WO2015057585A1 - Therapeutic compounds - Google Patents

Abstract

Compounds of formula (I): or salts thereof are disclosed. Also disclosed are pharmaceutical compositions comprising a compound of formula (I), processes for preparing compounds of formula (I), intermediates useful for preparing compounds of formula (I) and therapeutic methods for treating a cancer.

Description

THERAPEUTIC COMPOUNDS

Cross Reference to Related Applications

This application claims the benefit of and priority to U.S. Provisional Application Serial Nos. 61/890,789, filed October 14, 2013, and 61/890,754, filed October 14, 2013, which are incorporated by reference in their entirety.

Background

Tubulysins are naturally occurring antimitotic tetrapeptides with potent anticancer activity against multidrug-resistant (MDR) cancer cells, acting by inhibition of tubulin polymerization. Their first report was in 2000, when they were identified through a screen of the culture broths from myxobacteria Archangium gephyra and Angiococcus disciformis against mammalian cell lines (Sasse, F.; et al., Tubulysins, New Cytostatic Peptides from Myxobacteria Acting on Microtubuli Production, Isolation, Physico-chemical and Biological Properties. J Antibiot. 2000, 53, 879-885.). Tubulysins A (1), B (2), D (4), and E (5) possessed potent antiproliferative activity against L929 mouse fibroblasts, PtK₂ kidney cancer cells, and KB-3.1 cervix cancer cells (IC₅₀ = 20 pg/mL to 1 ng/mL, Sasse et al. 2000). Additionally, all retained their high potency against the MDR cervical cancer cell line KB- VI (IC₅₀ = 80 pg/mL to 1 ng/mL, Sasse et al. 2000). Investigation into the cellular structure of treated cells showed that the tubulysins function through disruption of the microtubule network, where microtubule decay was followed by complete solubilization beginning after 2 and 24 hours, respectively (Sasse et al. 2000).

Tubulysins follow a standard tetrapeptide template with a few key modifications to the core structure (Figure 1). Starting at the N-terminus, the four amino acid residues are N-methyl-D- pipecolinic acid (Mep), L-isoleucine (He), tubuvaline (Tuv), and tubuphenylalanine or tubutyrosine (Tup or Tut). Variations exist at the oxygen and nitrogen of tubuvaline, where substitution of a rare N, O-acetal is present in certain members (1-10), and at the phenyl ring of the C-terminus residue based on either a phenylalanine or tyrosine source for this residue. Full stereochemical

determination was found by analysis of fragmentation following acidic hydrolysis of tubulysin D (4) (Hofle, G.; et al., Semisynthesis and Degradation of the Tubulin Inhibitors Epothilone and Tubulysin. Pure Appl. Chem. 2003, 75, 167-178).

Tubulysins bind to β-tubulin at the peptide site of the Vinca alkaloid domain and exert their anticancer activity through inhibition and destabilization of microtubule polymerization (Khalil, M. W.; et al., Mechanism of Action of Tubulysin, an Antimitotic Peptide from Myxobacteria. ChemBioChem, 2006, 7, 678-683). Microtubules are long, polymeric cylinders made up of a- and β-tubulin heterodimers and are essential to normal cellular function. These functions include maintenance of the cellular structure and transportation of cellular components involved in cell signaling, mitosis and apoptosis. Microtubule cellular tasks are achieved through its frequent lengthening and shortening via polymerization/depolymerization of the heterodimeric tubulin subunits, termed 'dynamic instability' (Jordan, M. A.; et al., Microtubules as a Target for

Anticancer Drugs. Nat. Rev. Cancer 2004, 4, 253-265). Agents that affect the dynamic instability of microtubules have found success as anticancer agents. These include the taxanes and epothilones (inducing and stabilizing tubulin polymerization), and the Vinca alkaloids (disrupting and inhibiting microtubule polymerization).

The anticancer activity of both microtubule stabilizing and destabilizing molecules lies within their ability to suspend mitosis (Jordan et al., 2004). Mitosis is the multistage process of cell division, wherein a parent cell replicates into two genetically identical daughter cells. In contrast to the microtubule dynamics of interphase, the resting phase of the cell cycle, comparatively rapid dynamic instability is necessary for proper attachment and movement of the duplicated

chromosomes through each stage of mitosis (Jordan et al., 2004). Studies have shown that drugs targeting microtubules cause incomplete delivery of chromosomes to the metaphase plate, a process critical to cell division (Jordan and Wilson 2004). Even one chromosome not aligned at the metaphase plate will stop forward mitotic progress, trapping the cell mid-mitosis and eventually causing programmed cell death, or apoptosis (Jordan et al., 2004). These agents' effectiveness against malignant cells can be partially explained by the higher rate of mitosis cancer cells undergo compared to normal cells. An increased incidence of mitosis puts cancer cells in a position of vulnerability with drugs that arrest the mitotic cycle (Jordan et al., 2004).

Biochemical investigations into the tubulysin mode of action have revealed their ability to disrupt microtubule dynamics by depolymerizing or inhibiting the polymerization of the tubulin subunits onto microtubules (Sasse et al., 2000, Khalil et al., 2006). As a result, treated cells are halted mid-mitosis which leads to their eventual shift into apoptosis, as shown by classic markers such as DNA fragmentation and increased caspase-3 activity (Kaur, G.; et al., Biological

Evaluation of Tubulysin A: A Potential Anticancer and Antiangiogenic Natural Product. Biochem. J. 2006, 396, 235-242, Khalil et al., 2006). Additionally, cell angiogenesis was severely hindered upon treatment with tubulysin A, which may prove to be another venue in which these compounds exert their effects. Competitive binding studies have shown non-competitive inhibition of vinblastine with tubulysin A, similarly to the peptide antimitotics dolastatin 10 and phomopsin A (Khalil et al., 2006). This, together with NMR conformation studies showing that tubulysin A and epothilone A share a common tubulin binding site (Kubicek, K.; et al., The Tubulin-Bound

Structure of the Antimitotic Drug Tubulysin. Angew. Chem. Int. Ed, 2010, 49, 4809-4812), suggests that the tubulysins bind at the peptide site of the Vinca domain.

Naturally derived tubulysins have shown potent in vitro growth inhibitory activity against a variety of different cancer cell lines, including breast (Ranganathan et al. 2009), cervix (Sasse et al. 2000, Steinmetz, H.; et al., Isolation, Crystal and Solution Structure Determination, and

Biosynthesis of Tubulysins— Powerful Inhibitors of Tubulin Polymerization from Myxobacteria, Angew. Chem. Int. Ed. 2004, 43, 4888^1892), colon (Kaur et al., 2006), leukemia (Sasse et al. 2000), lung (Ullrich, A.; et al., Synthesis and Biological Evaluation of Pretubulysin and

Derivatives. Eur. J. Org. Chem. 2009b, 6367-6378), melanoma (Kaur et al., 2006), ovarian (Ranganathan et al. 2009) and prostate (Kaur et al., 2006) carcinomas, with IC ₀ values

representing a 20-1000 fold improvement over the epothilones, vinblastine and palitaxel (Steinmetz et al., 2004). A correlation exists between lipophilicity and in vitro activity, where tubulysins A through I follow a predictable tend of increased lipophilicity showing more potent antiproliferative activity (Steinmetz et al., 2004). This trend may be explained by the observation that increased cellular uptake of the more lipophilic tubulysins resulted in higher cellular concentrations in mouse fibroblast cells. The question of whether the mechanism of cellular uptake is through diffusion or active transport is still unknown (Steinmetz et al., 2004).

Preliminary animal studies have shown limited success using low doses of tubulysin A in hollow fiber assays of 12 human cancer cell lines (Kaur et al. 2006), and no success in xenograft mouse models, where both tubulysins A and B show no therapeutic window (Leamon, C. P.; et al., Folate Targeting Enables Durable and Specific Antitumor Responses from a Therapeutically Null Tubulysin B Analogue. Cancer Res. 2008, 68, 9839-9844, Schluep, T.; et al., J. Polymeric

Tubulysin-Peptide Nanoparticles with Potent Antitumor Activity. Clin. Cancer Res. 2009, 15, 181- 189, Reddy, J. A.; et al., In Vivo Structural Activity and Optimization Studies of Folate-Tubulysin Conjugates. Mol. Pharm. 2009, 6, 1518-1525). Targeted delivery of tubulysins by conjugation to cancer-specific substrates, on the other hand, has seen limited success in mouse studies. Over- expression of the folate receptor in cancer cells was taken advantage of by conjugation of tubulysins to folate, where the drug is released for its anticancer action follow cancer cell selective absorption of the complex (Vlahov, I. R.; et al., Design and Regioselective Synthesis of a New Generation of Targeted Chemotherapeutics. Part II: Folic Acid Conjugates of Tubulysins and their Hydrazides. Bioorg. Med. Chem. Lett. 2008, 18, 4558^561, Leamon et al. 2008, Reddy et al. 2009). This same strategy has also been used with cyclodextrin nanoparticle-tubulysin conjugates (Schluep et al. 2009) and prostate-specific antigen-tubulysin prodrugs (Kularatne, S. A.;et al., Synthesis and Biological Analysis of Prostate-Specific Membrane Antigen- Targeted Anticancer Prodrugs. J Med. Chem. 2010, 53, 7767-7777). The folate and nanoparticle bound conjugates have both shown an increased therapeutic window and prolonged survival when compared to unbound tubulysins in mice (Leamon et al. 2008, Reddy et al. 2009, Schluep et al. 2009).

Accordingly, there is a need for anti-cancer agents as well as anti-cancer agents that are effective against drug-resistant cancers. There is also a need for anti-cancer agents that disrupt microtubule dynamics (e.g., by inhibiting the polymerization of tubulin).

Summary of the Invention

Provided herein are compounds and methods for the treatment of cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers).

One embodiment provides a compound of formula I:

I

wherein:

R¹ is (d-Q alkyl;

each R² is independently H or (C_!-C₆)alkyl;

R³ is H or (C C₆)alkyl;

R⁴ is (d-C₆)alkyl;

R⁵ is H or (d-C₆)alkyl;

R⁶ is (C]-C₆)alkyl;

R⁷ is H or (C₁-C₆)alkyl, and R⁸ is H, -C(=0)(d-C₆)alkyl, -C(=0)(C₂- C₆)alkenyl, -C(=0)aryl, -C(=0)OR^a, or -C(=0)NR^bR^c, wherein any -C(=0)(d-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R⁸ is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R⁸ is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH, -0(d-C₃)alkyl, -C0₂H,

and -C(=0)NR^bR^c; or R⁷ and R⁸ together with the nitrogen to which they are attached form a 4-10 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from halogen, oxo, (C₁-C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

when R¹² is NR⁷R⁸ then R⁹ is H or (C₁-C₆)alkyl, and R¹⁰ is (C_rC₆)alkyl wherein the (d-

C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R⁹ and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo;

when R¹² is OR¹³ then R¹³ is R^13a, R⁹ is H or (d-C₆)alkyl, and R¹⁰ is (d-C₆)alkyl wherein the (C_!-C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R¹³ is R^13b, and R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (d- C₆)alkyl and oxo;

R¹¹ is aryl, wherein any aryl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH or -0(C₁-C₃)alkyl;

R¹² is NR⁷R⁸ or OR¹³;

R^13a is -(d-C₆)alkylO(d-C₆)alkyl, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂- C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(d-C6)alkylO(d- C₆)alkyl, -C(=0)(C₂-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R^13b is H, -(d-C^alkylOCd-Ce^lkyl, -C(=0)(d-C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl wherein any -(d-C₆)alkylO(d-C₆)alkyl, -C(=0)(C C₆)alkyl or -C(=0)(C₂- C₆)alkenyl of R^13a is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (d- C₃)alkyl, -OH and -0(C C₃)alkyl;

R^a is (d-C₆)alkyl;

R^b and R^c are each independently selected from H and (C₁-C₆)alkyl; or R^b and R^c together with the nitrogen to which they are attached form a 4, 5, 6 or 7 heterocycle wherein the heterocycle is optionally substituted with one or more groups selected from (C₁-C₆)alkyl and oxo; and

n is 1, 2, 3 or 4;

or a salt (e.g. pharmaceutically acceptable salt) thereof. One embodiment provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

One embodiment provides a method for treating cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers) in a mammal (e.g., a human), comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, to the mammal.

One embodiment provides a method for inhibiting cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers) cell growth comprising contacting the cancer cell in vitro or in vivo with a compound of formula I, or a pharmaceutically acceptable salt thereof.

One embodiment provides a method for inhibiting cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers) cell growth comprising contacting the cancer cell in vitro or in vivo with an amount of a compound of formula I, or a pharmaceutically acceptable salt thereof to provide an cancer inhibiting effect in the cell.

One embodiment provides a method for inhibiting tubulin polymerization in a mammal (e.g., a human), comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, to the mammal.

One embodiment provides a method for inhibiting tubulin polymerization in a mammal (e.g., a human) in need of such treatment, comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, to the mammal to provide a inhibition of tubulin polymerization effect.

One embodiment provides a method for inhibiting angiogenesis in a mammal (e.g., a human), comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, to the mammal.

One embodiment provides a method for inhibiting angiogenesis in a mammal (e.g., a human) in need of such treatment, comprising administering a compound of formula I or a pharmaceutically acceptable salt thereof, to the mammal to provide an angiogenesis inhibition effect. One embodiment provides a method for inhibiting tubulin polymerization in a cell (e.g., a cancer cell (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers), comprising contacting the cell in vitro or in vivo, with a compound of formula I or a salt (e.g., a pharmaceutically acceptable salt) thereof.

One embodiment provides a method for inhibiting tubulin polymerization in a cell (e.g., a cancer cell (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers), comprising contacting the cell in vitro or in vivo, with a compound of formula I or a salt (e.g., a pharmaceutically acceptable salt) thereof effective to inhibit tubulin polymerization in the cell.

One embodiment provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in medical therapy.

One embodiment provides a compound of formula I or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers).

One embodiment provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof, to prepare a medicament for treating cancer (e.g., kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer and prostate cancer as well as drug resistant forms (e.g., MDR) of these cancers) in an mammal (e.g., a human).

One embodiment provides processes and intermediates disclosed herein that are useful for preparing a compound of formula I or a salt thereof.

Brief Description of the Figures

Figure 1 shows the structures of naturally occurring tubulysins.

Detailed Description

The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.

The term "aryl" as used herein refers to a single all carbon aromatic ring or a multiple condensed all carbon ring system wherein at least one of the rings is aromatic. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Aryl includes a phenyl radical. Aryl also includes multiple condensed ring systems (e.g., ring systems comprising 2 or 3 rings) having about 9 to 20 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic (i.e., carbocycle). Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2 or 3) oxo groups on any carbocycle portion of the multiple condensed ring system. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the point of attachment of a multiple condensed ring system, as defined above, can be at any position of the ring system including an aromatic or a carbocycle portion of the ring. Typical aryl groups include, but are not limited to phenyl, indenyl, naphthyl, 1, 2, 3, 4-tetrahydronaphthyl, anthracenyl, and the like.

The term "heterocyclyl" or "heterocycle" as used herein refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms. It is to be understood that the point of attachment for a heterocycle or heterocycle can be at any suitable atom of the heterocycle including a carbon atom and a heteroatom (e.g., a nitrogen). The term

heterocycle also includes a heterocycle as defined above that is fused to a phenyl to form a fused bi cyclic heterocycle. In one embodiment the heterocycle is a 4-10 membered monocyclic or bicyclic heterocycle from about 1-9 carbon atoms and from about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. Exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl,

homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, imidazolidin-2-one and pyrrolidin-2-one.

The compounds disclosed herein can also exist as tautomeric isomers in certain cases.

Although only one delocalized resonance structure may be depicted, all such forms are

contemplated within the scope of the invention.

It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium ( H or D). As a non-limiting example, a - CH₃ group may be substituted with -CD₃.

It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

When a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities. When a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted. In one embodiment, the compound may be at least 51% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95 the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted.

Specific values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents

Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl or hexyl and (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1- butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2- hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl.

