOH (40 mL) solution of Compound 115 (2.1 g, 5.1 mmol). After 16 h TLC (30% EtO Ac-heptanes, R_f = 0.35) showed the reaction was complete. The mixture was concentrated in vacuo and the solid residue was stirred with CH₂C1₂ (50 mL) and then the slurry was filtered thru Celite and rinsed with CH₂C1₂ (1 x 50 mL). The filtrate was concentrated in vacuo and chromatography of the residue on silica gel using 25% to 60% EtOAc-heptane gave Compound 116 (1.39 g, 75%) as a clear oil: [a]u = -14.3° (c = 1, CH₂C1₂); FT-ER (CH₂C1₂ cast) 2976, 2822, 1698, 1456, 1420, 1211, 1172, 1046 cm^"1; 1H NMR (400 MHz, CDC1₃) δ 4.63 (q, = 7.2 Hz, 2H), 3.80 (ddq, J = 6.8, 13.4, 13.4 Hz, lH), 3.43 (dd, / = 5.2, 5.2 Hz, AH), 2.45 (app ddd, / = 2.8, 5.8, 8.2 Hz, 2H), 2.38 (ap dd, J = 4.8, 4.8 Hz, 4H), 1.90 (dddd, / = 6.6, 6.6, 8.2, 13.2 Hz, 1H), 1.78 (dddd, / = 6.5, 6.5, 8.7, 13.6 Hz, 1H), 1.45 (s, 9H), 1.42 (t, / = 7.4 Hz, 3H), 1.40 (d, / = 6.8 Hz, 3H); ¹³C NMR (125 MHz, CDC1₃) δ 214.5, 154.8, 79.6, 69.6, 55.7, 53.0, 44.0, 34.0, 28.4, 20.5, 13.8; MS (m/z) calcd for Ci₆H₃₁N₂0₃S₂ (M+H): 363.18; found: 363.57.

[0174] Example 16

[0175] Compound 117: Ethylenediamine (2.35 mL, 34.8 mmol) was added dropwise over 5 minutes to a solution of Compound 116 (1.05 g, 2.90 mmol) in EtOH (17 mL) at 0°C. The mixture was allowed to gradually warm to 23 °C over 30 minutes. After 1.5 h uPLC-MS showed the reaction was complete so it was concentrated in vacuo, diluted with CH₂C1₂ (100 mL), washed with H₂0 (1 x 20 mL) and brine (1 x 20 mL). The combined organic layers were dried with Na₂S0₄, filtered and concentrated in vacuo. Chromatography of the residue on silica gel using 30% to 80% EtOAc-heptane gave Compound 117 (790 mg, -100%) as a clear oil: Ή NMR (400 MHz, CDC1₃) δ 3.41-3.39 (m, 4H), 2.98 (ddq, / = 6.6, 6.6,13.4 Hz, 1H), 2.43 (ddd, / = 2.0,7.6, 7.6 Hz, 2H), 2.37-2.34 (m, AH), 1.78 (dddd, J = 5.7, 5.7, 7.8, 12.8 Hz, IH), 1.69-1.59 (m, 2H), 1.44 (s, 9H), 1.33 (d, / = 7.2 Hz, 3H).

[0176] Example 17

[0177] Compound 118: S-Methyl methanesulfonothiolate (460 mg, 3.64 mmol) in THF (22 mL) was added dropwise over 15 minutes to a 0°C solution of Compound 117 (800 mg, 2.91 mmol) in pH = 7.0 phosphate buffer (2.0 mL) and EtOH (15 mL). The mixture was allowed to gradually warm to 23°C over 30 minutes and after 1.5 h uPLC-MS showed the reaction was complete. The mixture was concentrated in vacuo and then diluted with CH₂C1₂ (100 mL), washed with H₂0 (1 x 10 mL) and brine (1 x 10 mL). The organic layers were dried with Na₂S0₄, filtered and concentrated in vacuo. Chromatography of the residue on silica gel using 8% to 30% EtOAc-heptane gave Compound 118 (830 mg, 89%) as a clear oil: [α]ο = 29.9° (c = 1, CH₂C1₂); FT-IR (CH₂C1₂ cast) 2974, 1697, 1420, 1365, 1245, 1172,1128 cm^" ';Ή NMR (400 MHz, CDC1₃) δ 3.40 (dd, / = 5.2, 5.2 Hz, AH), 2.93 (ddq, / = 6.8, 6.8, 13.4 Hz, 1H), 2.43 (dd, / = 7.4, 7.4 Hz, 2H), 2.40 (s, 3H), 2.38 (dd, / = 5.0, 5.0 Hz, AH), 1.87 (dddd, 7 = 7.2, 7.2, 7.2, 14.4 Hz, 1H), 1.66 (dddd, J = 7.3, 7.3, 7.3, 13.7 Hz, 1H), 1.57 (s, 9H), 1.34 (d, / = 6.8 Hz, 3H); ¹³C NMR (100 MHz, CDC1₃) δ 154.7, 79.6, 55.9, 53.0, 43.9, 33.0, 28.4, 24.5, 21.0; MS (m/z) calcd for Ci₄H₂₉N₂0₂S₂ (M+H): 321.1670; found:

321.1674.

[0178] Example 18

[0179] Compound 119: TFA (2 ml, 25.9 mmol) was added to a 0°C CH₂C1₂ (5 mL) solution (S)-tert-buty\ 4-(3-(methyldisulfanyl)butyl)piperazine-l-carboxylate (100 mg, 0.31 mmol). The mixture was stirred for 2 h at 0°C, warmed to 23°C over 0.5 h, diluted with 10 ml toluene (10 mL) concentrated under vacuum and kept under vacuum for 0.5 h. The residue was dissolved in water (10 mL) and cooled to 0°C and saturated NaHC0₃ was added to achieve pH = 8.0, extracted with CH₂C1₂ (3 x 20 ml), washed with brine, dried and concentrated in vacuo to yield the product Compound 119 (75 mg) as a clear oil: 1H NMR (400 MHz, CDCI3) δ 2.95-2.88 (m, 1H), 2.88 (dd, J = 5.0, 5.0 Hz, 4H), 2.46 (m, 6H), 2.40 (dddd, J = 7.2, 7,2, 7.2, 14.0 Hz, lH), 1.67 (dddd, 1 Η, / = 6.8, 6.8, 6.8, 13.2 Hz, 1H), 1.34 (d, / = 6.8 Hz, 3H) MS (m/z) calcd for C₉H₂o ₂S2 (M+H): 221.11 ; found: 221.08. Crude Compound 119 was used for the next step without any further purification.