A specific compound of formula I is a compound of formula la:

la

wherein:

R¹ is (C,-C₆)alkyl;

each R² is independently H or (C₁-C )alkyl;

R³ is H or (d-C₆)alkyl;

R⁴ is (C₁-C₆)alkyl;

R⁵ is H or (d-C₆)alkyl;

R⁶ is (C_!-C₆)alkyl;

R⁷ is H or (C_rC₆)alkyl, and R⁸ is H, -C(=0)(C₁-C₆)alkyl, -C(=0)(C₂-

C₆)alkenyl, -C(=0)aryl, -C(=0)OR^a, or -C(=0)NR^bR^c, wherein any -C(=0)(C₁-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R⁸ is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R⁸ is optionally substituted with one or more groups independently selected from halogen, (d-C3)alkyl, -OH, -0(C₁-C3)alkyl, -C0₂H,

and -C(=0)NR^bR^c; or R⁷ and R⁸ together with the nitrogen to which they are attached form a 4-10 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from halogen, oxo, (C₁-C3)alkyl, -OH and -0(C₁-C3)alkyl;

R⁹ is H or (d-C₆)alkyl, and R¹⁰is (d-C₆)alkyl wherein the (C C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R⁹ and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (d-C6)alkyl and oxo;

R¹¹ is aryl, wherein any aryl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (C₁-C3)alkyl, -OH or -0(d-C3)alkyl;

R^a is (d-C₆)alkyl;

R^b and R^c are each independently selected from H and (C]-C6)alkyl; or R^b and R^c together with the nitrogen to which they are attached form a 4, 5, 6 or 7 heterocycle wherein the heterocycle is optionally substituted with one or more groups selected from (d-C₆)alkyl and oxo; and

n is 1 , 2, 3 or 4;

or a pharmaceutically acceptable salt thereof. A specific group of compounds of formula I are compounds wherein R⁹ is H or (d- C₆)alkyl, and R¹⁰is (Ci-C₆)alkyl wherein the (C C₆)alkyl of R¹⁰ is substituted with one -C0₂H group.

A specific value for R⁹ is H.

A specific value for R¹⁰ is (C₂-C₄)alkyl substituted with one -C0₂H group.

A specific value for R¹⁰ is:

A specific value for R¹ is methyl.

A specific value for n is 3.

A specific value for R² is H.

A specific value for R is H.

A specific value for R⁴ is (C₃-C₅)alkyl.

A specific value for R⁴ is butyl.

A specific value for R⁴ is:

A specific value for R⁵ is H.

A specific value for R⁶ is (C₂-C4)alkyl.

A specific value for R⁶ is propyl.

A specific value for R⁶ is:

A specific value for R¹¹ is phenyl, wherein any phenyl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH and -0(C₁-C₃)alkyl.

A specific value for R¹¹ is phenyl.

A specific group of compounds of formula I are compounds wherein R⁷ is H, and R⁸ is -C(=0)(C₁-C₆)alkyl, -C(=0)(C₂-C₆)alkenyl, -C(0)aryl, -C(=0)OR^a or -C(=0)NR^bR^c, wherein any -C(=0)(C!-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R⁸ is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH, -0(CrC₃)alkyl, -C0₂H, and -C(=0)NR^bR^c.

A specific group of compounds of formula I are compounds wherein R⁷ is H, and R⁸ is -C(=0)(C₁-C₆)alkyl, -C(=0)aryl, -C(=0)OR^a, or -C(=0)NR^bR^c, wherein any -C(=0)(C C₆)alkyl of R is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH, -0(d-C₃)alkyl, -C0₂H, and -C(=0)NR^bR^c.

A specific group of compounds of formula I are compounds wherein R is H, and R g is -C(=0)OR^a, or -C(=0)NR^bR^c.

A specific group of compounds of formula I are compounds wherein R⁷ is H, and R⁸ is -C(=0)aryl, wherein any -C(=0)aryl of R⁸ is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH, -0(C_rC₃)alkyl, -C0₂H,

and -C(=0)NR^bR^c.

A R⁸ is:

A specific compound of formula I is:

or a pharmaceutically acceptable salt thereof.

A specific compound of formula I is a compound of formula lb:

lb

wherein:

R¹ is (d-C₆)alkyl;

each R² is independently H or (C₁-C₆)alkyl;

R³ is H or (d-Q alkyl;

R⁵ is H or (C C₆)alkyl;

R⁴ is (C]-C₆)alkyl;

R⁶ is (C₁-C₆)alkyl;

R¹³ is R^13a, R⁹ is H or (d-C₆)alkyl, and R¹⁰ is (d-C₆)alkyl wherein the (d-C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R¹³ is R^13b, and R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo;

R^,3a is -(d-C₆)alkylO(d-C₆)alkyl, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂- C₆)alkyl, -C(=0)(C -C₆)alkenyl or -C(=0)aryl, wherein any -(d-C₆)alkylO(d-

C₆)alkyl, -C(=0)(C₂-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R^13b is H, -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl wherein any -(d-C₆)alkylO(d-C₆)alkyl, -C(=0)(d-C₆)alkyl or -C(=0)(C₂-

C₆)alkenyl of R^13a is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (d- C₃)alkyl, -OH and -0(d-C₃)alkyl;

R¹¹ is aryl, wherein any aryl of R⁹ is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH or -0(C₁-C₃)alkyl; and

n is 1, 2, 3 or 4;

or a pharmaceutically acceptable salt thereof.

A specific compound of formula I is a compound of formula lc:

Ic

or a pharmaceutically acceptable salt thereof wherein:

R^13a is -(Q-C^alkylOCCrCe^lkyl, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂- C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkylO(C₁- C₆)alkyl, -C(=0)(C₂-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R⁹ is H or (d-C₆)alkyl; and

R¹⁰ is (C₁-C₆)alkyl, wherein the (CrC₆)alkyl is substituted with one -C0₂H group.

A specific value for R^13a is -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C₃-C₆)alkyl, -C(=0)(C₂- C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkyl0(C₁-C₆)alkyl, -C(=0)(C₃-C₆)alkyl or -C(=0)(C₂-C )alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^6a is optionally substituted with one or more groups independently selected from halogen, (C C₃)alkyl, -OH and -0(C₁-C₃)alkyl.

A specific value for R^13a is -C(=0)H, -C(=0)CH₂C1, - CH₂OCH₃, -C(=0)phenyl, -C(=0)CH₂CH₃, -C(=0)CH₂CH₂CH₃, -C(=0)CH₂CH(CH₃)₂, -C(=0)C( CH₃)₃, -C(=0)CH₂C1 or -C(=0)CH=CH₂.

A specific value for R⁹ is H.

A specific value for R¹⁰ is (C₂-C₄)alkyl substituted with one -C0₂H group.

A specific value for R¹⁰ is:

A specific com ound of formula I is a compound of formula Id:

Id

or a pharmaceutically acceptable salt thereof wherein:

R^13b is H, -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(Ci-C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)ar l wherein any -(CrC₆)alk lO(Ci-C₆)alkyl, -C(=0)(Ci-C₆)alk l or -C(=0)(C₂- C₆)alkenyl of R^6b is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R 13b i *s optionally substituted wi *th one or more groups independently selected from halogen, (C C₃)alkyl, -OH and -0(C_!-C₃)alkyl; and

R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (d-C^alkyl and oxo.

A specific value for R⁹and R¹⁰ together with the atoms to which they are attached form a 5 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo.

A specific value for R⁹ and R¹⁰ together with the atoms to which they are attached form a pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted with one or more groups independently selected from

or oxo.

A specific value for R⁹and R¹⁰ together with the atoms to which they are attached form the moiety:

A specific value for R is methyl,

A specific value for n is 3.

A specific value for R² is H.

A specific value for R³ is H.

A specific value for R⁴ is (C₃-C )alkyl.

A specific value for R⁴ is butyl,

A specific value for R⁴ is: A specific value for R⁵ is (C₂-C₄)alkyl.

A specific value for R⁶ is propyl.

A specific value for R⁶ is:

A specific value for R¹¹ is phenyl, wherein any phenyl of R¹¹ is optionally substituted with more groups independently selected from halogen, (d-C₃)alkyl, -OH and -0(C₁-C₃)alkyl. A specific value for R¹¹ is phenyl.

A specific compound is:

or a pharmaceutically acceptable salt thereof.

Processes for preparing compounds of formula I are provided as embodiments of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as given above unless otherwise qualified.

A compound of formula I, or a salt thereof can be prepared by converting compound A, or a salt thereof to the compound of formula I, or a salt thereof:

I

A compound of formula A, or a salt thereof can be prepared by converting compound B, or a salt thereof to the compound of formula I, or a salt thereof:

In one embodiment compound A is prepared from compound B, by the reaction of

O compound B with a compound of formula R⁸ C(=0)C1, (R^8'C(=0))₂0, or ^R

wherein R⁸ is (C!-C₆)alkyl, (C₂-C₆)alkenyl, aryl, -OR^a, or -NR^bR^c, wherein any (Q- C₆)alkyl or (C₂-C₆)alkenyl of R⁸ is optionally substituted with one or more groups selected from halogen and wherein any aryl of R⁸ is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH, -0(d-C₃)alkyl, -C0₂H,

and -C(=0)NR^bR^c.

A compound of formula I, or a salt thereof can be prepared by converting compound A to the com ound of formula I:

wherein R¹³ is R^13a' or R^13b', wherein; R^13a' is CH₂C1, (C₂-C₆)alkyl, (C₂-C₆)alkenyl or aryl, wherein any (C₂-C₆)alkyl or (C₂- C )alkenyl of R^13a is optionally substituted with one or more halogen and wherein any aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C_\- C₃)alkyl, -OH and -0(C C₃)alkyl; and

R^13b' is (Q-C_f alkyl, -(C₂-C₆)alkenyl or aryl wherein any (C₁-C₆)alkyl or (C₂-C₆)alkenyl of R^6b is optionally substituted with one or more halogen, and wherein any aryl of R^13b is optionally substituted with one or more groups independently selected from halogen, (Q-C^alkyl, -OH and -0(d-C₃)alkyl.

In one embodiment pyridine is used together with (R C(=0))₂0 in the conversion of A to the compound of formula I.

A compound of formula I, or a salt thereof can also be prepared by converting compound A to the com ound of formula I:

R^13a is CH₂C1, (C₂-C₆)alkyl, (C₂-C₆)alkenyl or aryl, wherein any (C₂-C₆)alkyl or (C₂- C₆)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (Q- C₃)alkyl, -OH and -0(C C₃)alkyl; and

R^13b' is d-C₆)alkyl, (C₂-C₆)alkenyl or aryl wherein any (C C₆)alkyl or (C₂-C₆)alkenyl of R^6b is optionally substituted with one or more halogen, and wherein any aryl of R¹³ is optionally substituted with one or more groups independently selected from halogen, (Q-C^alkyl, -OH and -0(C₁-C₃)alkyl.

In one embodiment DMAP (dimethyl aminopyridine) and DCC

(Ν,Ν'-dicyclohexylcarbodiimide) are used together with R C0₂H in the conversion of A to the compound of formula I. In cases where compounds are sufficiently basic or acidic, a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I. Additionally, administration of a compound of formula I as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts include organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate,

methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a- ketoglutarate, and a-glycerophosphate. Suitable inorganic acid addition salts may also be formed, which include a physiological acceptable anion, for example, chloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as

compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No.

4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day. (

The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the treatment of breast cancers, lung cancers, ovarian cancers, and Kaposi sarcoma. Examples of such agents include doxorubicin,

cyclophosphamide, 5-fluorouracil, epirubicin, carboplatin, cisplatin, trastuzumab, gemcitabine, and irinotecan. Accordingly, in one embodiment the invention also provides a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat breast cancers, lung cancers, ovarian cancers, and Kaposi sarcoma.

The ability of a compound of the invention to act as an anticancer agent may be determined using pharmacological models which are well known to the art, or using Test A below which describes cytotoxicity assays using various cancer cell lines.

Test A: Cytotoxicity assay

Cytotoxity assays were performed against various cancer cell lines using methods known in the literature. Representative experimental descriptions of one such assay using a human ovarian carcinoma cell line can be found at Raghavan, B., et al., Cytotoxic Simplified Tubulsis Analogs, J. Med. Chem., 2008, 51, 1530-1533. Cytotoxicity assays using different known cancer cell lines were also carried out using assays similar to those described in Ragnaven and Sani. Experimental results from Test A for representative compounds described herein are shown in Tables 1 and 2. (

Table 1

A. Cytotoxicity to Ca46 lymphoma cells. 3-day exposure, colorimetric Resazurin assay. B. Cytotoxicity to 1 A9 ovarian carcinoma cells. 4-day exposure, SRB assay.

C. Cytotoxicity to 1 A9 ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

D. Cytotoxicity to lA9-HTI-aS165P;R221H ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

E. RR - relative resistance (D/C)

F. Cytotoxicity to 1 A9-HTI- S172A(S1) ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

G. RR - relative resistance (F/C)

Table 2

102 >200 750 1000 >13000 >13000

A. Cytotoxicity to Ca46 lymphoma cells. 3-day exposure, colorimetric Resazunn assay. B. Cytotoxicity to 1 A9 ovarian carcinoma cells. 4-day exposure, SRB assay.

C. Cytotoxicity to 1 A9 ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

D. Cytotoxicity to lA9-HTI-aS165P;R221H ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

E. RR - relative resistance (D/C)

F. Cytotoxicity to 1A9-HTI-PS172A(S1) ovarian carcinoma cells, 4-day exposure, sulforhodamine assay.

G. RR - relative resistance (F/C)

The invention will now be illustrated by the following non-limiting Examples which describe the preparation of compounds described herein and intermediates to prepare the compounds described herein.

Examples

Example 1. Preparation of compound 6.

Preparation of ethyl 2-aminothiazole-4-carboxylate hydrochloride salt (3). A suspension of ethyl bromopyruvate (90%, 33 mL, 0.26 mol, 1 equiv), thiourea (30 g, 0.39 mol, 1.5 equiv) and absolute EtOH (500 mL) was heated to reflux. The resulting solution was refluxed for 18 h, concentrated under reduced pressure, and purified by recrystallization from EtOH/MeOH to afford the title compound as a white solid (56.0 g, 84% yield). 1H and ¹³C NMR of the product matched those previously reported (Kelly and Lang 1996).

Preparation of ethyl 2-bromothiazole-4-carboxylate (4). To a solution of thiazole 3 (20.1 g,

0.0794 mol, 1 equiv) and CuBr (26.6 g, 0.119 mol, 1.5 equiv) in acetonitrile (400 mL) at 0 °C was added t-BuN0₂ (90%, 15.7 mL, 0.119 mol, 1.5 equiv) dropwise. After warming to room temperature over 1 h, the reaction was quenched with 1 M aqueous HC1 (400 mL) and diluted with CH₂C1₂ (400 mL). The layers were separated, and the aqueous layer was extracted with CH₂C1₂ (2 x 200 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by recrystallization from hexanes afforded the title compound as a tan solid (13.2 g, 71% yield). R/= 0.3 (Si0₂, 10% EtOAc:hexanes); 1H and ¹³C NMR of the product matched those previously reported (Kelly and Lang 1996).

Preparation of (2-bromothiazol-4-yl)methanol (5). To a solution of ester 4 (10.0 g, 42.4 mmol, 1 equiv) in THF (200 mL) was added NaB¾ (4.81 g, 127 mmol, 3 equiv), LiCl (5.4 g, 130 mmol, 3 equiv) and H₂0 (40 mL). The resulting biphasic mixture was vigorously stirred for 2 h, after which TLC (20% EtOAc:hexanes) showed complete consumption of starting material. The reaction was quenched with saturated aqueous NH₄CI (200 mL), diluted with EtOAc (200 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (100 mL), and the organic (

layers were combined, dried (Na₂S0₄), filtered, and concentrated under reduced pressure to give the title compound as a yellow semi-solid (7.82 g, 95%). R_f= 0.2 (Si0₂, 20% EtOAcrhexanes); 1H and C NMR of the product matched those previously reported (Wipf and Wang 2007).

Preparation of 2-bromo-4-((tert-butyldiphenylsilyloxy)methyl)thiazole (6). To a solution of alcohol 5 (22.55 g, 0.116 mol, 1 equiv) in DMF (50 mL) was added imidazole (7.923 g, 0.116 mol, 1 equiv), DMAP (1.421 g, 0.0116 mol, 0.1 equiv), and TBDPSCl (30.5 mL, 0.116 mol, 1 equiv). After 48 h, TLC (20% EtOAc:hexanes) showed complete consumption of starting material. The reaction was quenched with H₂0 (330 mL), diluted with EtOAc (300 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (2 x 200 mL), and the combined organic layers were washed with H₂0 (330 mL), 1 M aqueous HC1 (330 mL), and saturated aqueous NaCl (330 mL), dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash

chromatography (0-10% EtOAc:hexanes) afforded the title compound as a white solid (42.45 g, 85%). R_f= 0.8 (Si0₂, 20% EtOAc:hexanes); 1H and ¹³C NMR of the product matched those previously reported (Raghavan et al. 2008).

Example 2. Preparation of compound 8.

7 8

Preparation of (R)-tert-butyl l-(methoxy(methyl)amino)-4-methyl-l-oxopentan-3-ylcarbamate (8). To a solution of Boc-L-valine 7 (10.5 g, 48.3 mmol, 1 equiv) in THF (100 mL) at -78 °C was added Et₃N (6.8 mL, 48 mmol, 1 equiv) and the reaction was stirred for 15 min. To this solution was added ethyl chloroformate (4.62 mL, 48.3 mmol, 1 equiv) causing a white precipitate to form. The reaction mixture stirred for 15 min, followed by removal of the stir bar. To the standing reaction mixture was added 75% of an ethereal diazomethane solution generated using Diazald (21.4 g, 100 mmol, 2 equiv) and KOH (16.8 g, 300 mmol, 6 equiv) (de Boer and Backer 1963). After 1 h, the remaining diazomethane solution and a stir bar were added to the reaction mixture, and the reaction stirred while warming to room temperature overnight. Separately, a mixture of (MeO)MeNH»HCl

(14.4 g, 145 mmol, 3 equiv) and Et₃N (20.4 mL, 145 mmol, 3 equiv) in THF (100 mL) was stirred at room temperature overnight. After 18 h, the diazomethane reaction was quenched with 0.5 M aqueous AcOH solution (60 mL), and the resulting clear yellow mixture was stirred for 30 min. The layers were separated, and the organic layer was washed with saturated aqueous NaHC0₃ (100 mL) and saturated aqueous NaCl (100 mL), dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The crude diazomethylketone was then dissolved in THF (100 mL), and the mixture containing the desalted (MeO)MeNH was added following salt removal by filtration through a fritted glass funnel. (CAUTION: Diazoketones have been reported to be explosive, and proper precaution should be used in the synthesis and handling of these compounds.) The resulting solution was cooled to -40 °C and the flask was wrapped in aluminum foil. To the reaction was added silver benzoate (1.6 g, 7.3 mmol, 0.15 equiv) as a solution in Et₃N (15 mL), and the reaction was stirred while warming to room temperature overnight. After 24 h, the reaction was diluted with CH₂C1₂ (200 mL) and washed with 0.1 M aqueous HC1 (100 mL). The aqueous layer was extracted with CH₂CI₂ (3 x 200 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (30% EtOAc:hexanes) afforded the title compound (9.8 g, 74%) as a colorless oil. R_f= 0.6 (Si0₂, 50% EtOAc:hexanes); ^]H and C NMR of the product matched those previously reported (Raghavan et al. 2008). Example 3. Preparation of compound 9.

6

Preparation of (R)-tert-bu y\ l-(4-((tert-butyldiphenylsilyloxy)methyl)thiazol-2-yl)-4-methyl- l-oxopentan-3-ylcarbamate (9). To a solution of bromo thiazole 6 (9.5 g, 22 mmol, 1.2 equiv) in THF (200 mL) at -78 °C was added «-BuLi (2.4 M in hexane, 11 mL, 26 mmol, 1.4 equiv) dropwise and the reaction was stirred for 90 min. Separately, to a solution of Weinreb amide 8 (5.0 g, 18 mmol, 1 equiv) in THF (200 mL) at -10 °C was added z^'-PrMgCl (1.7 M in THF, 11.2 mL, 19 mmol, 1.05 equiv) dropwise and the reaction stirred for 30 min. The Weinreb amide 8 solution was cooled to -78 °C and was added dropwise to the lithiated thiazole 7 solution via cannula over 30 min. The reaction was stirred at -78 °C for 200 minutes, warmed to 0 °C, and stirred for an additional 90 min. TLC (10% EtOAc:hexanes) showed the reaction was no longer progressing, so the reaction was quenched with saturated aqueous NaHC0₃ (200 mL). The reaction mixture was diluted with Et₂0 (200 mL) and the layers were separated. The organic layer was washed with saturated aqueous NaHC0₃ (100 mL) and the combined aqueous layers were extracted with Et₂0 (2 x 100 mL). The combined organic layers were washed with H₂0 (100 mL) and saturated aqueous NaCl (100 mL), dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash

chromatography (5% EtOAcrhexanes, then 50% EtOAc:hexanes) afforded the title compound (5.245 g, 51%, 58% brsm) as a viscous orange oil and recovered Weinreb amide 8 (0.650 g). Rf= 0.5 (Si0₂, 20% EtOAcrhexanes); 1H NMR (400 MHz, CDC1₃) δ 7.72-7.67 (m, 4H), 7.62 (s, 1H), 7.48-7.33 (m, 6H), 5.00-4.82 (m, 3H), 4.01-3.88 (m, 1H), 3.25 (dd, J = 15.6, 7.4 Hz, 1H), 3.16 (dd, J = 15.6, 4.0 Hz, 1H), 1.97-1.76 (m, 1H), 1.37 (s, 9H), 1.12 (s, 9H), 0.93 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 192.9, 166.8, 159.3, 155.6, 135.7, 133.1, 130.1, 128.0, 121.7, 79.1, 63.0, 53.3, 40.9, 32.2, 28.5, 27.0, 19.4, 19.4, 18.6; HRMS calcd for C₃₁H4₃N₂0₄SSi⁺ [M + H⁺] 567.2707, found 567.2714.

Example 4. Preparation of compound 10.

Preparation of tert-butyl ( 1 S,3R)- 1 -(4-((tert-butyldiphenylsilyloxy)methyl)thiazol-2-yl)- 1 - hydroxy-4-methylpentan-3-ylcarbamate (10). To a solution of (i?)-CBS (20 mg, 0.072 mmol, 0.2 equiv) in THF (10 mL) at 0 °C was added BH₃ ^«SMe₂ (2.0 M in THF, 0.21 mL, 0.42 mmol, 1.2 equiv) and the reaction stirred for 30 min. Ketone 9 (200 mg, 0.353 mmol, 1 equiv) was added as a solution in THF (6 mL, with 3 mL wash) dropwise. The reaction was stirred at 0 °C for 30 min and was then warmed to room temperature. After 3.5 h, TLC (20% EtOAc:hexanes) showed remaining starting material. The reaction was re-cooled to 0 °C and additional BH₃ ^»SMe₂ (2.0 M in THF, 0.21 mL, 0.42 mmol, 1.2 equiv) was added. The reaction was slowly warmed to room temperature overnight. After 24 h total, TLC showed complete consumption of starting material. The reaction was quenched with slow addition of MeOH (CAUTION: generates hydrogen gas) and concentrated under reduced pressure. Purification by flash chromatography (20% EtOAcrhexanes) afforded the title compound as a clear oil (123 mg, 61% yield). R_f= 0.2 (Si0₂, 20% EtOAc.hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.73-7.64 (m, 4H), 7.46-7.33 (m, 6H), 7.19 (s, 1H), 5.02 (ddd, J = 7.5, 5.4, 4.5 Hz, lH), 4.85 (d, J = 1.3 Hz, 2H), 4.61^.48 (m, 1H), 4.39 (d, J = 5.7 Hz, 1H), 3.68-3.59 (m, 1H), 2.28-2.17 (m, 1H), 1.97-1.86 (m, 1H), 1.86-1.76 (m, 1H), 1.41 (s, 9H), 1.10 (s, 9H), 0.93 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H); ¹³C MR (101 MHz, CDC1₃) δ 175.8, 156.9, 156.6, 135.7, 133.4, 129.9, 127.9, 1 13.7, 78.0, 71.3, 63.2, 53.7, 41.0, 32.6, 28.5, 27.0, 19.4, 19.1, 17.7; HRMS calcd for C₃₁H₄₅N₂0₄SSi⁺ [M + H⁺] 569.2863, found 569.2863.

Alternate preparation of compound 10.

Preparation of tert-Butyl (1S,3R)- and tert-Butyl (li?,3i?)-l-(4-((/ert- butyldiphenylsilyloxy)methyl)thiazol-2-yl)- 1 -hydroxy-4-methylpentan-3-ylcarbamate. To MeOH (45 mL) at 0 °C was added NaBHt (0.268 g, 7.1 mmol, 15 equiv) resulting in gas evolution. After 20 min, ketone 9 (0.268 g, 0.473 mmol, 1 equiv) was added as a solution in MeOH (35 mL, with 10 mL wash) precooled to 0 °C. After 4 h, TLC showed complete consumption of starting materials (20% EtOAc:hexanes). The reaction was quenched with saturated aqueous NH CI (50 mL) and MeOH was removed under reduced pressure. The remaining aqueous reaction mixture was diluted with enough H₂0 to dissolve all solids and was extracted with EtOAc (3 x 30 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. 1H NMR of the crude reaction mixture showed a 1.6:1 dr of the IS,3R and \R,3R diastereomers, respectively. Purification by flash chromatography (20% EtOAc:hexanes) afforded the title compounds as a yellow oil (\R, 3R diastereomer 86, 0.102 g, 38% yield) and a clear oil (\S, 3R diastereomer 55, 0.156 g, 58% yield). 10: 1H and ¹³C NMR of the product matched those reported above. \R,3R-1 : R_f= 0.4 (Si0₂, 20% EtOAc:hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.72-7.64 (m, 4H), 7.45-7.31 (m, 6H), 7.18 (s, 1H), 5.07-4.95 (m, 1H), 4.91 (d, J= 9.8 Hz, 1H), 4.85 (s, 2H), 4.57 (d, J= 9.6 Hz, 1H), 3.78-3.66 (m, 1H), 1.97-1.86 (m, 1H), 1.83-1.75 (m, 1H), 1.75-1.66 (m, 1H), 1.43 (s, 9H), 1.10 (s, 9H), 0.94 (d, J= 4.2 Hz, 3H), 0.92 (d, J= 4.3 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 175.4, 158.0, 156.5, 135.7, 133.4, 129.9, 127.8, 1 13.4, 80.4, 69.2, 63.2, 52.5, 46.2, 42.1 , 32.3, 28.5, 27.0, 19.4, 18.5; HRMS calcd for C₃₁H4₅N₂0₄SSi⁺ [M + H⁺] 569.2863, found 569.2859.

To a solution of alcohol \R,3R-10 (48 mg, 0.084 mmol) in CH₂C1₂ (5 mL) was added Mn0₂

(88% activated, 10 equiv, 0.84 mmol) and the reaction was stirred vigorously overnight. After 20 h, TLC (20% EtOAc:hexanes) showed complete consumption of starting material. The solvent was removed under reduced pressure, and the crude product was mixed with EtOAc (10 mL). The resulting slurry was filtered through a tightly packed plug of Celite and washed with EtOAc (3 x 10 mL). The combined washings were concentrated under reduced pressure to give ketone 9 (42.5 mg, 89% yield) that was identical to that prepared in Example 1. Ketone 9 is then suitable for reduction to alcohol 10 as described.

Example 5. Preparation of compound 13.

12 12A 13

Preparation of tert-butyl (li?,3i?)-l-(4-((tert-butyldiphenylsilyloxy)methyl)thiazol-2-yl)-l-(l,3- dioxoisoindolin-2-yl)-4-methylpentan-3-ylcarbamate (11). To a solution of alcohol 10 (0.590 g, 1.04 mmol, 1 equiv) in THF (5.0 mL) at 0 °C was added PPh₃ (0.345 g, 1.35 mmol, 1.3 equiv) and phthalimide (0.199 g, 1.35 mmol, 1.3 equiv) with only partial dissolution of solutes. To this mixture was added DIAD (94%, 0.28 mL, 1.4 mmol, 1.3 equiv) dropwise which resulted in an orange reaction solution. After 2 h, the reaction had turned yellow and TLC (20% EtOAc:hexanes using an aliquot partitioned between H₂0 and ether) showed complete consumption of starting material. The reaction was quenched with ice cold H₂0 (20 mL) and THF was removed under reduced pressure. The remaining aqueous reaction mixture was extracted with Et₂0 (2 x 20 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (10% EtOAc :hexanes) afforded the title compound as a white solid (0.316 g, 50% yield). R_f= 0.4 (Si0₂, 20% EtOAc:hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.83 (dd, J = 5.4, 3.1 Hz, 2H), 7.71 (dd, J = 5.4, 3.1 Hz, 2H), 7.68-7.63 (m, 4H), 7.45-7.30 (m, 6H), 7.17 (s, 1H), 5.76 (dd, J = 12.2, 4.5 Hz, 1H), 4.83 (s, 2H), 4.37 (d, J = 10.5 Hz, 1H), 3.51-3.40 (m, 1H), 3.14— 3.04 (m, 1H), 2.13 (ddd, J = 14.3, 12.3, 4.5 Hz, 1H), 1.77-1.63 (m, 1H), 1.39 (s, 9H), 1.08 (s, 9H), 0.89 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 169.5, 167.9, 156.8, 155.7, 135.7, 134.1, 133.4, 132.1, 129.9, 127.9, 123.6, 114.1 , 79.4, 63.2, 52.3, 50.2, 34.2, 33.3, 28.5, 27.0, 19.4, 19.2, 18.1; HRMS calcd for C₃9H4₈N₃0₅SSi⁺ [M + H⁺] 698.3084, found 698.3099.

Preparation of fert-butyl (li?,3i?)-l-(l,3-dioxoisoindolin-2-yl)-l-(4-(hydroxymethyl)thiazol-2- yl)-4-methylpentan-3-ylcarbamate (12). To a solution of silyl ether 11 (202 mg, 0.290 mmol, 1 equiv) in THF (2.0 mL) at 0 °C was added TBAF (1.0 M in THF, 0.32 mL, 0.32 mmol, 1.1 equiv). After 1.5 h, TLC (50% EtOAc:hexanes) showed complete consumption of starting material. The solvent was removed under reduced pressure, and the reaction was purified by flash chromatography (50% EtOAc:hexanes) affording the title compound as a clear oil (105 mg, 79% yield). R/= 0.3 (Si0₂, 50% EtOAc:hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.83 (dd, J = 5.4, 3.1 Hz, 2H), 7.71 (dd, J = 5.4, 3.1 Hz, 2H), 7.10 (s, 1H), 5.79 (dd, J = 12.2, 4.4 Hz, 1H), 4.70 (s, 2H), 4.52 (d, J = 10.5 Hz, 1H), 3.52-3.38 (m, 1H), 3.16-3.02 (m, 1H), 2.77 (br s, 1H), 2.23-2.10 (m, 1H), 1.77-1.63 (m, 1H), 1.39 (s, 9H), 0.88 (apparent t, J = 7.5 Hz, 6H); ¹³C NMR (101 MHz, CDC1₃) δ 170.3, 167.9, 156.2, 155.8, 134.2, 132.0, 123.6, 115.2, 79.4, 61.0, 52.3, 50.1, 34.2, 33.2, 28.5, 19.2, 18.1 ; HRMS calcd for C₂₃H₃oN₃0₅S⁺ [M + H^1"] 460.1906, found 460.1925.