[0180] Example 19

(120) (121)

[0181] Compound 121: K₂C0₃ (11.80 g, 85.348 mmol) was added to 3-mercapto-3-methyl- butan- l-ol (Compound 120) (5.13 g, 42.674 mmol) in acetonitrile (100 mL) followed by benzyl bromide (6.35 mL, 53.3 mmol) at room temperature. The reaction mixture was stin-ed overnight under an atmosphere of nitrogen at which point the mixture was concentrated in vacuo and flash chromatography on silica gel using 0% to 50% EtO Ac-heptane to give

Compound 121 (8.2 g, 91%) as a colorless oil: 1H NMR (400 MHz, CDC1₃) δ 7.34-7.20 (m, 5 x ArH), 3.83 (t, J = 6.3 Hz, 2H), 3.77 (s, 2H), 2.22 (s, br, OH), 1.85 (t, J = 6.3 Hz, 2H), 1.36 (s, 6H).

[0182] Example 20

(121) (122)

[0183] Compound 122: Et₃N (4.12 mL, 29.5 mmol) was added to a 0°C CH₂C1₂ (100 mL) solution of 3-(benzylthio)-3-methylbutan-l-ol (2.07 g, 9.84 mmol followed by

methanesulfonyl chloride (0.92 mL, 11.8 mmol). The resulting mixture was stirred at 0°C for 2.5 h and then water (20 mL) was added to quench the reaction. The mixture was extracted with CH₂C1₂ (2 x 100 mL), the combined organic phases were washed with water (1 x 40 mL), brine (1 x 20 mL), dried over Na₂S0₄, filtered and concentrated in vacuo to give crude Compound 122 (2.9 g, 100%) as a colorless oil: Ή NMR (400 MHz, C₆D₆) δ 7.18 (d, 2 H, J = 7.4 Hz, 2 x ArH), 7.07-7.03 (m, 2 Η, 2 x ArH), 6.98-6.95 (m, 1 x ArH), 4.10 (t, J = 7.4 Hz, 2H), 3.37 (s, 2H), 2.09 (s, 3H), 1.61 (t, J = 7.4 Hz, 2H), 0.96 (s, 6H).

[0184] Example 21

[0185] Compound 123: Et₃N (4.504 mL, 32.314 mmol) was added to a THF (50 mL) the solution of 3-(benzylthio)-3-methylbutyl methanesulfonate (2.33 g, 8.078 mmol) followed by tert-butyl piperazine-l-carboxylate (4.257 g, 22.854 mmol). The reaction mixture was heated to 70°C overnight at which point DMF (20 mL) was added and the now clear mixture was heated for a further 5 h at 75°C. The THF was removed in vacuo and the residue was diluted with MTBE (100 mL), washed with water (2 x 50 mL) and brine (1 x 50 mL), dried over Na₂S0₄ concentrated. The crude residue was purified on silica gel using 0% to 80% EtO Ac- Heptane to afford Compound 123 (886 mg, 29%) as a pale yellow oil: Ή NMR (400 MHz, C₆D₆) δ 7.24-7.22 (d, / = 7.42, 2 x ArH), 7.08-7.04 (m, 2 x ArH), 6.99-6.97 (m, 11 x ArH), 3.48 (s, 2H), 3.41 (br s, 4H), 2.30-2.26 (m, 2H), 2.06-1.93 (t, / = 4.69, 4H), 1.52-1.48 (m, 2H), 1.44 (s, 9H), 1.11 (s, 6H).

[0186] Example 22

(123) (124)

[0187] Compound 124: Liquid ammonia (-50 mL) was condensed into a 250 mL of 3- necked flask and cooled at -78°C. Sodium metal was added to the ammonia in small portions until the solution remained deep blue. Tert-butyl 4-(3-(benzylthio)-3-methylbutyl)piperazine- 1-carboxylate (1.02 g, 2.69 mmol) was added as a solution in THF (13 mL, 158.6 mmol). Excess sodium was added to maintain the blue color. The mixture was stirred at -78°C for 1 hr. Solid NH4CI was added portion-wise until the blue color disappeared. The cold bath was removed and the mixture was stirred at room temperature for 0.5 h, then N₂ was bubbled through the mixture for another 0.5 h. The residue was dissolved in water (100 mL) and EtO Ac (250 mL), the organic layer was seperated and aqueous phase was extracted with EtOAc (1 x 100 mL). The combined organic phases were washed with brine (1 x 50 mL), dried over Na₂S0₄, filtered and concentrated to give Compound 124 as an opaque colorless oil (875 mg, 100%). This crude product was carried forward to the next step directly without further purification: 1H NMR (400 MHz, C₆D₆) δ 3.43 (dd, J = 4.8, 4.8 Hz, 4H), 2.54-2.50 (m, 2H), 2.40 (app dd, J = 4.8, 4.8 Hz, AH), 1.80-1.76 (m, 2H), 1.45 (s, 9H), 1.39 (s, 6H).

[0188] Example 23

(124) (125)

[0189] Compound 125: 5-Methyl methanesulfonothioate (0.305 mL, 3.23 mmol) in THF (5 mL) was added to an ethanol (10 mL, 171.3 mmol)-water (5 mL, 277.5 mmol)-phosphate buffer pH=7.0 (5 mL) solution of tert-butyl 4-(3-mercapto-3-methylbutyl)piperazine-l- carboxylate (777 mg, 2.69 mmol) at 23 °C and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated NaHC0₃ (5 mL), extracted with CH₂C1₂ (2 x 50 mL). The combined organic phased were concentrated in vacuo. The residue was redissolved in CH₂C1₂, dried over Na₂S0₄, filtered and concentrated. The crude was purified on silica gel using 0% to 50% EtOAc-heptane to afford Compound 125 (848 mg, 94%) as a colorless oil: FT-IR (CH₂C1₂ cast) 2971, 2928, 1695, 1418.7, 1364, 1246, 1173, 1005 cm^"1; Ή NMR (500 MHz, CDC1₃) δ 3.43 (dd, / = 4.5, 4.5 Hz, 4H), 2.46-2.40 (m, 6H), 2.41 (s, 3Η), 1.82-1.79 (m,2 H), 1.46 (s, 9H), 1.31 (s, 6H); ¹³C NMR (125 MHz, CDC1₃) δ 154.7, 79.6, 54.4, 53.2, 50.2, 37.9, 28.4, 27.9, 25.2; MS (m/z) calcd for Ci₅H₃oN₂0₂S₂ (M+): 334.17; found: 334.81.