Preparation of tert-Butyl ( 1 R,3R)- 1 -( 1 ,3 -dioxoisoindolin-2-yl)- 1 -(4-formylthiazol-2-yl)-4- methylpentan-3-ylcarbamate (12A). To a solution of alcohol 12 (53 mg, 0.12 mmol, 1 equiv) in CH₂C1₂ (5 mL) was added Mn0₂ (88% activated, 114 mg, 1.15 mmol, 10 equiv) and the reaction was stirred vigorously overnight. After 20 h, TLC (50% EtOAc:hexanes) showed complete consumption of starting material. The solvent was removed under reduced pressure, and the crude solid was mixed with EtOAc (10 mL). The resulting slurry was filtered through a tightly packed plug of Celite and washed with EtOAc (3 x 10 mL). The combined washings were concentrated under reduced pressure to give the title compound as a white/yellow solid (45 mg, 0.098 mmol, 85% yield) in sufficient purity for the next reaction. An analytically pure sample can be prepared by flash chromatography (20% EtOAc :hexanes). R_f= 0.6 (Si0₂, 50% EtOAc :hexanes); ^!H NMR (400 MHz, CDC1₃) δ 9.96 (s, 1H), 8.10 (s, 1H), 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.72 (dd, J = 5.4, 3.0 Hz, 2H), 5.87 (dd, J = 12.2, 4.6 Hz, 1H), 4.45 (d, J = 10.5 Hz, 1H), 3.52-3.40 (m, 1H), 3.19-3.04 (m, 1H), 2.22 (ddd, J = 14.2, 12.2, 4.7 Hz, 1H), 1.78-1.64 (m, 1H), 1.38 (s, 9H), 0.89 (apparent t, J = 7.3 Hz, 6H); C NMR (101 MHz, CDC1₃) δ 184.9, 171.5, 167.8, 155.8, 154.7, 134.5, 132.0, 128.0, 123.8, 79.6, 52.3, 50.2, 34.4, 33.3, 28.5, 19.3, 18.2; HRMS calcd for C₂₃H₂₈N₃0₅S⁺ [M + H⁺] 458.1750, found 458.1769.

Preparation of 2-((li?,3i?)-3-(tert-Butoxycarbonylamino)-l-(l,3-dioxoisoindolin-2-yl)-4- methylpentyl)thiazole-4-carboxylic acid (13). To a solution of 12A (0.138 g, 0.302 mmol, 1 equiv) and 2-methyl-2-butene (1.76 mL, 16.6 mmol, 55 equiv) in t-BuOH (12 mL) was added a solution of NaC10₂ (80%, 0.306 g, 2.71 mmol, 9 equiv) and NaH₂P0₄-H₂0 (0.291 g, 2.11 mmol, 7 equiv) in H₂0 (6 mL) dropwise. The reaction color changed from yellow to colorless over 2 h and TLC (50% EtOAc:hexanes) showed complete consumption of starting material. t-BuOH was removed under reduced pressure, and the resulting slurry was partitioned between H₂0 (30 mL) and CH₂C1₂ (30 mL). The layers were separated, and the aqueous layer was acidified to pH 3 with 5% aqueous KHS0₄ and extracted with CH₂C1₂ (2 x 30 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (10% MeOH:CH₂Cl₂ with 0.5% AcOH), followed by removal of AcOH by addition and removal of benzene (3 x 20 mL) under reduced pressure, afforded the title compound as a white solid (0.138 g, 98%). R/= 0.4 (Si0₂, 10% MeOH:CH₂Cl₂ with 0.5% AcOH); 1H NMR (400 MHz, CDC1₃) δ 8.20 (s, 1H), 7.86 (dd, J = 3.1, 5.4 Hz, 2H), 7.75 (dd, J = 3.1, 5.4 Hz, 2H), 5.87 (dd, J = 4.7, 11.9 Hz, 1H), 4.40 (d, J = 10.5 Hz, 1H), 3.56-3.44 (m, 1H), 3.15-3.01 (m, 1H), 2.28-2.15 (m, 1H), 1.78-1.63 (m, 1H), 1.40 (s, 9H), 0.92 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 175.5, 170.9, 167.7, 161.3, 155.8, 134.5, 131.9, 128.5, 123.8, 79.7, 52.3, 50.0, 34.6, 33.2, 28.5, 19.3, 18.0; HRMS calcd for C₂₃H₂6N₃0₆S^" [M - H] 472.1548, found 472.1549.

Example 6. Preparation of compound 18.

Preparation of compound 16. A solution of benzyl ester 14 (Raghavan, B., et al., 2008) (160 mg, 0.40 mmol, 1.4 equiv) in HCl (4.0 M in dioxane, 5.0 mL) was stirred at room temperature for 2 h, after which TLC (50% EtOAc:hexanes) showed complete consumption of starting material. The reaction solution was concentrated under reduced pressure, and the resulting solid was dissolved in CH₂C1₂ (5 mL) and concentrated under reduced pressure; this was repeated twice to afford the crude HCl salt 15 as a white solid. Separately, to a solution of acid 13 (138 mg, 0.29 mmol, 1 equiv) in CH₂C1₂ (10 mL) at -15 °C was added Et₃N (0.12 mL, 0.87 mmol, 3 equiv) and ethyl chloroformate (33 μί, 0.35 mmol, 1.2 equiv), and the resulting solution stirred for 35 min. To this was added a solution of the crude HCl salt in CH₂C1₂ (10 mL) at -15 °C via cannula. After 1 h, TLC (10% MeOH:CH₂Cl₂ with 0.5% AcOH) showed complete consumption of starting material. The reaction was quenched with saturated aqueous NH₄CI (15 mL), the layers were separated, and the aqueous layer was extracted with CH₂C1₂ (2 x 15 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (30% EtOAc:hexanes) afforded the title compound 16 as a white solid (202.4 mg, 92% yield). R = 0.3 (Si0₂, 40% EtOAc:hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.98 (s, 1H), 7.83 (dd, J= 5.4, 3.1 Hz, 2H), 7.72 (dd, J= 5.4, 3.1 Hz, 2H), 7.35-7.07 (m, 11H), 5.76 (dd, J= 12.1, 4.7 Hz, 1H), 5.10 (d, J= 12.5 Hz, 1H), 5.05 (d, J= 12.4 Hz, 1H), 4.48^1.35 (m, 2H), 3.54-3.42 (m, 1H), 3.14-3.03 (m, 1H), 2.91 (A of ABX, dd, J= 13.7, 6.0 Hz, 1H), 2.85 (B of ABX, dd, J= 13.7, 6.6 Hz, 1H), 2.69-2.57 (m, 1H), 2.11 (ddd, J= 14.1, 12.4, 4.8 Hz, 1H), 2.00 (ddd, J= 13.7, 9.2, 4.2 Hz, 1H), 1.77-1.65 (m, 1H), 1.60 (ddd, J= 14.2, 9.8, 4.6 Hz, 1H), 1.40 (s, 9H), 1.16 (d, J= 7.1 Hz, 3H), 0.92 (d, J= 6.8 Hz, 3H), (

0.89 (d, J- 6.8 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 176.1, 169.6, 167.8, 160.5, 155.8, 149.8, 137.7, 136.3, 134.4, 132.0, 129.7, 128.7, 128.6, 128.22, 128.18, 126.7, 123.9, 123.7, 79.6, 66.5, 52.3, 49.9, 48.6, 41.4, 37.8, 36.8, 34.4, 33.3, 28.5, 19.4, 18.1, 17.8; HRMS calcd for C₄₂H₄₉N₄0₇S⁺ [M + H⁺] 753.3322, found 753.3311.

Preparation of compound 17. A solution of compound 16 (95.6 mg, 0.127 mmol, 1 equiv) in

HC1 (4.0 M in dioxane, 4.0 mL) was stirred at room temperature for 2 h, after which TLC (50% EtOAc:hexanes) showed complete consumption of starting material. The reaction solution was concentrated under reduced pressure, and the resulting solid was dissolved in CH₂C1₂ (5 mL) and concentrated under reduced pressure; this was repeated twice to afford the crude HC1 salt as a white solid. Separately, to a solution of Boc-L-Ile (41 mg, 0.18 mmol, 1.4 equiv) in DMF (1.5 mL) at -15 °C was added HATU (68 mg, 0.18 mmol, 1.4 equiv) and N-methylmorpholine (56 μί, 0.51 mmol, 4 equiv), and the resulting solution was stirred for 20 min. To this solution was added a solution of the crude HC1 salt in DMF (1.5 mL, with 0.5 mL wash) at -15 °C dropwise via pipette, and the reaction stirred while warming to room temperature overnight. After 18 h, the reaction was concentrated under reduced pressure and partitioned between CH₂C1₂ (30 mL) and 5% aqueous KHS0₄ (15 mL). The aqueous layer was extracted with CH₂C1₂ (15 mL), and the combined organic layers were washed with H₂0 (15 mL), saturated aqueous NaHC0₃ (15 mL), H₂0 (15 mL) and saturated aqueous NaCl (15 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (30% EtOAc:hexanes) afforded the title compound 17 as a white solid (99 mg, 90% yield). R_f= 0.2 (Si0₂, 40% EtOAc:hexanes); 1H NMR (400 MHz, CDC1₃) δ 7.96 (s, 1H), 7.86 (dd, J= 5.4, 3.1 Hz, 2H), 7.74 (dd, J= 5.4, 3.1 Hz, 2H), 7.36-7.07 (m, 10H), 6.09 (d, J= 9.9 Hz, 1H), 5.64 (dd, J= 11.7, 4.2 Hz, 1H), 5.10 (d, J= 12.4 Hz, 1H), 5.04 (d, J= 12.4 Hz, 1H), 4.97-4.78 (br m, 1H), 4.49-4.35 (m, 1H), 3.90-3.75 (br m, 2H), 3.10 (ddd, J= 14.5, 11.9, 2.9 Hz, 1H), 2.91 (A of ABX, dd, J= 13.7, 6.1 Hz, 1H), 2.85 (B of ABX, dd, J = 13.7, 6.6 Hz, 1H), 2.68-2.57 (m, 1H), 2.22 (ddd, J= 14.5, 11.6, 4.5 Hz, 1H), 2.06-1.63 (m, 4H), 1.58 (ddd, J= 14.3, 9.9, 4.5 Hz, 2H), 1.42 (s, 9H), 1.16 (d, J= 7.1 Hz, 3H), 1.13-1.03 (m, 1H), 1.00 (d, J= 6.7 Hz, 3H), 0.95 - 0.84 (m, 9H); ¹³C NMR (101 MHz, CDC1₃) δ 176.0, 171.9, 169.4, 167.7, 160.4, 149.9, 137.6, 136.3, 134.5, 131.8, 129.7, 128.59, 128.55, 128.3, 128.2, 128.0, 126.6, 123.83, 123.76, 80.4, 66.4, 59.8, 50.8, 50.0, 48.6, 41.4, 37.93, 36.8, 35.4, 34.2, 32.6, 28.4, 24.8, 19.2, 18.0, 16.0, 11.3; HRMS calcd for C₄8H₆₀N₅O₈S⁺ [M + H⁺] 866.4163, found 866.3153.

Preparation of compound 18. A solution of tripeptide 17 (41.5 mg, 0.0479 mmol, 1 equiv) in HC1 (4.0 M in dioxane, 3.0 mL) was stirred at room temperature for 1 h, after which TLC (50% EtOAc:hexanes) showed complete consumption of starting material. The reaction solution was concentrated under reduced pressure, and the resulting solid was dissolved in CH₂CI2 (5 mL) and concentrated under reduced pressure; this was repeated twice to afford the crude HC1 salt as a white/yellow solid. Separately, to a mixture of N-Me-D-Pip (20 mg, 0.14 mmol, 3 equiv) in DMF (4 mL) at 0 °C was added HATU (53 mg, 0.14 mmol, 3 equiv) and Et₃N (27 μΐ, 0.19 mmol, 4 equiv), and the resulting suspension was stirred for 1 h. To this suspension was added a solution of the crude HC1 salt in DMF (2.0 mL, with 0.5 mL wash) at 0 °C dropwise via pipette, and the reaction stirred while warming to room temperature overnight. After 18 h, the reaction was concentrated under reduced pressure and partitioned between CH₂C1₂ (30 mL) and H₂0 (15 mL). The aqueous layer was extracted with CH₂C1₂ (15 mL), and the combined organic layers were washed with saturated aqueous NaHC0₃ (15 mL), H₂0 (15 mL) and saturated aqueous NaCl (15 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (3.5% MeOF CEbCL.) afforded the title compound 18 as a yellow solid (41.0 mg, 96% yield). R_f= 0.3 (Si0₂, 5% MeOH:CH₂Cl₂); 1H NMR (400 MHz, CDCI3) δ 7.96 (s, 1H), 7.86 (dd, J= 5.5, 3.0 Hz, 2H), 7.74 (dd, J= 5.5, 3.1 Hz, 2H), 7.36-7.09 (m, 9H), 7.03 (d, J= 8.4 Hz, 1H), 6.34 (d, J= 10.0 Hz, 1H), 5.65 (dd, J= 11.7, 4.3 Hz, 1H), 5.10 (d, J= 12.4 Hz, 1H), 5.04 (d, J= 12.4 Hz, 1H), 4.48-4.36 (m, 1H), 4.09 (t, J= 8.4 Hz, 1H), 3.90-3.78 (m, 1H), 3.08 (ddd, J= 14.4, 11.9, 2.8 Hz, 1H), 2.97-2.79 (m, 3H), 2.69-2.56 (m, 1H), 2.50 (dd, J= 1 1.1, 3.0 Hz, 1H), 2.22 (s, 3H), 2.09-1.29 (m, 15H), 1.19-1.12 (ovlp m, 1H), 1.16 (d, J= 7.1 Hz, 3H), 1.03 (d, J= 6.7 Hz, 3H), 0.95-0.81 (m, 9H); ¹³C NMR (101 MHz, CDC1₃) δ 176.1, 175.4, 171.3, 169.3, 167.7, 160.4, 150.0, 137.7, 136.3, 134.5, 131.8, 129.6, 128.6, 128.5, 128.3, 128.2, 126.6, 123.83, 123.78, 100.1, 69.8, 66.4, 58.1, 55.5, 50.7, 50.1, 48.7, 45.1, 41.4, 37.9, 36.8, 34.7, 33.9, 32.5, 31.0, 25.2, 25.0, 23.4, 18.9, 18.1, 18.0, 16.2, 10.9; HRMS calcd for C₅₀H₆₃N₆O₇S⁺ [M + H⁺] 891.4474, found 891.4481.

Example 7. Preparation of compound 20.

Preparation of compound 20. To a solution of compound 18 (14 mg, 0.016 mmol, 1 equiv) in MeOH (2.6 mL) was added hydrazine monohydrate (39

0.79 mmol, 50 equiv) and the solution was stirred for 3 h, after which TLC (10% MeOH:CH₂Cl₂) showed complete consumption of starting material to the intended product and the open chain intermediate 19. The reaction was heated to reflux and stirred overnight. After 18 h, TLC showed full conversion to the desired product. The reaction was concentrated under reduced pressure, 0.1 M aqueous HC1 (20 mL) was added and the resulting aqueous solution was extracted with Et₂0 (3 x 10 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash

chromatography (5% MeOH:CH₂Cl₂) afforded the title compound 20 as a white/yellow solid (11.2 mg, 94% yield). R_f= 0.2 (19), 0.4 (20) (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (400 MHz, MeOD) δ 8.01 (s, 1H), 7.36-7.12 (m, 10H), 5.08 (d, J= 12.3 Hz, 1H), 5.01 (d, J= 12.3 Hz, 1H), 4.44-^.33 (m, lH), 4.19 (d, J= 8.9 Hz, 1H), 4.13 (dd, J= 10.3, 3.3 Hz, 1H), 4.04-3.95 (m, 1H), 2.99 (d, J= 11.7 Hz, 1H), 2.90 (dd, J= 13.6, 7.2 Hz, 1H), 2.84 (dd, J= 13.7, 6.7 Hz, 1H), 2.72 (dd, J= 11.1, 1.8 Hz, 1H), 2.69-2.59 (m, 1H), 2.26 (s, 2H), 2.25-2.15 (m, 1H), 2.03 (apparent dddd, J= 27.7, 13.8, 9.9, 3.7 Hz, 2H), 1.93-1.49 (m, 10H), 1.40-1.25 (m, 3H), 1.25-1.18 (m, 1H), 1.16 (d, J= 7.1 Hz, 3H), 0.95 (d, J= 6.7 Hz, 8H), 0.89 (t, J= 7.4 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 178.1, 177.5, 174.8, 174.1, 163.1, 150.6, 139.4, 137.5, 130.5, 129.5, 129.4, 129.2, 129.1, 127.4, 124.5, 70.3, 67.4, 59.3, 56.6, 52.8, 52.5, 50.3, 44.5, 42.4, 42.0, 39.0, 38.0, 37.4, 33.8, 31.4, 26.0, 25.9, 24.1, 20.0, 18.7, (

18.3, 16.2, 11.0; HRMS calcd for C₄₂H₆₁N₆0₅S⁺ [M + Ff] 761.4424, found 761.4419.

Example 8. Preparation of compound 21.

To a solution of compound 18 (10.6 mg, 11.9 μηιοΐ) in THF (1.5 mL) and H₂0 (0.5 mL) was added LiOH*H₂0 (20 mg, 0.48 mmol, 40 equiv) and the reaction was stirred overnight. After 72 h, HPLC showed complete consumption of starting material, and THF was removed under reduced pressure. The aqueous reaction mixture was acidified to pH 2 with 1.0 M aqueous HC1, extracted with CH2CI2 (4 x 15 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by HPLC (C₁₈, 150 x 10 mm, 32% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 10 min, 90% MeCN/25 mM aqueous NiL_tOAc, pH 4.78 for 5 min, 5 mL/min) afforded the title compound as a white solid (4.6 mg, 47% yield). HPLC rt = 5.5 min; R/= 0.1 (Si0₂, 10% MeOH:CH₂Cl₂ with 1% AcOH); 1H NMR (400 MHz, D₂0) δ 8.41 (d, J= 9.1 Hz, 1H), 8.00 (s, 1H), 7.70-7.52 (m, 4H), 7.30 (d, J= 4.3 Hz, 3H), 7.28-7.21 (m, 1H), 5.28 (dd, J= 11.3, 3.5 Hz, 1H), 4.33-4.25 (m, 1H), 4.22 (d, J= 8.5 Hz, 1H), 4.06-3.96 (m, 1H), 3.86 (d, J= 9.9 Hz, 1H), 3.54 (d, J= 12.8 Hz, 1H), 3.17-3.06 (m, 1H), 3.02 (dd, J= 13.2, 5.3 Hz, 1H), 2.84 (dd, J= 13.9, 8.7 Hz, 1H), 2.77 (s, 2H), 2.61-2.52 (m, 1H), 2.42-2.15 (m, 4H), 2.10-1.84 (m, 5H), 1.85-1.65 (m, 4H), 1.65-1.42 (m, 3H), 1.34-1.18 (m, 2H), 1.16 (d, J= 7.1 Hz, 3H), 0.95 (d, J= 6.7 Hz, 6H), 0.86 (t, J= 7.3 Hz, 3H); ¹³C NMR (214 MHz, D₂0) δ 183.2, 175.1, 173.6, 172.8, 172.4, 168.8, 162.8, 148.3, 138.4, 136.8, 133.8, 130.7, 129.7, 129.5, 128.4, 128.2, 127.6, 126.5, 124.5, 66.9, 59.0, 55.1, 51.9, 50.0, 49.8, 41.9, 40.7, 40.4, 38.1, 37.9, 35.6, 32.2, 28.7, 24.4, 22.6, 20.7, 18.1, 17.6, 17.5, 15.0, 9.9; HRMS calcd for C₄₃H₅₉N₆0₈S ⁺ [M + H⁺] 819.4115, found 819.4137.

Preparation of compound 22. A solution of compound 18 (37.9 mg, 0.0425 mmol) in pyrrolidine (2.0 mL) was stirred for 5 h, after which TLC (5% MeOH:CH₂Cl₂, using an aliquot partitioned between 1.0 M aqueous HC1 and EtO Ac) showed complete consumption of starting material. The reaction was poured into 1.0 M aqueous HC1 (75 mL) and the resulting aqueous solution was extracted with EtO Ac (30 mL, then 2 x 20 mL). The combined organic layers were washed with 1.0 M aqueous HC1 (2 x 25 mL) and saturated aqueous NaCl (25 mL), dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by flash chromatography (5%— 10% MeOH:CH₂Cl₂) afforded the title compound as an off white solid (37.8 mg, 92% yield). R_f= 0.4 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (400 MHz, CDC1₃) δ 7.96 (s, 1H), 7.91-7.75 (m, 1H), 7.60- 7.39 (m, 3H), 7.39-7.12 (m, 11H), 5.48 (s, 1H), 5.12 (d, J= 12.4 Hz, 1H), 5.06 (d, J= 12.4 Hz, 1H), 4.54-^.37 (m, 1H), 4.05 (d, J= 39.3 Hz, 2H), 3.70-3.58 (m, 1H), 3.57-3.48 (m, 1H), 3.29-3.13 (m, 2H), 3.09 (dd, J= 14.6, 7.3 Hz, 1H), 3.03-2.78 (m, 3H), 2.75-2.64 (m, 1H), 2.64-2.11 (m, 5H), 2.05 (ddd, J= 13.7, 9.6, 4.0 Hz, 2H), 1.99-1.31 (m ,15 H), 1.19 (d, J= 7.1 Hz, 3H), 1.15-1.01 (m, 2H), 0.93 (dd, J= 14.7, 6.7 Hz, 7H), 0.88-0.69 (m, 6H); ¹³C NMR (101 MHz, CDC1₃) δ 176.6, 176.1, 174.9, 171.6, 171.3, 169.6, 167.7, 160.7, 150.4, 138.0, 136.3, 133.0, 131.0, 129.6, 129.5, 128.57, 128.55, 128.5, 128.2, 128.1, 128.0, 126.5, 123.1, 69.8, 66.4, 57.7, 55.5, 51.2, 49.6, 49.3, 48.8, 46.1, 45.1, 41.6, 38.0, 36.9, 36.8, 35.9, 32.1, 30.9, 26.0, 25.3, 24.6, 23.4, 22.8, 19.4, 18.1, 16.0, 14.3, 10.9; HRMS calcd for C₅₄H₇₂N₇0₇S ⁺ [M + H⁺] 962.5214, found 962.5197. (

Preparation of compound 23. To a solution of compound 22 (37.8 mg, 0.0393 mmol) in THF (2.0 mL), H₂0 (0.40 mL) and MeOH (0.40 mL) was added LiOH«H₂0 (33 mg, 0.79 mmol, 20 equiv), and the resulting mixture was stirred overnight. After 18 h, the reaction had turned an orange color, and TLC (10% MeOH:CH₂Cl₂) showed complete consumption of starting material to the intended product and 21. The organic solvents were removed under reduced pressure, and the resulting aqueous reaction mixture was diluted with H₂0 (3 mL) and acidified to pH 2 with 1.0 M aqueous HC1. The resulting aqueous reaction solution was extracted with CH₂C1₂ (5 x 5 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by HPLC (C₁₈, 150 x 10 mm, 32% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 10 min, 90% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 5 min, 5 mL/min) afforded the title compound as a white solid (5.10 mg, 15%) and 21 (18.85 mg, 59%) as an acetate salt. HPLC rt = 8.4 min; R_f= 0.3 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (400 MHz, D₂0) δ 8.51-8.35 (m, 1H), 8.02 (s, 1H), 7.81 (d, J= 7.4 Hz, 1H), 7.78-7.59 (m, 2H), 7.44 (d, J= 7.1 Hz, 1H), 7.41-7.17 (m, 4H), 5.24 (dd, J= 11.0, 3.2 Hz, 2H), 4.33 (br s, 1H), 4.21 (d, J= 8.4 Hz, 1H), 4.10 (br s, 1H), 3.97-3.66 (m, 3H), 3.55 (d, J= 11.9 Hz, 1H), 3.47-3.30 (m, 2H), 3.24-2.97 (m, 3H), 2.95-2.75 (m, 4H), 2.66 (br s, 1H), 2.46-2.33 (m, 1H), 2.33-2.16 (m, 2H), 2.14-1.42 (m, 13H), 1.40-1.26 (m, 2H), 1.20 (d, J= 6.9 Hz, 3H), 1.08-0.90 (m, 7H), 0.90-0.76 (m, 3H); ¹³C NMR (214 MHz, D₂0) δ 185.1, 181.1, 172.8,

170.4, 168.8, 162.7, 158.7, 148.3, 138.3, 135.8, 132.1, 131.3, 129.8, 129.4, 128.4, 127.9, 127.0,

126.5, 124.6, 66.9, 59.0, 55.2, 51.6, 49.6, 49.5, 48.9, 45.9, 41.9, 40.7, 37.7, 36.7, 35.5, 35.0, 32.5, 28.7, 25.1, 24.3, 24.0, 22.6, 20.7, 18.2, 17.4, 17.1, 15.1, 9.9; HRMS calcd for C₄₇H₆₆N₇0₇S ⁺ [M +

H⁺] 872.4744, found 872.4738. Compound 23 is also a compound of the invention.

Preparation of compound 24. A solution of compound 23 (5.0 mg, 5.7 μιηοΐ) in HC1 (4.0 M in dioxane, 3 mL) was heated to 60 °C and stirred for 48 h, after which TLC (10% MeOH:CH₂Cl₂) showed complete consumption of starting material. The reaction solution was concentrated under reduced pressure, and the resulting solid was dissolved in CH₂C1₂ (5 mL) and concentrated under reduced pressure; this was repeated twice to afford the crude HC1 salt as a white/yellow solid.

Purification by flash chromatography (10% MeOH:CH₂Cl₂) afforded the title compound as a white/yellow solid (2.5 mg, 54% yield). R_f= 0.4 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (400 MHz, D₂0) δ 8.31 (d, J= 9.4 Hz, 1H), 8.03 (s, 1H), 7.99-7.84 (m, 3H), 7.30-7.09 (m, 4H), 5.60 (dd, J= 11.8, 3.2 Hz, 1H), 4.24 (d, J= 7.8 Hz, 2H), 3.90-3.59 (m, 5H), 3.53 (d, J= 12.2 Hz, 1H), 3.36 (s, 1H), 3.12 (dd, J= 28.8, 13.3 Hz, 2H), 2.94 (dd, J= 13.3, 4.2 Hz, 1H), 2.74 (s, 3H), 2.59-2.45 (m, 1H), 2.45-2.32 (m, 1H), 2.28-2.15 (m, 1H), 1.99-1.45 (m, 10H), 1.36-1.16 (m, 2H), 1.11 (d, J = 6.9 Hz, 2H), 1.04 (d, J- 6.7 Hz, 2H), 0.90 (dd, J= 14.4, 6.9 Hz, 8H); ^liC NMR (214 MHz, D₂0) δ 181.0, 172.9, 169.8, 168.83, 168.76, 162.4, 147.9, 137.9, 135.4, 130.8, 129.4, 128.3, 126.5, 125.4, 123.8, 66.9, 59.0, 55.2, 52.1, 49.9, 49.4, 41.9, 40.5, 37.5, 36.6, 35.7, 32.6, 32.3, 28.7, 24.2, 22.5, 20.7, 18.1, 17.6, 17.1, 15.0, 10.0; HRMS calcd for C₄₃H₅₇N₆0₇S ⁺ [M + H⁺] 801.4009, found 801.4000.

Example 10. Preparation of compound 25.

Preparation of compound 25. To a solution of compound 24 (9.5 mg, 0.012 mmol) in MeOH

(5 mL) was added Η₂ΝΝΗ₂·Η₂0 (6.0 uL, 0.12 mmol, 10 equiv) and the reaction stirred at room temperature over 72 h, where crude MS showed major mass peaks for the starting material and the hydrazine added intermediate, and a minor product mass peak. The reaction was heated to 40 °C and stirred for an additional 48 h, where crude MS showed the reaction had not progressed significantly. The reaction was cooled to room temperature, additional Η₂ΝΝΗ₂·Η₂0 (30 μί, 0.62 mmol, 50 equiv) was added and the reaction was heated to 40 °C. After an additional 24 h, crude MS showed that starting material had been consumed and a small portion of the intermediate remained. After an additional 24 h, no significant reaction progress was observed, so the reaction was concentrated under reduced pressure. Purification by HPLC (C₁₈, 150 x 10 mm, 10-50% MeCN/25 mM aqueous N¾OAc, pH 4.78 over 10 min, 90% MeCN/25 mM aqueous NFLtOAc, pH 4.78 for 5 min, 5 mL/min) afforded the acetate salt of the title compound as a white/yellow solid (1.99 mg, 25% yield) as a mixture with the methyl ester of the product. HPLC rt = 7.4 min; R_f= 0.2 (Si0₂, 10%

MeOH:CH₂Cl₂); 1H NMR (400 MHz, MeOD) δ 8.04 (s, 1H), 7.23 (d, J= 3.8 Hz, 4H), 7.19-7.10 (m, 1H), 4.41^1.29 (m, 1H), 4.26-4.14 (m, 2H), 4.02-3.93 (m, 1H), 3.16-3.05 (m, 1H), 3.03-2.82 (m, 3H), 2.63-2.48 (m, 1H), 2.46-2.31 (m, 4H), 2.16-2.06 (m, 1H), 1.95 (s, 3H), 2.05-1.52 (ovlp m, 12H), 1.41 (ddd, J= 16.3, 8.5, 3.8 Hz, 1H), 1.31-1.18 (m, 1H), 1.18-1.10 (m, 3H), 1.06-0.86 (m, 11H); ¹³C NMR (214 MHz, MeOD) δ 182.3, 177.4, 176.6, 174.2, 173.8, 162.9, 150.8, 139.7, 130.5, 129.3, 127.4, 124.5, 69.8, 59.6, 56.4, 53.0, 52.3, 51.0, 44.1, 42.6, 41.7, 41.6, 39.0, 37.4, 33.7, 31.1, 26.1, 25.5, 23.7, 22.2, 19.9, 18.8 (2C), 16.2, 11.0; HRMS calcd for C₃₅H55N₆0₅S ⁺ [M + H⁺] 671.3955, found 671.3960.

Example 11. Preparation of compound 26.

To a solution of compound 20 (2.9 mg, 3.8 μηιοΐ) in CH2CI2 (1 mL) at 0 °C was added Et₃N (4.2 μί, 30 μιηοΐ, 8 equiv) as a solution in CH₂C1₂ (100 μΕ) and AcCl (1.1 \ L, 15 μπιοΐ, 4 equiv) as a solution in CH₂C1₂ (100 iL), and the reaction stirred while warming to room temperature overnight. After 18 h, TLC (2 x 5% MeOH:CH₂Cl₂ with 1% Et₃N) showed complete consumption of starting material. The reaction was concentrated under reduced pressure and purification by HPLC (C₁₈, 250 x 10 mm, 0-20% MeCN/0.04% aqueous HCl over 2 min, 20% MeCN/0.04% aqueous HCl for 2 min, 20-90% MeCN/0.04% aqueous HCl over 35 min, 90% MeCN/0.04% aqueous HCl for 5 min, 90-10% MeCN/0.04% aqueous HCl over 5 min, 3 mL/min) afforded the benzyl ester intermediate as a white solid (1.3 mg, 42 % yield) as confirmed by HRMS. The intermediate was brought forward without further characterization. HPLC rt = 21.9 min; R/= 0.5 (Si0₂, 5% MeOH:CH₂Cl₂ with 1% Et₃N); HRMS calcd for C₄₄H₆₃N₆0₆S⁺ [M + H⁺] 803.4524, found 803.4511.

To a solution of the intermediate (1.3 mg, 1.6 μηιοΐ) in THF (1.2 mL) was added an aqueous LiOH solution (0.2 M in H₂0, 0.4 mL, 0.08 mmol, 50 equiv) and the reaction stirred overnight.

After 18 h, HPLC trace analysis showed complete consumption of starting material, and a crude MS confirmed the presence of the product mass peak. The reaction mixture was acidified to pH 2 with 1.0 M aqueous HCl, and was concentrated under reduced pressure. Purification by HPLC (C₁₈, 250 x 10 mm, 0-20% MeCN/0.04% aqueous HCl over 2 min, 20% MeCN/0.04% aqueous HCl for 2 min, 20-90% MeCN/0.04% aqueous HCl over 35 min, 90% MeCN/0.04% aqueous HCl for 5 min, 90- 10% MeCN/0.04% aqueous HCl over 5 min, 3 mL/min) afforded the title compound as a white solid (0.73 mg, 63 % yield, 27% yield over two steps). HPLC rt = 15.9 min; R_f= 0.2 (Si0₂, 10%

MeOH:CH₂Cl₂); 1H NMR (400 MHz, D₂0) δ 7.96 (s, 1H), 7.38-7.21 (m, 5H), 5.09 (dd, J= 11.3, 3.2 Hz, 1H), 4.35 - 4.25 (m, 1H), 4.22 (d, J= 8.4 Hz, 1H), 3.94-3.82 (m, 2H), 3.55 (br d, J= 13.3 Hz, 1H), 3.20-3.07 (m, 1H), 3.00 (dd, J= 13.5, 5.4 Hz, 1H), 2.85 (dd, J= 13.8, 8.3 Hz, 1H), 2.78 (s, 3H), 2.72 - 2.61 (m, 1H), 2.28-2.13 (m, 2H), 2.10 (s, 3H), 2.07-1.69 (m, 8H), 1.67-1.45 (m, 3H), 1.26-1.16 (ovlp m, 1H), 1.19 (d, J = 7.0 Hz, 3H), 0.97 (d, J- 6.7 Hz, 3H), 0.92 (ovlp d, J= 6.7 Hz, 5H), 0.88 (ovlp t, J= 7.5 Hz, 4H); ¹³C NMR (214 MHz, D₂0) δ 181.0, 174.2, 174.1, 172.8, 168.8, 162.8, 148.4, 138.3, 129.5, 128.4, 126.5, 124.3, 66.9, 59.0, 55.2, 51.5, 49.6, 49.4, 41.9, 40.8, 37.5, 36.6, 35.6, 32.4, 28.7, 24.3, 22.6, 21.7, 20.7, 18.3, 17.3, 17.0, 15.0, 9.9; HRMS calcd for

C₃₇H₅₇N₆0₆S⁺ [M + H⁺] 713.4060, found 713.4055.