[0190] Example 24

[0191] Compound 126: TFA (2 ml, 25.9 mmol) was added to a 0°C CH₂C1₂ (5 ml) solution of (5)-tert-butyl 4-(3-(methyldisulfanyl)butyl)piperazine-l-carboxylate (102 mg, 0.265 mmol) over 5-10 min. The reaction mixture was stirred for 1 h at 0°C and then warmed to 23°C over 0.5 h, diluted with toluene (10 mL) and the reaction mixture was concentrated in vacuo and kept under vacuum for 0.5 h. The residue was dissolved in water (5 mL) and cooled to 0°C and saturated NaHC0₃ (~3 mL) was added to achieve pH = 8.0, extracted with CH₂C1₂ (3 x 20 ml), washed with brine (1 x 10 mL), dried over Na₂S0₄ and concentrated in vacuo to yield the desired product Compound 126 (67 mg) as a clear oil which was used for the next step without any further purification: 1H NMR (400 MHz, CDCI3) δ 2.89 (dd, / = 4.8, 4.8 Hz, AH), 2.44-2.39 (m, 6H), 2.40 (s, 3H), 1.91 (br s, 1H), 1.82-1.78 (m, 2H), 1.31 (s, 6H).

[0192] Example 25

(127) (128)

[0193] Compound 128: A solution of 2-(2-mercaptophenyl)acetic acid (2.00 g, 11.9 mmol) in EtOH (21 mL) was slowly added to a heterogeneous mixture of l,2-di(pyridin-2- yl)disulfane (7.86 g, 35.7 mmol) in EtOH (7 mL) and AcOH (0.60 mL, 10.6 mmol) at 0 °C. The reaction mixture was gradually warmed to room temperature and stirred overnight. The solvent was removed in vacuo and chromatography of the residue on silica gel using 0% to 80% EtOAc-heptane gave Compound 128 (1.66 g, 50%) as a clear oil: FT-IR (CH₂C1₂ cast) 3427, 1705, 1416, 1337, 1219, 747 cm^"1; 1H NMR (400 MHz, CDC1₃) δ 8.47-8.46 (m, 1H), 7.69-7.65 (m, 1H), 7.61-7.58 (m, 2H), 7.26-7.22 (m, 3H), 7.09 (ddd, J = 2.6, 5.0, 6.0 Hz, 1H), 3.98 (s, 2H); ¹³C NMR (100 MHz, CDCI₃) δ 171.2, 158.9, 149.6, 137.6, 136.1, 134.9, 131.2, 129.2, 128.2, 127.9, 121.3, 119.6, 38.5; MS (m/z) calcd for C[₃H₁₂N0₂S₂ (M+H): 278.02; found: 278.06.

[0194] Example 26

[0195] Compound 129: 2-(2-(pyridin-2-yldisultanyi)phenyi)acetic acid (365 mg, 1.32 mmol) was dissolved in THF (6 mL), the solution was cooled to 0°C and DCC (434 mg, 2.11 mmol) was added followed by l-hydroxypyriOlidine-2,5-dione (227 mg, 1.97 mmol). The reaction mixture was warmed to room temperature and stirred for 16h. The crude reaction mixture was filtered through a pad of celite and the filter cake washed with a mixture of EtO Ac/heptane (5 mL, 1 : 1) and the solvent was removed in vacuo . Chromatography of the residue on silica gel using 0% to 80% EtO Ac-heptane gave Compound 129 (362 g, 73%) as a light yellow oil: FT-IR (CH₂C1₂ cast) 2945, 1814, 1783, 1739, 1574, 1418, 1205, 1073 cm^"

1H NMR (400 MHz, CDC1₃) δ 8.47 - 8.46 (m, 1H), 7.70-7.68 (m, lH), 7.61-7.60 (m, 2H), 7.36-7.33 (m, 1H), 7.28-7.24 (m, 2H), 7.11-7.05 (m, 1H), 4.29 (s, 2H), 2.82 (s, 4H) ; ¹³C NMR (100 MHz, CDC1₃) δ 168.9, 166.3, 158.9, 149.6, 137.3, 136.3, 131.6, 130.7, 130.3, 129.1, 128.4, 121.1, 120.1, 35.8, 25.6; MS (m/z) calcd for C₁₇H₁₅N₂0₄S₂ (M+H): 375.0473; found: 374.0469.

[0196] Example 27

[0197] The reaction scheme for Example 27 is shown in FIG. 9.

[0198] Chlorotoxin having SEQ ID NO: 1 (Compound 5) (4.00 g, 1.00 mmol) was dissolved in a mixture of water (60 mL) and DMF (120 ml), cooled to 0°C and triethylamine (0.837 mL, 6.00 mmol) was added dropwise, followed by 2,5-dioxopyrrolidin-l-yl 2-(2-(pyridin-2- yldisulfanyl)phenyl)acetate (330 mg, 1.00 mmol). After 2 h the reaction appeared to proceed about 50% (based on uPLC-MS). More NHS-ester (45 mg) in DMF (10 mL) was added and reaction mixture was stirred for additional lh at 0°C. uPLC-MS analysis indicated that about equal amounts of chlorotoxin (SEQ ID NO: 1)-Lys₂₃ and chlorotoxin (SEQ ID NO: 1)- Lys₂₇ analogs were formed along with some bis-addition product. The crude material was purified on preparative HPLC (C-18 column, gradient 15-30% MeCN and water with 0.1% formic acid, run time lOmin). Desired fractions were concentrated under high vacuum at 30°C and residue was lyophilized to give Compound 130 (183.5 mg, 14%) as a white solid: MS (m/z) calcd for Ci₇iH₂₅₈N₅₄0₄₈S₁₃ (M+H): 4254.5859 found 4254.5728. The same process using chlorotoxin having SEQ ID NO: 2 as a starting material may be used to produce chlorotoxin analog (SEQ ID NO: 2)-Lys₂₇-CH₂-SS-Py.