Exam le 12. Standard Procedure for the preparation of compounds 27-31 from compound 20.

27; R = -tbu

28; R = -CF₃

29; R = OCH₃

30; R = NHCH₃

31 ; R = N(CH₃)₂ a. Standard Acylation Procedure:

To a solution of compound 20 (1 equiv) in CH₂C1₂ (5.0 mL) at 0 °C was added base (15-50 equiv) and acylating agent (10-25 equiv), and the reaction was stirred for up to 2 h. When TLC showed complete consumption of starting material, the reaction was quenched with 1.0 M aqueous HC1 (3 mL), and the layers were separated. The aqueous layer was extracted with CH₂C1₂ (3 x 3 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The crude intermediate was brought forward without purification,

b. Standard LiOH Hydrolysis Procedure:

To a solution of the intermediate in THF (2.5 mL), MeOH (0.5 mL), and H₂0 (0.5 mL) was added LiOH«H₂0 (21 mg) to make a 0.14 M reaction solution. If complete consumption of starting material was not observed by TLC after 18 h, additional LiOH»H₂0 (21 mg) was added. After TLC and crude MS showed loss of starting material, the organic solvents were removed under reduced pressure and the remaining aqueous mixture was diluted with additional H₂0 (3 mL). The aqueous mixture was acidified to pH 2 with 1.0 M aqueous HC1 solution and extracted with CH₂C1₂ (5 x 3 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced (

pressure. Purification by HPLC afforded the title compounds.

Example 13. Preparation of compound 27.

Compound 27 was obtained using compound 20 (5.2 mg, 6.8 μπιοΐ, 1 equiv), pyridine (14 μί, 0.17 mmol, 25 equiv) and trimethylacetyl chloride (21 μί, 0.17 mmol, 25 equiv) following the standard procedure outlined in Example 12 and after purification by HPLC (C₁₈, 150 x 10 mm, 30- 35% MeCN/25 mM aqueous N¾OAc, pH 4.78 over 3 min, 35% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 7 min, 90% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 5 min, 5 niL/min). White solid (1.68 mg, 33% over 2 steps); HPLC rt = 8.6 min; R_f= 0.5 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (601 MHz, MeOD with N^OAc) δ 7.98 (s, 1H), 7.25-7.19 (m, 4H), 7.17-7.11 (m, 1H), 5.14 (dd, J= 11.5, 3.3 Hz, 1H), 4.36-4.28 (m, 1H), 4.19 (d, J= 8.7 Hz, 1H), 3.93 (ddd, J= 11.5, 4.9, 3.0 Hz, 1H), 3.00-2.94 (m, 2H), 2.91 (dd, J= 13.6, 7.2 Hz, 1H), 2.67 (dd, J= 11.1, 2.8 Hz, 1H), 2.53- 2.46 (m, 1H), 2.27 (ddd, J= 14.7, 11.7, 3.4 Hz, 1H), 2.22 (s, 3H), 2.20-2.11 (m, 2H), 2.00 (ddd, J= 14.2, 9.4, 4.7 Hz, 1H), 1.88-1.71 (m, 4H), 1.69-1.53 (m, 5H), 1.36-1.27 (m, 2H), 1.25 (s, 9H), 1.22- 1.16 (m, 1H), 1.15 (d, J= 7.0 Hz, 3H), 0.97 (d, J= 6.8 Hz, 3H), 0.95 (d, J= 4.8 Hz, 3H), 0.94 (d, J= 4.8 Hz, 3H), 0.91 (t, J= 7.4 Hz, 3H); ¹³C NMR (226 MHz, MeOD with N¾OAc) 6 184.3, 181.2, 179.9, 176.1, 175.1, 173.7, 162.8, 151.0, 139.8, 130.6, 129.2, 127.3, 123.9, 70.4, 59.5, 56.6, 52.3, 51.4, 50.7, 44.5, 41.5, 40.6, 39.8, 39.6, 37.4, 36.5, 34.1, 31.4, 27.8, 26.0, 24.1, 23.9, 19.5, 19.4, 18.5, 16.4, 11.0; HRMS calcd for C₄₀H₆₃N₆O₆S⁺ [M + H⁺] 755.4530, found 755.4546. Example 14. Preparation of compound 28.

28

Compound 28 was obtained using compound 20 (2.2 mg, 2.9 μπιοΐ, 1 equiv), pyridine (11 L, 0.14 mmol, 50 equiv), and TFAA (4 μί, 0.03 mmol, 10 equiv) following the standard procedure outlined in Example 12 and after purification by HPLC (C₁₈, 150 x 10 mm, 10-50% MeCN/25 mM aqueous NFLtOAc, pH 4.78 over 10 min, 90% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 5 min, 5 mL/min). White solid (0.22 mg, 10% over 2 steps); HPLC rt = 9.9 min; R_f= 0.2 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (900 MHz, MeOD) δ 8.08 (d, J= 3.0 Hz, 1H), 7.31-7.23 (m, 4H), 7.20- 7.15 (m, 1H), 5.24 (dd, J= 11.3, 3.4 Hz, 1H), 4.41-4.35 (m, 1H), 4.23 (d, J= 8.4 Hz, 1H), 3.93 (ddd, J= 11.4, 5.4, 2.9 Hz, 1H), 3.28-3.25 (m, 1H), 3.19-3.10 (m, 1H), 2.98-2.92 (m, 2H), 2.62-2.47 (m, 2H), 2.44 (s, 3H), 2.32 (ddd, J= 14.6, 11.6, 3.4 Hz, 1H), 2.26 (ddd, J= 14.4, 1 1.4, 2.9 Hz, 1H), 2.02 (ddd, J= 13.9, 9.5, 4.3 Hz, 1H), 1.97-1.80 (m, 4H), 1.77 (d, J- 13.6 Hz, 1H), 1.72-1.59 (m, 4H), 1.50-1.41 (m, 1H), 1.26-1.20 (m, 1H), 1.18 (d, J = 7.1 Hz, 3H), 1.02 (d, J= 6.8 Hz, 3H), 0.98 (t, J= 7.3 Hz, 6H), 0.96-0.92 (m, 3H); ¹³C NMR (226 MHz, MeOD) δ 173.8, 172.9, 162.7, 160.7, 159.2, 151.2, 139.7, 130.5, 129.3, 127.4, 124.6, 69.6, 59.8, 56.3, 52.4, 51.1, 50.9, 43.9, 41.6, 38.9, 37.5,

36.7, 33.8, 31.0, 25.9, 25.3, 23.5, 19.5, 19.4, 18.9, 18.5, 16.2, 11.1; HRMS calcd for C₃₇H₅₄F₃N₆0₆S⁺ [M + H⁺] 767.3778, found 767.3776.

Example 15. Preparation o

29

Compound 29 was obtained using compound 20 (4.9 mg, 6.4 μπιοΐ, 1 equiv), Et₃N (13 iL, 0.094 mmol, 15 equiv) and methyl chloroformate (12 μί, 0.16 mmol, 25 equiv) following the standard procedure outlined in Example 12 and after purification by HPLC (C₁₈, 150 x 10 mm, 20- 25% MeCN/0.05% aqueous formic acid over 5 min, 25% MeCN/0.05% aqueous formic acid for 10 min, 90% MeCN/0.05% aqueous formic acid for 5 min, 5 mL/min). Off-white solid (0.69 mg, 15% over 2 steps); HPLC rt = 10.3 min; R_f= 0.3 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (900 MHz, MeOD with NH₄0Ac) δ 7.98 (s, 1H), 7.25-7.19 (m, 4H), 7.16-7.10 (m, 1H), 4.32-4.27 (m, 1H), 4.21 (d, J = 8.7 Hz, 1H), 4.00-3.92 (m, 1H), 3.66 (s, 3H), 2.98 (dd, J= 13.8, 5.6 Hz, 1H), 2.92 (dd, J= 13.3, 8.1 Hz, 2H), 2.55 (dd, J= 11.2, 2.5 Hz, 1H), 2.50-2.44 (m, 1H), 2.21-2.13 (ovlp m, 1H), 2.17 (s, 3H), 2.09-1.97 (m, 3H), 1.83-1.72 (m, 3H), 1.66-1.52 (m, 5H), 1.36-1.25 (m, 3H), 1.24-1.16 (m, 1H), 1.14 (d, J= 7.0 Hz, 3H), 1.00 (d, J= 6.7 Hz, 3H), 0.96-0.90 (m, 9H); ¹³C NMR (226 MHz, MeOD with NILtOAc) δ 185.0, 176.5, 175.7, 173.9, 162.8, 159.0, 151.4, 139.9, 130.6, 129.2, 127.2, 123.9, 70.7, 59.3, 56.7, 52.8, 52.4, 52.3, 51.8, 44.7, 41.3, 41.2, 39.8, 37.5, 34.1, 31.6, 30.8, 26.2, 26.0, 24.4, 19.6, 19.5, 18.5, 16.3, 11.0; HRMS calcd for C₃₇H₅₇N₆0₇S⁺ [M + H⁺] 729.4009, found 729.4013.

Example 16. Preparation of compound 30.

30

To a solution of compound 20 (5.3 mg, 7.0 μπιοΐ, 1 equiv) in MeCN (1.0 mL) was added imidazolium 33 (2.8 mg, 10 μπιοΐ, 1.5 equiv) and Et₃N (1 drop) and the resulting solution was stirred overnight. After 18 h, TLC (5% MeOH:CH₂Cl₂) showed complete consumption of starting material. The reaction was concentrated under reduced pressure, partitioned between C¾C1₂ (3 mL) and 1.0 M aqueous HC1 solution (3 mL), and the layers were separated. The aqueous layer was extracted with CH2CI2 (3 x 1 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The resulting crude product was hydrolyzed using the standard LiOH hydrolysis procedure outlined in Example 12. Purification by HPLC (C₁₈, 150 x 10 mm, 10- 50% MeCN/25 mM aqueous N^OAc, pH 4.78 over 10 min, 90% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 5 min, 5 mL/min) afforded the title compound as a white solid (0.86 mg, 17% yield over 2 steps). HPLC rt = 8.3 min; R_f= 0.2 (Si0₂, 10% MeOH:CH₂Cl₂); HRMS calcd for C₃₇H₅₈N₇0₆S⁺ [M + H⁺] 728.4169, found 728.4164. Example 17. Preparation of compound 33.

32 33

Preparation of N-methyl-lH-imidazole-l-carboxamide (32). To a suspension of CDI (3.57 g, 22.0 mmol, 1.1 equiv) in CH₂C1₂ (20 mL) at 0 °C was added a solution of methylamine (2.0 M in THF, 10 mL, 20 mmol, 1 equiv) dropwise and the resulting orange solution was stirred while warming to room temperature overnight. After 72 h, the reaction was quenched with H₂0 (20 mL) and the layers were separated. The aqueous layer (pH 8) was extracted with CH₂C1₂ (4 x 10 mL), and the combined organic layers were dried (Na₂S04), filtered, and concentrated under reduced pressure. The resulting orange/white solid (1.363 g) was a 1 :1 mixture of 32:imidazole by 1H NMR. A portion of this crude intermediate was purified by partitioning between CH₂C1₂ (15 mL) and saturated aqueous NH4CI (15 mL), and the layers were separated. The aqueous layer was extracted with CH₂C1₂ (3 x 10 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The resulting white solid was exclusively 32 by ^!H NMR and was used without further purification. 1H NMR (400 MHz, CDC1₃) δ 8.20 (s, 1 H), 7.34 (s, 1 H), 7.10 (s, 1H), 5.92 (br s, 1H), 3.05 (d, J= 4.8 Hz, 3H).

Preparation of 3 -methyl- l-(methylcarbamoyl)-lH-imidazol-3-ium iodide (33). To a solution of 32 (39.8 mg, 0.318 mmol, 1 equiv) in MeCN (3 mL) was added Mel (80 μί, 1.3 mmol, 4 equiv) dropwise. After 72 h, the reaction was concentrated under reduced pressure, and the resulting crude was triturated with CH₂C1₂ (3 x 3 mL) leaving the title compound (5.6 mg, 7% yield) as a yellow solid. 1H NMR (400 MHz, CDC1₃) δ 10.97 (s, 1H), 9.21 (br s, 1H), 8.10 (s, 1H), 7.24 (s, 1H), 4.09 (s, 3H), 3.06 (d, J= 4.6 Hz, 3H).

Example 18. Preparation of compound 31.

31 To a solution of compound 20 (5.6 mg, 7.4 μπιοΐ, 1 equiv) in MeCN (1.0 mL) was added imidazolium 35 (4.1 mg, 15 μηιοΐ, 2 equiv) and Et₃N (1 drop) and the resulting solution was stirred overnight. After 48 h, no reaction had occurred by TLC (3 x 5% MeOH:CH₂Cl₂) or crude MS. Additional 35 (21 mg, 75 μπιοΐ, 10 equiv) was added as a solution in MeCN (1.0 mL) and the resulting solution was stirred overnight. After an additional 40 h, no reaction had occurred by crude MS, so a catalytic amount of DMAP was added and the resulting solution was heated to reflux and stirred overnight. After an additional 48 h, crude MS showed some reaction progress. Additional 35 (98 mg, 350 μπιοΐ, 47 equiv) was added to the reaction and the solution was heated to reflux and stirred overnight. After an additional 24 h, TLC showed complete consumption of starting material. The reaction was concentrated under reduced pressure, taken up in 1.0 M aqueous HC1 (3 mL), and the resulting aqueous mixture was extracted with CH₂C1₂ (3 x 3 mL). The combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The resulting crude product was hydrolyzed using the standard LiOH hydrolysis procedure of Example 12. Purification by HPLC (C₁₈, 150 x 10 mm, 10-50% MeCN/25 mM aqueous NH₄OAC, pH 4.78 over 10 min, 90% MeCN/25 mM aqueous NH4OAC, pH 4.78 for 5 min, 5 mL/min) afforded the title compound as a white solid (0.55 mg, 10% yield over 2 steps). HPLC rt = 8.7 min; R_f= 0.2 (Si0₂, 10%

MeOH:CH₂Cl₂); 1H NMR (850 MHz, MeOD) δ 7.97 (s, IH), 7.28-7.20 (m, 4H), 7.19-7.12 (m, IH), 5.02 (dd, J= 11.1, 3.6 Hz, IH), 4.38^1.30 (m, IH), 4.19 (d, J= 8.7 Hz, IH), 4.00 (ddd, J= 10.9, 5.0, 3.2 Hz, IH), 3.95-3.69 (m, 2H), 3.03 (d, J- 11.8 Hz, IH), 2.95 (s, 6H), 2.88-2.83 (m, IH), 2.57 - 2.51 (m, IH), 2.30 (s, 3H), 2.23 (ddd, J= 14.6, 11.2, 3.8 Hz, IH), 2.13 (ddd, J= 14.3, 11.2, 3.1 Hz, IH), 2.00 (ddd, J= 13.9, 9.3, 4.6 Hz, IH), 1.90-1.54 (m, 10H), 1.40-1.31 (m, IH), 1.24-1.17 (m, IH), 1.15 (d, J= 7.1 Hz, 3H), 0.98 (d, J - 6.8 Hz, 3H), 0.95 (d, J= 6.9 Hz, 3H), 0.94 (d, J= 6.8 Hz, 3H), 0.91 (t, J= 7.4 Hz, 3H); ^,3C NMR (214 MHz, MeOD) δ 183.1, 178.9, 177.9, 174.3, 173.7, 163.0, 160.0, 150.9, 139.8, 130.5, 129.3, 127.3, 123.9, 70.1, 69.0, 59.6, 56.4, 52.5, 52.3, 51.3, 44.3, 41.6, 39.7, 39.2, 37.5, 37.4, 36.7, 33.8, 31.2, 26.1, 25.7, 23.9, 23.3, 19.6, 19.1, 18.4, 16.3, 11.0; HRMS calcd for C₃₈H₆₀N₇O₆S⁺ [M + H⁺] 742.4326, found 742.4310.