[0199] Example 28

(112) (131)

[0200] Compound 131: Hiinig's base (0.50 mL, 2.86 mmol) was added to a solution of Compound 112 (200 mg, 0.286 mmol) in CH₂C1₂ (3.0 mL) at -20°C followed by methanesulfonyl chloride (0.18 mL, 2.29 mmol). The mixture was allowed to gradually warm to 0°C over 90 minutes and after 1 hour at 0°C uPLC-MS showed the reaction was complete. While at 0°C, the mixture was diluted with CH₂C1₂ (15 mL) and quenched with saturated aqueous NaHC0₃ (5 mL). The mixture was transfeiTed to a seperatory funnel and the aqueous layer was extracted with CH₂C1₂ (2 x 50 mL). The combined organic layers were washed with brine (1 x 50 mL) and the organic layer was dried with Na₂S0₄, filtered and concentrated in vacuo. Chromatography of the residue on silica gel using 30% to 80% EtO Ac-heptane gave Compound 131 (205 mg, 92%) as an oil: [a]_D = +2.2° (c = 1.0, CH₂C1₂); FT-rR (CHC1₃ cast) 3396, 3297, 2961 , 1741, 1647, 1505, 1352, 1173, 975 cm^"1 ; Ή NMR (400 MHz, CDC1₃) δ 7.41 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 1.6, 8.0, 1H), 6.93 (bd, / = 9.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.73 (ddd, / = 4.0,11.2, 14.8 Hz, 1H), 5.88 (t, / = 7.6 Hz, 1H), 5.69 (d, J = 10.4 Hz, 1H), 5.50-5.49 (m, 1H), 5.28-5.26 (m, lH), 5.24 (app s, 2H), 4.64 (ddd, / = 5.2, 7.2, 7.2 Hz, 1H), 4.43 (ddd, J = 5.6, 9.2, 9.2 Hz, 1H), 3.87 (s, 3H), 3.69 (d, / = 2.0 Hz, 1H), 3.46 (dd, / = 9.2, 13.2 Hz, 1H), 3.13 (bd, / = 12.4 Hz, 1H), 3.07 (dd, / = 4.8, 14.4 Hz, 1H), 2.97 (s, 3H), 2.89 (dd, J = 7.2, 2.0 Hz, 1H), 2.54 (bd, / = 12.4 Hz, 1H), 2.48-2.36 (m, 1H), 1.80 (ddd, J = 10.8, 17.6, 17.6 Hz, 1H), 1.62 (br s, 1H), 1.58-1.49 (m, 1H), 1.48-1.31 (m, 2H), 1.17 (s, 3H), 1.14-1.12 (m, 6Η), 0.86 (s, 3H), 0.85 (s, 3 H); ¹³C NMR (100 MHz, CDC1₃) δ 177.8, 173.1, 170.5, 164.6, 154.1, 141.6, 138.2, 133.7, 130.8, 129.3, 129.2, 128.2, 126.1, 125.0, 122.5, 112.4, 74.9, 70.8, 63.3, 58.4, 56.1 , 54.8, 50.4, 46.8, 43.1, 41.2, 40.3, 38.3, 36.7, 35.5, 24.9, 24.8, 22.7, 22.5, 21.6, 13.3; MS (m/z) calcd for C₃₈H₅iClN₃O₁₀S (M+H): 776.2984.; found:

776.2975.

[0201] Example 29

(131) (134)

[0202] Compound 134: Hunig's base (0.23 mL, 1.29 mmol) was added to a solution of Compound 119 Please provide explanation (46.5 mg, 0.211 mmol) in DMF (4 mL) at 0°C followed by Compound 131 (100 mg, 0.129 mmol) in THF (4 mL). The mixture was allowed to gradually warm to 23 °C over 2 hours and after 18 hours uPLC-MS showed the reaction was complete. The mixture was diluted with CH₂C1₂ (30 mL) and quenched with saturated aqueous NaHC0₃ (5 mL). The mixture was transferred to a seperatory funnel and the aqueous layer was extracted with CH₂C1₂ (1 x 20 mL). The combined organic layers were washed with H₂0 (1 x 5 mL) and brine (1 x 5 mL) and the organic layer was dried with Na₂S0₄, filtered and concentrated in vacuo. Flash chromatography of the residue on silica gel using 30% to 80% EtOAc-heptane and then 2% to 20% MeOH-CH₂Cl₂ gave Compound 134 (105 mg, 91%) as an oil: [<x]_D = +14.6° (c = 0.1, CH₂C1₂); FT-IR (CH₂C1₂ cast) 3301, 2956, 1742, 1651, 1504, 1258 cm^"1; Ή NMR (400 MHz, CDC1₃) δ 7.29 (d, 7 = 8.4 Hz, 2H), 7.16-7.14 (m, 3H), 7.02 (dd, 7 = 1.6, 8.0 Hz, 1H), 6.95 (bd, 7 = 9.2 Hz, 1H), 6.83 (d, / = 8.4 Hz, 1H), 6.72 (ddd, 7 = 3.6, 10.8, 14.8 Hz, 1H), 5.89 (d, / = 8.8 Hz, 1H), 5.70 (dd, J = 1.2, 16 Hz, 1H), 5.64-5.59 (m, 1H), 5.27-5.23 (m, 1H), 4.63 (dt, 7 = 7.6, 7.6 Hz, 1H), 4.42 (ddd, 7 = 4.8, 8.8, 8.8 Hz, 1H), 3.86 (s, 3H), 3.64 (s, 1H), 3.49 (s, 2H), 3.45 (dd, / = 12.4, 16.0 Hz, 1H), 3.14-3.05 (m, 2H), 2.98-2.96 (m, 3H), 2.54-2.37 (m, 10H), 2.38 (s, 3H), 1.86 (ddd, 7 = 6.8, 14.0, 14.0 Hz, 1H), 1.76 (dt, 7 = 6.8, 6.8 Hz, 1H), 1.67 (ddd, 7 =8.1 , 8.1 , 16.1 Hz, 1H), 1.56-1.50 (m, 1H), 1.50-1.27 (m, 2H), 1.33 (d, 7 = 6.8 Hz, 3H), 1.15 (s, 3H), 1.23-1.02 (m, 3H), 1.11 (s, 3H), 0.84-0.82 (m, 6H); ^{l 3}C NMR (100 MHz, CDC1₃) δ 177.8, 173.1, 170.4, 164.6, 154.2, 141.8, 138.8, 135.4, 130.7, 129.5, 129.2, 128.1 , 125.6, 124.9, 122.6, 112.5, 75.0, 63.0, 62.6, 58.9, 56.1, 56.0, 54.7, 53.2, 53.0, 50.4, 46.7, 44.1, 43.1, 41.2, 40.5, 36.7, 35.6,33.1, 24.9, 24.8, 24.4, 22.6, 22.6, 21.5, 21.0, 13.4; MS (m/z) calcd for C₄₆H₆₇C1N₅0₇S₂ (M+H): 900.4170 found 900.4171.

[0203] Example 30

[0204] The reaction scheme for Example 30 is shown in FIG. 10.

[0205] Compound 1: Ammonium carbonate (3.2 mL, 200 mM in water) was added to a 0°C THF (5 mL) solution of Compound 134 (40 mg, 0.044 mmol). This was followed by the addition of 3,3',3"-phosphinetriyltripropanoic acid hydrochloride (56.4 mg, 0.197 mmol) in water (1.4 mL). The solution was stirred for 30 min at 0°C, allowed to warm up to room temperature and stirred for additional 0.5 h. CH₂C1₂ (25 mL) was added to dilute the reaction mixture and then it was quenched with saturated NaHC0₃ (6 mL). The organic layer was seperated and aqueous layer was extracted with CH₂C1₂ ( 1 x 5 mL). The combined organic layers were washed with saturated NaHC0₃ (4 x 6 mL), brine (1 5 mL), dried over Na₂S0₄ and concentrated in vacuo to yield the desired product (39 mg, 97%) as a white solid which was used as is for the coupling with chlorotoxin (SEQ ID NO: l)-Lys₂₇-CH₂-SS-Py (Compound 130). Compound 130 (47 mg, 11 μπιοΐ) was dissolved in acetate buffer (pH 5.5 buffer, 2.0 mL), cooled to 0°C and crude thiol (9.7 mg, 11 μηιοΐ) in DMF (1.7 mL) was added and the mixture kept at 0°C. All the starting material was consumed after one hour. The reaction mixture was purified on HPLC (C-18 column, gradient 20-45% MeCN and water with 0.1 % formic acid, run time 10 min) the collected material was lyophilized to give Compound 1 (25 mg, 45%) as a white powder: MS (m/z) calcd for C₂nH₃i₇ClN₅80₅₅Si₃ (M+H): 4997.9922, found 4997.9790. This process may also be used for coupling of chlorotoxin analog (SEQ ID NO: 2)-Lys₂₇-CH₂-SS-Py with cryptophycin amide.

[0206] Example 31

(131) (133)

[0207] Compound 133: Hunig's base (0.27 mL, 1.55 mmol) was added to a 0°C solution of Compound 126 (68.2 mg, 0.247 mmol) in DMF (6 mL) at 0°C. This was followed by the addition of Compound 131 (120 mg, 0.155 mmol) in THF (6 mL). The mixture was warmed to 23 °C over 1 h and after 14 hours uPLC-MS showed the reaction was complete. The mixture was diluted with CH₂C1₂ (20 mL) and quenched with saturated aqueous NaHC0₃ (10 mL). The mixture was transferred to a seperatory funnel and the aqueous layer was extracted with CH₂C1₂ (2 x 30 mL). The combined organic layers were washed with brine (1 x 5 mL) and the organic layer was dried over Na₂S0₄, filtered and concentrated in vacuo.

Chromatography of the residue on silica gel using 30% to 90% EtO Ac-heptane and then 2% to 10% MeOH-CH₂Cl₂ gave Compound 133 (138 mg, 98%) as an oil: [a]_D = +7.1 (c = 0.1, CH₂C1₂); FT-I (CH₂C1₂ cast) 3491, 3301, 2958, 2870, 1741, 1651, 1504, 1258, 1180 cm^"1 ; 1H NMR (400 MHz, CDC1₃) δ 7.29 (app d, 7 = 8.4 Hz, 2H), 7.15 (d, 7 = 4.8 Hz, ArH), 7.01 (dd, 7 = 2.0, 8.4 Hz, 1 x ArH), 6.95 (dd, / = 2.4, 9.2 Hz, 1 x ArH), 6.82 (d, 7 = 8.0 Hz, 1H), 6.71 (ddd, 7 = 4.0, 10.8, 14.8 Hz, 1 x ArH), 5.87 (d, 7 = 9.2 Hz, 1H), 5.70 (dd, 7 = 1.6, 15.2 Hz, 1H), 5.56 (br s, 1H), 5.28-5.20 (m, 1H), 4.64 (ddd, J = 5.1, 7.8, 7.8 Hz, 1H), 4.43 (ddd, / = 5.5, 9.7 Hz, 1H), 3.87 (s, 3H, OCHj), 3.65 (d, 7 = 2.0 Hz, 1H), 3.49 (s, 1H), 3.46 (dd, / = 4.0, 13.2 Hz, 1H), 3.13 (dd, 7 = 2.0, 12.8 Hz, 1H), 3.08 (dd, 7 = 4.8, 19.6, Hz, 1H), 2.95 (dd, 7 = 8.0, 22.4, Hz, 1H), 2.90 (dd, 7 = 2.0, 8.0 Hz, 1H), 2.59-2.34 (br m, 10H), 1.86-1.72 (m, 2H), 1.58-1.46 (m, 1H), 1.45-1.31 (m, 2H), 1.15 (s, CH₃), 1.12 (s, CHj), 1.11 (s, CHj), 0.84 (d, 7 = 6.3 Hz, CHj), 0.82 (d, 7 = 6.3 Hz, CH₃); ¹³C NMR (100 MHz, CDC1₃ δ 177.8, 173.1 , 170.4, 164.6, 154.2, 141.7, 135.4, 130.8, 129.5, 129.2, 128.1, 125.6, 124.9, 122.6, 112, 5, 75.0, 63.0, 62.5, 58.9, 56.1, 54.8, 54.3, 53.3, 50.4, 50.2, 46.7, 43.1, 41.2, 40.5, 36.7, 35.6, 27.9, 27.9, 25.2, 24.9, 24.8, 22.6, 22.6, 21.6, 13.4; MS (m/z) calcd for C₄7H₆₈C1N₅0₇S₂ (M+H): 914.43; found: 914.21.

[0208] Example 32

[0209] The reaction scheme for Example 32 is shown in FIG. 11.

[0210] Compound 133 (40 mg, .044 mmol) was dissolved in THF (5 mL), cooled to 0°C, ammonium carbonate (3.2 mL, 200 mM in water) was added, followed by 3,3',3"- phosphinetriyl tripropanoic acid hydrochloride (56.4 mg, 0.197 mmol) in water (1.4 mL). The solution was stirred for 0.5 h at 0°C , allowed to warm up to room temperature and stirred for an additional 0.5 h. CH₂C1₂ (25 mL) was added to the reaction mixture and it was then quenched with saturated NaHC0₃ (6 mL). The organic layer was separated and aqueous layer was extracted with CH₂C1₂ (2 x 10 mL). The combined organic layers were washed with saturated NaHC0₃ (4 x 6 mL), brine (1 x 10 mL), dried over Na₂S0₄ and concentrated in vacuo to yield the desired product (39 mg, 97%) as a white solid which was used for the coupling with chlorotoxin(SEQ ID NO: 1)-S-S-Py without any further purification.