Example 19. Preparation of compound 35.

34 35 (

Preparation of N,N-Dimethyl-lH-imidazole-l-carboxamide (34). To a suspension of CDI (3.57 g, 22.0 mmol, 1.1 equiv) in CH₂C1₂ (20 mL) at 0 °C was added Na₂C0₃ (2.12 g, 20.0 mmol, 1 equiv) and dimethyl amine hydrochloride (1.63 g, 20.0 mmol, 1 equiv), and the resulting suspension was stirred while warming to room temperature overnight. After 72 h, the reaction was quenched with H₂0 (20 mL), diluted with CH₂C1₂ (20 mL), and the layers were separated. The aqueous layer (pH 11) was extracted with CH₂C1₂ (4 x 10 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The resulting yellow liquid (2.63 g) was a 2: 1 mixture of 34: imidazole by 1H NMR. A portion of this crude intermediate was purified by partitioning between CH₂C1₂ (15 mL) and saturated aqueous N¾C1 (15 mL), and the layers were separated. The aqueous layer was extracted with CH₂C1₂ (3 x 10 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The resulting orange liquid, which solidified to an orange solid upon standing, was exclusively 34 by ^!H NMR and was used without further purification. 1H NMR (400 MHz, CDC1₃) δ 7.90 (s, 1H), 7.24 (br s, 1H), 7.09 (s, 1H), 3.11 (s, 6H).

Preparation of 1 -(dimethylcarbamoyl)-3 -methyl- lH-imidazol-3-ium iodide (35). The same procedure for the preparation of compound 33 of Example 18 was followed using compound 34 (161.5 mg, 1.16 mmol) to afford the title compound (325.1 mg, 99.7% yield) as an orange oil that solidified upon standing. 1H NMR (400 MHz, CDC1₃) δ 10.72 (s, 1H), 7.65 (s, 1H), 7.33 (s, 1H), 4.30 (s, 3H), 3.32 (br s, 6H).

Example 20. Pre aration of compound 102.

(

To a solution of the compound 101 (5.1 mg, 6.7 μπιοΐ, 1 equiv) (Balasubramanian, R., et al., Total Synthesis and Biological Evaluation of Tubulysin U, Tubulysin V, and Their Analogues, J Med. Chem., 2009, 52, 238-240) in CH₂C1₂ (5 mL) at 0 °C was added chloromethyl methyl ether (20 μί, 0.27 mmol, 40 equiv) and DIPEA (50 μί, 0.29 mmol, 40 equiv). After 2 h, TLC (5%

MeOH:CH₂Cl₂) showed complete consumption of starting material, and the reaction was quenched with 0.1 M aqueous HCl (5 mL) resulting in a biphasic mixture with a white precipitate. The layers were separated, the aqueous layer was extracted with CH₂C1₂ (2 x 5 mL), and the combined organic layers were washed with saturated aqueous NaCl (2 x 5 mL). The resulting clear organic layer was dried (Na₂S0₄), filtered, and concentrated under reduced pressure. The crude intermediate was brought forward without purification and subjected to the standard LiOH hydrolysis procedure.

Purification by HPLC (C₁₈, 150 x 10 mm, 10-50% MeCN/25 mM aqueous NH₄OAC, pH 4.78 over 10 min, 90% MeCN/25 mM aqueous NHUOAc, pH 4.78 for 5 min, 5 mL/min) afforded the title compound (0.52 mg, 11% over 2 steps) as a white solid. HPLC rt = 10.8 min; R_f= 0.2 (Si0₂, 10% MeOH:CH₂Cl₂); HRMS calcd for C₃₇H₅₈N₅0₇Si⁺ [M + H⁺] 716.4057, found 716.4051.

Example 21. Preparation of compound 103.

Compound 103 was prepared from compound 104 using the standard procedure in Example 22 below at room temperature instead of -20 °C. 1H NMR (400 MHz, D₂0) δ 8.28 (d, J= 9.7 Hz, 1H), 7.96 (s, 1H), 7.34-7.10 (m, 5H), 5.85 (d, J- 10.9 Hz, 1H), 4.96 (q, J= 6.8 Hz, 1H), 4.24 (d, J= 7.8 Hz, 1H), 4.01-3.91 (m, 1H), 3.87 (dd, J= 12.3, 2.7 Hz, 1H), 3.55 (d, J= 12.2 Hz, 1H), 3.19-2.95 (m, 3H), 2.78 (s, 3H), 2.43-2.26 (m, 2H), 2.23 (s, 3H), 2.20-1.44 (m, 12H), 1.28-1.17 (m, 1H), 1.14 (d, J- 6.9 Hz, 3H), 1.00 (d, J= 6.7 Hz, 3H), 0.92 (t, J= 5.8 Hz, 9H); HPLC (C₁₈, 250 x 10 mm, 0- 20% MeCN/0.04% aqueous HCl over 2 min, 20% MeCN/0.04% aqueous HCl for 2 min, 20-90% MeCN/0.04% aqueous HCl over 35 min, 90% MeCN/0.04% aqueous HCl for 5 min, 90-10% MeCN/0.04% aqueous HCl over 5 min, 3 mL/min) rt = 21.3 min; low resolution MS calcd for C₃₇H₅₄N₅0₆Si⁺ [M + H⁺] 696.38, found 696.31. (

Example 22. Standard procedure for compound 104 acylation using anhydrides in pyridine.

To a solution of compound 4 (1 equiv) (Balasubramanian, R., et al., Total Synthesis and Biological Evaluation of Tubulysin U, Tubulysin V, and Their Analogues, J Med. Chem. , 2009, 52, 238-240) in pyridine (0.035 M) at -20 °C was added the anhydride (100 equiv) and the solution was placed in a -20 °C freezer overnight. After 24-48 h, TLC (10% MeOH:CH₂Cl₂, using an aliquot partitioned between 1.0 M aqueous HC1 and EtOAc) showed complete consumption of starting material. The reaction was poured directly into 1.0 M aqueous HC1 (30 mL) and extracted with EtOAc (15 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 x 5 mL), and the combined organic layers were dried (Na₂S0₄), filtered, and concentrated under reduced pressure. Purification by HPLC afforded the title compound.

Example 23. Standard procedure for compound 104 acylation using DCC activated acids.

a solution of the acid (10 equiv) in CH₂C1₂ (5 mL) at 0 °C was added DMAP (1 equiv) and (

DCC (5 equiv) and the reaction was stirred while warming to room temperature over 4.5 h. The resulting mixture was re-cooled to 0 °C and cannulated into a flask pre-loaded with 104 (1 equiv) and the reaction stirred while warming to room temperature overnight. After 12—48 h, TLC (10% MeOH:CH₂Cl₂) showed complete consumption of starting material. The reaction was concentrated under reduced pressure, the crude solid was mixed with MeCN (3 mL), and filtered through a plug of cotton. The solution was concentrated under reduced pressure and purified by HPLC to afford the title compound.

Example 24. Preparation of compound 105.

After subjecting compound 104 (5.9 mg, 8.8 μπιοΐ) and formic acetic anhydride (55 μί, 0.88 mmol, Krimen, 1. 1., Acetic Formic Anhydride, Org. Synth, 1970, 50, 1-3) to the standard procedure for compound 104 acylation using anhydrides in pyridine, low resolution MS showed complete consumption of starting material after 24 h with exclusive production of the title compound following aqueous reaction workup. During HPLC purification (C₁₈, 150 x 10 mm, 32% MeCN/25 rnM aqueous NIL_tOAc, pH 4.78 for 10 min, 90% MeCN/25 mM aqueous NHjOAc, pH 4.78 for 5 min, 5 mL/min), partial degradation back to compound 104 occurred, followed by full degradation of purified 105 back to 104 upon heated centrifugal evaporation of the HPLC fractions, as shown by ^lH NMR, ¹³C NMR and low resolution MS. HPLC rt = 9.2 min; R_f= 0.25 (Si0₂, 10% MeOH:CH₂Cl₂); low resolution MS calcd for C₃₆H₅₄N₅0₇S⁺ [M + H⁺] 700.37, found 700.21.

Example 25. Preparation of compound 106.

After subjecting compound 104 (5.2 mg, 7.7 μιηοΐ) and benzoic anhydride (175 μί, 0.77 mmol) to the standard procedure for compound 104 acylation using anhydrides in pyridine, TLC analysis showed complete consumption of starting material after 24 h. Following aqueous reaction workup using the standard protocol, the title compound was obtained as a mixture with compound 4 and the intramolecularly cyclized lactams of 106 and 104. Low resolution MS calcd for

C₄₂H₅₈N₅0₇S⁺ [M + If"] 776.41, found 776.24.

Example 26. Preparation of compound 107.

The title compound was obtained using compound 104 (4.8 mg, 7.1 μιηοΐ) and butyric anhydride (0.12 mL, 0.71 mmol) after 18 h following the standard procedure for compound 104 acylation using anhydrides in pyridine and after purification by HPLC (C₁₈, 150 x 10 mm, 50% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 10 min, 90% MeCN/25 mM aqueous NtLOAc, pH 4.78 for 5 min, 5 mL/min). White solid (3.10 mg, 58% yield); HPLC rt = 2.7 min; R_f= 0.35 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (400 MHz, MeOD) δ 8.07 (s, 1 H), 7.23 (d, J = 4.2 Hz, 4H), 7.19- 7.11 (m, 1H), 5.92 (dd, J= 10.8, 2.7 Hz, 1H), 4.41^1.29 (m, 1H), 4.21 (d, J= 8.4 Hz, 1H), 3.96 (ddd, J= 8.3, 5.2, 2.7 Hz, 1H), 3.11 (d, J= 11.8 Hz, 1H), 3.05 (dd, J= 11.2, 2.6 Hz, 1H), 2.92 (d, J= 6.7 Hz, 2H), 2.60-2.37 (ovlp m, 4H), 2.40 (s, 3H), 2.24 (ddd, J= 14.0, 10.9, 2.9 Hz, 1H), 2.18-2.08 (m, 1H), 1.99 (ddd, J= 13.9, 9.7, 4.1 Hz, 1H), 1.93-1.53 (m, 11H), 1.48-1.34 (m, 1H), 1.26-1.18 (m, 1H), 1.16 (d, J= 7.1 Hz, 3H), 1.02-0.88 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ 174.2, 173.7,

173.4, 171.9, 162.7, 151.0, 139.7, 130.5, 129.3, 127.4, 124.9, 71.2, 69.7, 59.6, 56.4, 52.0, 51.1, 44.0, 41.7, 39.1, 38.0, 37.6, 36.8, 33.8, 31.1, 25.9, 25.4, 23.5, 22.4, 19.4, 19.3, 18.5, 16.2, 14.1, 11.1;

HRMS calcd for C₃₉H₆₀N₅O₇S⁺ [M + H⁺] 742.4213, found 742.4198. Example 27. Preparation of compound 108.

The title compound was obtained using compound 104 (4.5 mg, 6.7 μπιοΐ), propionic acid (5 67 μιηοΐ), DMAP (1 mg, 8 μηιοΐ) and DCC (6.8 mg, 33 μπιοΐ) after 48 h following the standard procedure for compound 104 acylation using DCC activated acids and after purification by HPLC (Ci8, 150 x 10 mm, 50% MeCN/25 mM aqueous NH₄OAC, pH 4.78 for 10 min, 90% MeCN/25 mM aqueous N¾OAc, pH 4.78 for 5 min, 5 mL/min). White solid (4.3 mg, 89% yield); HPLC rt = 2.4 min; R_f= 0.3 (Si0₂, 10% MeOH:CH₂Cl₂); 1H NMR (600 MHz, MeOD with NH₄OAc) δ 8.07 (s, 1H), 7.23 (d, J= 4.2 Hz, 4H), 7.18-7.12 (m, 1H), 5.92 (dd, J= 11.0, 2.5 Hz, 1H), 4.38-4.31 (m, 1H), 4.22 (d, J= 8.3 Hz, 1H), 3.99-3.94 (m, 1H), 3.12 (d, J= 12.0 Hz, 1H), 3.05 (dd, J= 1 1.1, 2.6 Hz, 1H), 2.92 (d, J= 6.8 Hz, 2H), 2.57-2.41 (m, 4H), 2.40 (s, 3H), 2.24 (ddd, J= 13.9, 1 1.2, 2.7 Hz, 1H), 2.13 (ddd, J= 14.4, 11.9, 2.6 Hz, 1H), 2.00 (ddd, J= 13.8, 9.4, 4.2 Hz, 1H), 1.91-1.53 (m, 9H),

I.45-1.36 (m, 1H), 1.24-1.18 (m, 1H), 1.18-1.12 (m, 6H), 0.99 (d, J= 6.7 Hz, 3H), 0.95 (d, J= 7.0 Hz, 6H), 0.92 (d, J= 7.4 Hz, 3H); ¹³C NMR (151 MHz, MeOD with NH₄OAC) δ 182.3, 177.8, 175.0, 173.7, 173.4, 171.9, 162.7, 151.0, 139.7, 130.5, 129.3, 127.3, 124.8, 71.2, 69.7, 59.6, 56.3, 52.0,

51.1, 44.1, 42.5, 41.8, 39.2, 38.0, 37.6, 33.8, 31.1, 28.3, 25.9, 25.4, 23.5, 22.5, 19.5, 19.0, 18.6, 16.3,

I I.1, 9.3; HRMS calcd for CasHssNsC^ [M + H⁺] 728.4057, found 728.4076.

Example 28. Preparation of compound 109.

After subjecting compound 104 (4.8 mg, 7.1 μηιοΐ), isovaleric acid (7.9 μΐ,, 71 μηιοΐ), DMAP (0.9 mg, 7 μπιοΐ) and DCC (7.4 mg, 36 μπιοΐ) to the standard procedure for compound 104 acylation using DCC activated acids, TLC analysis showed complete consumption of starting material after 24 h and low resolution MS showed exclusive production of the title compound. MS calcd for C₄oH₆₂N₅0₇S⁺ [M + H⁺] 756.44, found 756.52.

Example 29. Preparation of compound 110.

Modification to the standard procedure for compound 104 acylation using DCC activated acids was performed with compound 104 (4.7 mg, 7.0 μη οΐ), pivalic acid (7.1 mg, 70 μπιοΐ), DMAP (0.85 mg, 7 μπιοΐ) and DCC (7.2 mg, 35 μπιοΐ): After 24 h, no reaction progress had occurred by low resolution MS, so the reaction was heated to 45 °C. After an additional 24 h, no reaction progress had occurred by low resolution MS. To a separate container with a solution of pivalic acid (36 mg, 50 equiv) in CH₂C1₂ (1.5 mL) at 0 °C was added DMAP (1 mg, 1 equiv) and DCC (36 mg, 25 equiv), resulting immediately in a white precipitate. After 15 min, to this mixture was added the heated compound 104 mixture. After an additional 30 h, no reaction progress had occurred by low resolution MS, so DMF (6 mL) was added to the reaction and the mixture was heated to 80 °C.

After an additional 36 h, TLC analysis showed complete consumption of starting material and low resolution MS showed exclusive production of the title compound. MS calcd for C₃₅H₅₂N₅0₅S⁺ [M + H⁺] 654.37, found 654.

Example 30. Preparation of com ound 111.

Modification to the standard procedure for compound 104 acylation using DCC activated acids was performed with compound 104 (5.2 mg, 7.7 μπιοΐ), chloroacetic acid (7.3 mg, 77 μπιοΐ), DMAP

(0.94 mg, 7.7 μπιοΐ) and DCC (7.9 mg, 39 μπιοΐ): After 48 h, no reaction progress had occurred by

TLC analysis. To a separate container with a solution of chloroacetic acid (37 mg, 50 equiv) in (

CH₂C1₂ (3 mL) at 0 °C was added DMAP (1 mg, 1 equiv) and DCC (40 mg, 25 equiv), resulting in a white precipitate and effervescence to immediately form. After 15 min, to this mixture was added the compound 104 mixture. After an additional 30 h, TLC analysis showed complete consumption of starting material and low resolution MS showed exclusive production of the title compound. MS calcd for C₃₇H₅₅C1N₅0₇S⁺ [M + H⁺] 748.35, found 748.42.