[0211] Chlorotoxin (SEQ ID NO: l)-Lys₂₇-CH₂-SS-Py (97 mg, 0.023 mmol) was dissolved in acetate buffer (pH 5.5 buffer, 4 mL), cooled to 0°C and crude thiol Compound 136

(35,10R,165,E)-10-(3-chloro-4-methoxybenzyl)-3-isobutyl-16-((5)-l-((2R, 3R)-3-(4-((4-((5)- 3-mercaptobutyl)piperazin-l-yl)methyl)phenyl)oxiran-2-yl)ethyl)-6,6-dimethyl-l-oxa-4,8,l l- triazacyclohexadec-13-ene-2,5,9,12-tetraone (19.4 mg, 23.0 μπιοΐ) in DMF (3.4 mL, 34.0 μπιοΐ) was added and the mixture kept at 0°C. All the starting material was consumed after one hour. The reaction mixture was purified on HPLC (C-18 column, gradient 20-45% MeCN and water with 0.1% formic acid, run time 10 min) the collected material was lyophilized to give Compound 2 (44.2 mg, 39%) as a white powder: MS (m/z) calcd for C₂i₂H₃₁₉ClN₅₈0₅₅S₁₃ (M+H): 5010.0078 found 5009.9971

[0212] Example 33 - Biological Activity

[0213] This example reports antitumor activity of Compounds 2 and 4 in a subcutaneous human prostate cancer PC-3 xenograft model.

Protocol: • PC-3 human prostate cancer cells (ATCC CRL- 1435) were grown in RPMI- 1640 medium supplemented with 10% FBS.

• For inoculation, 2 x 10⁶ PC-3 cancer cells were injected subcutaneously into mice near the right axillary area using a 26-gauge needle in a volume of 0.1 mL. Mice were immune-compromised NU/NU females, approximately 6 weeks old from Charles River Labs.

• Tumors were measured at least twice weekly using calipers and mice were

randomized into treatment groups based on tumor size when the average tumor reached approximately 180 mm³.

• All treatments were initiated 13 days following tumor implantation.

• The experiment consisted of vehicle, Compounds 2 and 4-treated groups (n=5).

• All drugs were administered in the following vehicle: 10% EtOH, 5% Tween80 and 85% saline.

• Animals were treated intravenously on a Q4Dx3 schedule. Tumor size and body weight were measured twice per week.

Results and Conclusion:

• Results are shown in FIG. 4. Both Compound 2 and 4 treatments resulted in dose- responsive anticancer activity: tumor regression was observed at 1.2 mg/kg, whereas inhibition of tumor growth occurred at the 0.6 mg kg dose.

• Tumor-free mice were observed: 1, 1, 4 and 1 in Compound 2 at 1.2 and 0.6 and Compound 4 at 1.2 and 0.6 mg/kg treatment groups respectively on day 38 of study.

• No significant body weight loss or other signs of toxicity were observed in any study animals.

[0214] Example 34 - Biological Activity

[0215] This example reports antitumor activity of Compounds 1, 2 and 4 in a subcutaneous human pancreatic cancer MIA-PaCa2 xenograft model.

Protocol:

• MIA PaCa-2 human pancreatic cancer cells (ATCC CRL-1420) were grown in RPMI- 1640 medium supplemented with 10% FBS. • For inoculation, 5 x 10⁶ MIA PaCa-2 cancer cells were injected subcutaneous ly into mice near the right axillary area using a 26-gauge needle in a volume of 0.1 mL. Mice were immune-compromised NU/NU females, approximately 6 weeks old from Charles River Labs.

• Tumors were measured at least twice weekly using calipers and mice were

randomized into treatment groups based on tumor size.

• All treatments were initiated 14 days following tumor implantation when the average tumor size was approximately 400 mm³.

• The experiment consisted of a vehicle-treated group and drug-treated groups (n=6).

• Animals were treated intravenously on a Q4Dx3 schedule. Tumor size and body weight were measured twice a week.

Results and Conclusion:

• Results are shown in FIG. 5. Treatment with Compounds 2, 4 and 1 (2.5, 2.5 and 1.5 mg kg respectively) resulted in tumor regression and led to durable cures of tumors in all mice. Tumors did not re-grow during the monitoring period; >3 weeks post-last treatment.

• Dose responsive antitumor activity was observed for Compound 2 with tumor regression and tumor cures at 2.5 mg/kg versus minimal tumor growth inhibition at 0.6 mg/kg dose.

[0216] Example 35 - Biological Activity

[0217] This example shows antitumor activity of Compound 1 in an orthotopic human pancreatic cancer MIA-PaCa2 xenograft model.

Table 1

Vehicle +* +* +* +* + + + + -

Compound 1 f.d. f.d. f.d. - - - - - - +

+ Indicates presence of tumor

- Indicates absence of tumor

*Mice with clinical symptoms euthanized on Day 43

# Mice with clinical symptoms euthanized on Day 78

f.d. Mice found dead on Day 16, following 3^rd dose: acute event, likely drug-delivery related Protocol:

• MIA PaCa-2 human pancreatic cancer cells (ATCC CRL-1420) were grown in RPMI- 1640 medium supplemented with 10% FBS.

• For inoculation, 5 x 10⁶ MIA PaCa-2 cancer cells were directly injected into the mouse pancreas. Mice were immune-compromised NU/NU females, approximately 6 weeks old from Charles River Labs.

• All treatments were initiated 8 days after tumor orthotopic implantation.

• The experiment consisted of a vehicle-treated group and Compound 1 -treated group (n=10).

• Animals were treated intravenously on a Q4Dx3 schedule.

• Mice were monitored for general health daily and those with clinical symptoms of disease were euthanized and examined for presence of tumors.

• At 90 days post-orthotopic implantation the study was terminated and all remaining animals were euthanized for necropsy.

Results and Conclusion:

• Results are shown in Table 1. Five of the 10 vehicle-treated mice required euthanasia due to clinical symptoms of disease prior to study termination on Day 90. All 5 mice had ascites and large tumors. • Tumors were observed in 9 of 10 vehicle-treated mice. Tumors harvested from mice euthanized on Day 90 were weighed and ranged in size from 1.9 to 3.6 g.

• On Day 90, 6 of 7 Compound 1 -treated mice were tumor-free. These mice bore no clinical symptoms of disease. One mouse did have a tumor which weighed l.lg.

• Treatment with Compound 1 significantly reduced tumor growth in this model of orthotopic pancreatic cancer. In addition, the majority of treated mice appeared tumor free upon study termination.

[0218] Example 36 - Biological Activity

[0219] This example shows antitumor activity of Compound 2 in a subcutaneous human breast cancer MDA-MB-231 xenograft model.

Protocol:

• MDA-MB-231 human breast cancer cells (ATCC HTB-26) were grown in RPMI- 1640 medium supplemented with 10% FBS.

• For inoculation, 5 x 10⁶ MDA-MB-231 cells were mixed 1: 1 (v/v) with matrigel (BD Biosciences) and injected subcutaneously into mice near the right axillary area using a 26- gauge needle in a volume of 0.1 mL. Mice were immune-compromised NU/NU females, approximately 6 weeks old from Charles River Labs.

• Tumors were measured at least twice weekly using calipers and mice were randomized into treatment groups based on tumor size.

• All treatments were initiated 15 days following tumor implantation when the average tumor size was approximately 130 mm³.

• The experiment consisted of a vehicle-treated group and drug- treated groups (n=5).

Results and Conclusion: • Results are shown in FIG. 6. Compound 2 dosed at 2.3 mg/kg resulted in tumor regression and led to 4 of 5 tumor-free mice at study termination.

• Inhibition of tumor growth was also observed at lower doses of Compound 2, although no significant difference in antitumor activity was observed between the 1.2 and 0.6 mg kg doses.

• No significant body weight loss or other signs of toxicity was observed in Compound 2-treated animals.

[0220] Example 37 - Biological Activity

[0221] This example shows antitumor activity of Compound 2 in a subcutaneous primary human pancreatic cancer (PDx) xenograft model.

Protocol:

• This study was performed at Molecular Response.

• Primary human pancreatic cancer cells (2008120310 p2) were thawed and prepared for inoculation into mice: 95,950 viable cells per 100 μL· in cold PBS.

• For inoculation, cells were mixed 1: 1 (v/v) with cultrex ECM and injected subcutaneously into mice in the rear flank using a 26-gauge needle in a volume of 0.2 mL.

• Mice were immune-compromised NOD-SCID females from Harlan Labs.

• Tumors were measured 3 times per week using calipers and mice were randomized into treatment groups based on tumor size.

• All treatments were initiated at 41 days following tumor implantation (Day 1 on study), when the average tumor size was approximately 178-190 mm .

• The experiment consisted of a vehicle-treated group and drug-treated groups (n=10).

• Animals were treated intravenously on a Q4Dx3 schedule and were administered a second Q4Dx3 treatment cycle approximately 3 weeks later.

• Tumor size and body weight were measured twice a week.

Results and Conclusion: • Results are shown in FIG. 7. An anticancer effect was determined for Compound 2 in the pancreatic patient-derived xenograft model. The effect was dose responsive with a much greater effect at the 2.3 mg kg dose.

• Administration of a second treatment cycle slowed re- growth of tumors.

[0222] Example 38 - Biological Activity

[0223] This example shows antitumor activity of Compound 2 in a subcutaneous human glioblastoma U-87 MG xenograft model.

Protocol:

• U-87 MG human glioblastoma cells (ATCC HTB - 14) were grown in DMEM medium supplemented with 10% FBS.

• For inoculation, 5 x 10⁶ U-87 MG cells in IX HBSS were mixed 1 : 1 (v/v) with matrigel (BD Biosciences) and injected subcutaneously into mice near the right axillary area using a 26-gauge needle in a volume of 0.1 mL. Mice were immune-compromised Ncr nude females, approximately 6 weeks old from Taconic.

• All treatments were initiated 21 days following tumor implantation when the average tumor size was approximately 280 mm .

• The experiment consisted of a vehicle-treated group and Compound 2-treated groups (n=6).

• Compound 2 was administered in vehicle: 10% EtOH, 5% Tween80 and 85% saline.

Results and Conclusion:

• Results are shown in FIG. 8. Compound 2 treatments resulted in dose-responsive anticancer activity: tumor regression was observed at 2.3 mg/kg whereas inhibition of tumor growth occurred at the 0.6 and 1.2 mg/kg doses. • Tumor-free mice were observed: 3, 0 and 1 of 6 animals per group in the 2.3, 1.2 and 0.6 mg/kg Compound 2-treatment groups respectively on the final day of study.

[0224] Example 39 - Biological Activity

[0225] This example shows antitumor activity of compounds 1, 2, 3 and 4 in a subcutaneous human colon cancer COLO 320DM xenograft model.

Protocol:

• COLO 320DM human colon cancer cells (ATCC CCL-220) were grown in RPMI medium supplemented with 10% FBS.

• For inoculation, 5 x 10⁶ COLO 320DM cells were injected subcutaneously into mice near the right axillary area using a 26-gauge needle in a volume of 0.1 mL. Mice were immune-compromised NOD.SCID females, approximately 6 weeks old from Charles River Labs.

• All treatments were initiated 7 days following tumor implantation when the average tumor size was approximately 135 mm .

Results and Conclusion: • Antitumor activity was observed for Compounds 1, 3 and 4 dosed at 1.5, 1.5 and 2.3 mg/kg respectively; a comparable level of tumor growth inhibition was observed for all 3 compounds.

• P-glycoprotein expression is high in COLO 320 DM cells which likely accounts for the reduced compound potency observed in this model versus other xenografts.

• Modest, yet statistically significant tumor growth inhibition was observed for Compound 2 dosed at 1.2 mg/kg, (one-way ANOVA followed by Tukey's multiple comparisons test on Day 18).

[0226] No significant body weight loss or other signs of toxicity were observed in any study animals, however 1 animal was found dead on Day 15 and 1 on Day 17 in groups treated with Compound 1 and 3 respectively. Since nude mice were generally tolerant of the compounds at these doses, reduced tolerability may result from an inherent increased sensitivity of the immune-compromised NOD.SCID strain.

[0227] Example 40

[0228] One of skill in the art will also recognize that Compound 110 is also able to be prepared by the reactions presented in this prophetic example. Each of the reactions presented in this example may occur in either acidic or basic conditions. Any of a number of catalysts may be used. These include but are not limited to DCC (Ν,Ν'- Dicyclohexylcarbodiimide), EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide), HBTU (2-(lH-benzotriazol- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate), HOBT

(hydroxybenzotriazole), DPPF (l,l'-Bis(diphenylphosphino)feri cene), and N- hydroxysuccinimide (NHS) esters.

[0229] In one alternative, Compound 137, 5-((2^)-2-((55,65,E)-5-(((^)-2-amino-4- methylpentanoyl)oxy)-6-((^5)-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl- l,3-dioxolan-4-yl)hept-2-enamido)-3-(3-chloro-4-methoxyphenyl)propanamido)-2,2- dimethylpropanoic acid, is subjected to lactamization under acceptable conditions to prepare Compound 110.

[0230] In a further embodiment, Compound 138, (, )-2-(3-((R)-3-(3-chloiO-4- methoxyphenyl)-2-((5^,65,E)-5-hydroxy-6-((^R,5R)-5-(4-(((4- methoxybenzyl)oxy)methyl)phenyl)-2,2-dimethyl- l ,3-dioxolan-4-yl)hept-2- enamido)propanamido)-2,2-dimethylpropanamido)-4-methylpentanoic acid, is subjected to lactonization under acceptable conditions to prepare Compound 110.