Example 31. Preparation of compound 112 and compound 113.

113

Modification to the standard procedure for compound 104 acylation using DCC activated acids was performed with compound 104 (6.5 mg, 9.7 μπιοΐ), acrylic acid (6.6 μΐ,, 97 μηιοΐ), DMAP (1.2 mg, 9.7 μηιοΐ) and DCC (9.9 mg, 48 μπιοΐ): After 72 h, partial reaction progress had occurred by low resolution MS, which showed peaks corresponding to the starting material and compound 112 exclusively. To a separate flask with a solution of acrylic acid (6.6 μί, 10 equiv) in CH₂C1₂ (1.5 mL) at 0 °C was added DMAP (1 mg, 1 equiv) and DCC (9.9 mg, 5 equiv), and after 15 min, to this solution was added the compound 104 mixture. After an additional 36 h, TLC analysis showed complete consumption of starting material and low resolution MS showed production of the title compounds. MS calcd for C₃₈H₅₆N₅0₇S⁺ [112 + H⁺] 726.39, found 726.47; low resolution MS calcd for C₃₈H₅₄N₅0₆S⁺ [113 + H⁺] 708.38, found 708. (

Example 32. The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X'), for therapeutic or prophylactic use in humans.

(Ϊ) Tablet 1 mg/tablet

Compound X= 100.0

Lactose 77.5

Povidone 15.0

Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 1Q

300.0

(u) Tablet 2 mg/tablet

Compound X= 20.0

Microcrystalline cellulose 410.0

Starch 50.0

Sodium starch glycolate 15.0

Magnesium stearate 5

500.0

Cm) Capsule mg/capsule

Compound X= 10.0

Colloidal silicon dioxide 1.5

Lactose 465.5

Pregelatinized starch 120.0

Magnesium stearate M

600.0

(iv) Injection 1 (1 mg/ml) mg/ml

Compound X= (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide solution

(pH adjustment to 7.0-7.5) q.s.

Water for injection q.s. ad 1

(v) Injection 2 Π0 mg/ml) mg/ml

Compound X= (free acid form) 10.0

Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

1.0 N Sodium hydroxide solution

(pH adjustment to 7.0-7.5) q.s.

Water for injection q.s. ad 1 mL (

(νϊ) Aerosol mg/can

Compound X= 20.0

Oleic acid 10.0

Trichloromonofluoromethane 5,000.0

Dichlorodifluoromethane 10,000.0

Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures well known in the pharmaceutical art.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

is claimed is: A compound of formula I:

I

wherein:

R¹ is (d-C₆)alkyl;

each R is independently H or (C₁-C₆)alkyl;

R³ is H or (CrQ alkyl;

R⁴ is (Q-Q alkyl;

R⁵ is H or (C_rC₆)alkyl;

R⁶ is (d-Q alkyl;

R⁷ is H or (C₁-C₆)alkyl, and R⁸ is H, -C(=0)(C_!-C₆)alkyl, -C(=0)(C₂- C₆)alkenyl, -C(=0)aryl, -C(=0)OR^a, or -C(=0)NR^bR^c, wherein any -C(=0)(C₁-C₆)alkyl

o

or -C(=0)(C₂-C₆)alkenyl of R is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (C_rC₃)alkyl, -OH, -0(C_!-C₃)alkyl, -C0₂H, and -C(=0)NR^bR^c; or R and R together with the nitrogen to which they are attached form a 4-10 membered

heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from halogen, oxo,

-OH and -0(C₁-C₃)alkyl;

when R¹² is NR⁷R⁸ then R⁹ is H or (C C₆)alkyl, and R¹⁰ is (C C₆)alkyl wherein the (C C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo;

when R¹² is OR¹³ then R¹³ is R^13a, R⁹ is H or (Ci-C₆)alkyl, and R¹⁰ is (C C₆)alkyl wherein the (C C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R¹³ is R^13b, and R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo;

R¹¹ is aryl, wherein any aryl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (Ci-C3)alkyl, -OH or -0(C!-C3)alkyl;

NR⁷R⁸ or OR 13.

R ^a is -(C_rC₆)alkylO(Ci-C6)alk l, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂- C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C₂- C₆)alkyl or -C(=0)(C₂-C6)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R^13b is H, -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(Ci-C₆)alk l, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C₁-C₆)alkyl or -C(=0)(C₂- C₆)alkenyl of R^13a is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C]- C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R^a is (C C₆)alkyl;

R^b and R^c are each independently selected from H and (C₁-C₆)alkyl; or R^b and R^c together with the nitrogen to which they are attached form a 4, 5, 6 or 7 heterocycle wherein the heterocycle is optionally substituted with one or more groups selected from (Ci-C₆)alkyl and oxo; and

n is 1, 2, 3 or 4;

or a salt thereof.

2. The compound of claim 1 which is a compound of formula la:

la

wherein:

R is (C_!-C₆)alkyl;

each R is independently H or (CrC₆)alkyl;

R³ is H or (CrQ alkyl; (

R⁴ is (Ci-Qdalkyl;

R⁵ is H or (d-C₆)alkyl;

R⁶ is (d-C₆)alkyl;

R⁷ is H or (C C₆)alkyl, and R⁸ is H, -C(=0)(d-C₆)alkyl, -C(=0)(C₂- C₆)alkenyl, -C(=0)aryl, -C(=0)OR^a, or -C(-0)NR^bR^c, wherein any -C(=0)(Cj-C₆)alkyl

or -C(=0)(C2-C₆)alkenyl of R is optionally substituted with one or more groups selected from

o

halogen and wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH, -0(C₁-C₃)alkyl, -C0₂H, and -C(=0)NR^bR^c;

1 ft

or R and R together with the nitrogen to which they are attached form a 4-10 membered

heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from halogen, oxo, (C₁-C₃)alkyl, -OH and -0(d-C₃)alkyl;

R⁹ is H or (d-C₆)alkyl, and R¹⁰ is (C₁-C₆)alkyl wherein the (C₁-C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R⁹ and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (d-C )alkyl and oxo;

R¹¹ is aryl, wherein any aryl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH or -0(C_!-C₃)alkyl;

R^a is (d-C₆)alkyl;

R^b and R^e are each independently selected from H and (C₁-C6)alkyl; or R^b and R^c together with the nitrogen to which they are attached form a 4, 5, 6 or 7 heterocycle wherein the heterocycle is optionally substituted with one or more groups selected from (C₁-C₆)alkyl and oxo; and

n is 1 , 2, 3 or 4;

or a pharmaceutically acceptable salt thereof. 3. The compound of claim 1 wherein R⁹ is H or (C]-C₆)alkyl, and R¹⁰ is (d-C₆)alkyl wherein the (d-C₆)alkyl of R¹⁰ is substituted with one -C0₂H group.

4. The compound of claim 1 or claim 2, wherein R⁹ is H. 5. The compound of any one of claims 1 -4, wherein R¹⁰ is (C₂-C₄)alkyl substituted with one -C0₂H group. The compound of any one of claims 1-4, wherein R is:

The compound of any one of claims 1-6, wherein R is methyl.

The compound of any one of claims 1-7, wherein n is 3.

The compound of any one of claims 1-8, wherein each R is H.

The compound of any one of claims 1-9, wherein R is H.

The compound of any one of claims 1-10, wherein R⁴ is (C₃-C₅)alkyl. The compound of any one of claims 1-10, wherein R⁴ is butyl.

The compound of any one of claims 1-10, wherein R⁴ is:

The compound of any one of claims 1-13, wherein R⁵ is H.

The compound of any one of claims 1-14, wherein R⁶ is (C₂-C4)alkyl. The compound of any one of claims 1-14, wherein R⁶ is propyl.

The compound of any one of claims 1-14, wherein R⁶ is:

(

18. The compound of any one of claims 1-17, wherein R¹¹ is phenyl, wherein any phenyl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (Ci- C₃)alkyl, -OH and -0(d-C₃)alkyl. 19. The compound of any one of claims 1-17, wherein R¹¹ is phenyl.

20. The compound of any one of claims 1-19, wherein R⁷ is H, and R⁸ is -C(=0)(d- C₆)alkyl, -C(=0)(C₂-C₆)alkenyl, -C(=0)aryl, -C(=0)OR^a or -C(=0)NR^bR^c, wherein any -C(=0)(Ci-

Q

C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of R⁸ is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH, -0(CrC₃)alkyl, -C0₂H, and -C(=0)NR^bR^c.

21. The compound of any one of claims 1-19, wherein R⁷ is H, and R⁸ is -C(=0)(C]- C₆)alkyl, -C(=0)aryl, -C(=0)OR^a, or -C(=0)NR^bR^c, wherein any -C(=0)(C_!-C₆)alkyl of R⁸ is optionally substituted with one or more groups selected from halogen and wherein any -C(=0)aryl of

R 8 is optionally substi *tuted wi ·th one or more groups independently selected from halogen, (Q- C₃)alkyl, -OH, -0(C₁-C₃)alkyl, -C0₂H, and -C(=0)NR^bR^c.

22. The compound of any one of claims 1-19, wherein R⁷ is H, and R⁸ is -C(=0)OR^a, or -C(=0)NR^bR^c.

23. The compound of any one of claims 1-19, wherein R⁷ is H, and R⁸ is -C(=0)aryl, wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH, -0(C₁-C₃)alkyl, -C0₂H, and -C(=0)NR^bR^c.

24. The

The compound of claim 1 which is a compound of formula lb:

R¹ is (C_!-C₆)alkyl;

each R is independently H or (C₁-C₆)alkyl;

R³ is H or (C₁-C₆)alkyl;

R⁵ is H or (C C₆)alkyl;

R⁴ is (C,-C₆)alkyl;

R⁶ is (d-C₆)alkyl;

R¹³ is R^13a, R⁹ is H or (d-C^alkyl, and R¹⁰ is (C_!-C₆)alkyl wherein the (d-C₆)alkyl of R¹⁰ is substituted with one -C0₂H group; or R¹³ is R^13b, and R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C₁-C₆)alkyl and oxo;

R^13a is -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂- C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C₂- C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R^I3a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C C₃)alkyl, -OH and -0(C₁-C₃)alkyl;

R^13b is H, -(Ci-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(d-C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C_!-C₆)alkyl or -C(=0)(C₂- C₆)alkenyl of R^13a is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (d- C₃)alkyl, -OH and -0(d-C₃)alkyl;

R¹¹ is aryl, wherein any aryl of R⁹ is optionally substituted with one or more groups independently selected from halogen, (d-C₃)alkyl, -OH or -0(C₁-C₃)alkyl; and

n is 1, 2, 3 or 4;

or a pharmaceutically acceptable salt thereof. The com ound of claim 26 which is a compound of formula 1

Ic

or a pharmaceutically acceptable salt thereof wherein:

R^13a is -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)H, -C(=0)CH₂C1, -C(=0)(C₂-

C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl, -C(=0)(C₂- C₆)alkyl or -C(=0)(C₂-C )alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^13a is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH and -0(C_!-C₃)alkyl;

R⁹ is H or (d-C^alkyl; and

R¹⁰ is (C₁-C₆)alkyl, wherein the (Ci-C₆)alkyl is substituted with one -C0₂H group.

28. The compound of claim 26 or claim 27 wherein R^13a is

C₆)alkyl, -C(=0)(C₃-C₆)alkyl, -C(=0)(C₂-C₆)alkenyl or -C(=0)aryl, wherein any -(C₁-C₆)alkylO(C₁- C₆)alkyl, -C(=0)(C₃-C₆)alkyl or -C(=0)(C₂-C₆)alkenyl of R^13a is optionally substituted with one or more halogen and wherein any -C(=0)aryl of R^6a is optionally substituted with one or more groups independently selected from halogen, (C₁-C₃)alkyl, -OH and -0(C₁-C₃)alkyl.

29. The compound of claim 26 or claim 27 wherein R^13a is -C(=0)H, -C(=0)CH₂C1, - CH₂OCH₃, -C(=0)phenyl, -C(=0)CH₂CH₃, -C(=0)CH₂CH₂CH₃, -C(=0)CH₂CH(CH₃)₂, -C(=0)C(C H₃)_3, -C(=0)CH₂Cl or -C(=0)CH=CH₂.

30. The compound of any one of claims 26-29 wherein R⁹ is H. 31. The compound of any one of claims 26-30 wherein R¹⁰ is (C₂-C₄)alkyl substituted with one -C0₂H group.

32. The compound of any one of claims -30, wherein R¹⁰ is

The com ound of claim 26 which is a compound of formula Id:

or a pharmaceutically acceptable salt thereof wherein:

R^13b is H, -(C₁-C₆)alkylO(Ci-C₆)alkyl,

or -C(=0)aryl wherein any -(C₁-C₆)alkylO(C₁-C₆)alkyl,

or -C(=0)(C₂- C₆)alkenyl of R^6b is optionally substituted with one or more halogen, and wherein any -C(=0)aryl of R is optionally substituted with one or more groups independently selected from halogen, (Cj- C₃)alkyl, -OH and -0(C C₃)alkyl; and

R⁹and R¹⁰ together with the atoms to which they are attached form a 5, 6 or 7 membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C]-C₆)alkyl and oxo.

34. The compound of claim 26 or claim 33 wherein R⁹and R¹⁰ together with the atoms to which they are attached form a 5-membered heterocycle, wherein the heterocycle is optionally substituted with one or more groups independently selected from (C C₆)alkyl and oxo.

35. The compound of claim 26 or claim 33 wherein R⁹ and R¹⁰ together with the atoms to which they are attached form a pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted with one or more groups independently selected from (Q-C^alkyl or oxo. 36. The compound of claim 26 or claim 33 wherein R⁹and R¹⁰ together with the atoms to which they are attached form the moiety:

37. The compound of any one of claims 26-36 wherein R¹ is methyl.

38. The compound of any one of claims 26-37 wherein n is 3.

39. The compound of any one of claims 26-38 wherein each R is H.

40. The compound of any one of claims 26-39 wherein each R is H.

The compound of any one of claims 26-40 wherein R⁴ is (C3-C₅)alkyl

The compound of any one of claims 26-40 wherein R is butyl.

43. The compound of any one of claims 26-40 wherein R is:

44. The compound of any one of claims 26-43, wherein R⁵ is (C₂-C₄)alkyl.

45. The compound of any one of claims 26-44, wherein R⁶ is propyl.

46. The compound of any one of claims 26-44, wherein R⁶ is:

47. The compound of any one of claims 26-46 wherein R¹¹ is phenyl, wherein any phenyl of R¹¹ is optionally substituted with one or more groups independently selected from halogen, (Q- C₃)alkyl, -OH and -0(C₁-C₃)alkyl.

70

or a pharmaceutically acceptable salt thereof.

50. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1-49, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

51. A method for treating cancer in a mammal, comprising administering a therapeutically effective amount of a compound of formula I as described in any one of claims 1-49, or a pharmaceutically acceptable salt thereof to the mammal.

52. The method of claim 51 wherein the cancer is kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer or prostate cancer. 53. The method of claim 51 or claim 52 wherein the cancer is an multidrug resistant cancer.

54. A method for inhibiting cancer cell growth, comprising contacting the cancer cell in vitro or in vivo with a compound of formula I as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof.

55. A method for inhibiting tubulin polymerization in a mammal, comprising administering a compound of formula I as described in any one of claims 1-49, or a pharmaceutically acceptable salt thereof, to the mammal.

56. A compound of formula I as described in any of claims 1-49, or a pharmaceutically acceptable salt thereof for use in medical therapy. 57. A compound of formula I as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof, for the prophylactic or therapeutic treatment of cancer.

58. The compound of claim 57 wherein the cancer is kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer or prostate cancer.

59. The use of a compound as described in any one of claims 1-49 or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating cancer in a mammal.

60. The use of claim 59 wherein the cancer is kidney cancer, cervical cancer, breast cancer, colon cancer, leukemia, lung cancer, melanoma, ovarian cancer or prostate cancer.