[0231] In a still further embodiment, Compound 139, (55,55,.5)-5-(((5)-2-(3-(( e)-2-amino-3- (3-chloro-4-methoxyphenyl)propanamido)-2,2-dimethylpropanamido)-4- methylpentanoyl)oxy)-6-((4R,5R)-5-(4-(((4-methoxybenzyl)oxy)methyl)phenyl)-2,2- dimethyl-l,3-dioxolan-4-yl)hept-2-enoic acid, is subjected to lactamization under acceptable conditions to prepare Compound 110.

Claims

We claim:

1. A protein-drug conjugate of Formula I:

Ctx-L-Cp (I) or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ctx is chlorotoxin or a chlorotoxin analog;

Cp is a cryptophycin amide having the following formula:

and L is a linker having the following formula:

wherein the linker is bound to Ctx at the X₂ moiety of the linker and at one of a lysine group and the N-terminus of the chlorotoxin or chlorotoxin analog; q is an integer from 0-8 and m is an integer from 0-8; Xi is selected from the group consisting of -NH-, -N(CH₃) -, -CH₂ -, -(OR₄NX₃) and -(O- CH₂CH₂-)_P, wherein when Xi is -(0-CH₂CH₂-)p, m is 1 and p is an integer from 1-8, and wherein R₄ is Ci-C₆ alkyl and X₃ is selected from the group consisting of Ci-C₆ alkyl and aryl;

X₂ is selected from the group consisting of a bond, Ci-C₆ alkyl, and -(OCH₂CH₂-)_r, wherein r is an integer from 1-8;

Ri and R₂ are selected from the group consisting of -H, lower alkyl, lower alkyl and - CH₂CH₂-(OCH₂CH₂)_n-0-R5, wherein n is an integer from 1-6 and R₅ is lower alkyl, or Ri and R₂ together may form a ring selected from the group consisting of a 3 to 6-membered alkyl ring with optional N-Methyl substituent, morpholinyl, and furanyl, wherein Ri and R₂ may not simultaneously be -H; and

R₃ is at least one substituent independently selected from the group consisting of -H, lower alkyl, aryl, heteroaryl, hydroxy-(lower alkyl), amino-(lower alkyl), N-(lower aikyl)amino- (lower alkyl), N,N-di(lower alkyl)amino-(lower alkyl), N-arylamino-(lower alkyl), N,N- diarylamino-(lower alkyl), N-(heteroaryl)amino-(lower alkyl), N,N-di(heteroaryl)amino- (lower alkyl), hydroxylamino, 0-(lower alkoxy)amino, O-aryloxyamino, O- heteroaryloxyamino, fluoro, chloro, bromo, nitro, hydroxyl, lower alkoxy, aryloxy, hydroxycarbonyl, lower alkanoyl, lower alkanoyloxy, amino, N-(lower alkyl)amino, N,N- di(lower alkyl)amino, formylamino, N-acylamino, NN-diacylamino, hydrazido, N-(lower alkyl)hydrazido, N,N-di(lower alkyl)hydrazido, N-arylhydrazido, NN-diarylhydrazido, N- (heteroaryl)hydrozido, N,N-di(heteroaryl)hydrazido, carbamoyl, N-(lower alkyl)carbamoyl, NN-(lower alkyl)carbamoyl, N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl, NN-di(heteroaryl)carbamoyl, hydroxysulfonyl, (lower alkoxy)sulfonyl, aryoxysulfonyl, heteroaryloxysulfonyl, hydroxysulfonyl- (lower alkyl), (lower alkoxy)sulfonyl-(lower alkyl), aryoxysulfonyl-(lower alkyl), heteroaryloxysulfonyl-(lower alkyl), (lower alkyl)sulfonyl, arenesulfonyl, and heteroarenesulfonyl; wherein R5 is selected from the group consisting of -H and -CH₃; and wherein the disulfide bond is ortho, meta, or para to the substituent at a.

2. The protein-drug conjugate of claim 1, wherein Ctx consists of chlorotoxin having the amino acid sequence of SEQ ID NO: 1.

3. The protein-drug conjugate of claim 1, wherein Ctx consists of a chlorotoxin analog having the amino acid sequence of SEQ ID NO: 2.

4. The protein-dmg conjugate of claim 1, wherein Ri is selected from the group consisting of -H and -CH₃, wherein R₂ is selected from the group consisting of -H and -CH₃, and wherein R_\ and R₂ may not both be -H.

5. The protein-drug conjugate of claim 4, wherein Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1 ; q is 1 ; m is 1 ; X_\ is -CH₂-; X₂ is a bond; wherein one of Ri and R₂ is -CH₃ and the other is -H; Ri is -CH₃; R₂ is H; R₃ is H; the linker is bound to chlorotoxin at an L₂₇ lysine residue and the disulfide bond of the linker is ortho to the substituent at position a.

6. The protein-drug conjugate of claim 4, wherein Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 1 ; q is 1; m is 1 ; Xi is -CH₂-; X₂ is a bond; Ri is -CH₃; R₂ is -CH₃; R₃ is H; the linker is bound to chlorotoxin at an L₂₇ lysine residue; and the disulfide bond of the linker is ortho to the substituent at position a.

7. The protein-drug conjugate of claim 4, wherein Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 2; q is 1 ; m is 1 ; Xi is -CH₂-; X₂ is a bond; wherein one of Ri and R₂ is -CH₃ and the other is -H; R_\ is -CH₃; R₂ is H; R₃ is H; the linker is bound to chlorotoxin at an L₂₇ lysine residue and the disulfide bond of the linker is ortho to the substituent at position a.

8. The protein-drug conjugate of claim 4, wherein Ctx is a chlorotoxin having the amino acid sequence of SEQ ID NO: 2; q is 1 ; m is 1 ; Xi is -CH₂-; X₂ is a bond; Ri is -CH₃; R₂ is -CH₃; R₃ is H; the linker is bound to chlorotoxin at an L₂₇ lysine residue; and the disulfide bond of the linker is ortho to the substituent at position a.

9. A method for treating cancer in a subject, comprising administering to the subject the protein-drug conjugate of claim 1 in an amount sufficient to treat the subject for cancer.

10. The method of claim 9, wherein the subject is a human.

11. The method of claim 9, wherein the cancer is pancreatic cancer.

12. The method of claim 9, wherein the cancer is prostate cancer.

13. The method of claim 9, wherein the cancer is breast cancer.

14. The method of claim 9, wherein the cancer is glioblastoma.

15. The method of claim 9, wherein the cancer is colon cancer.