COMPOUNDS AND METHODS FOR USE IN TREATING NEOPLASIA AND

CANCER

FIELD OF THE INVENTION

The present invention relates to a novel method for the treatment of neoplasia, including cancer and other diseases and conditions in animals, including mammals, especially humans. More particularly, in preferred aspects, the present invention provides a method for the use of a novel class of chemical agents which are inhibitors of isoprenylcysteine methyltransferase, for the treatment of both neoplasia and cancer, and a number of

hyperproliferative disorders, among others.

CLAIM OF PRIORITY AND GOVERNMENT RIGHTS

This application claims the benefit of priority of United States provisional application no. 61/511,698, filed July 26, 2011, of identical title, the entire contents of which are incorporated by reference herein.

This invention was made with government support under CA1 12483 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is a disease of abnormal cell growth often leading to death. Cancer is treated by three principal means; surgical removal of the tumor, therapeutic radiation, and treatment with anti-tumor chemical compounds. Treatment with chemical compounds, termed chemotherapy, is often hindered by the inherent toxicity of the chemicals to the patient and resistance of the tumor to the chemical treatment. Therefore the identification of less toxic anti-tumor agents capable of inhibiting growth of resistant tumors is of great importance.

Ras proteins and many other important signal transduction proteins must undergo significant post-translational modification in order to be functional in the eucaryotic cell. These proteins possess a signature carboxyl -terminal CaaX box motif (See Figure 1), with is recognized by one of the two prenyltransferases, FTase (protein-farnesyltransferase) or GGTase I (protein-geranylgeranyltransferase I). FTase transfers the 15-carbon farnesyl moiety to the cysteine residue in certain CaaX sequences, while GGTase I transfers the 20- carbon geranylgeranyl moiety to different CaaX boxes. Ras proteins and certain other proteins are farnesylated, but the majority of naturally-occuring CaaX proteins are geranylgeranylated by GGTase I. Subsequent to prenylation, CaaX motif proteins are subjected to removal of the aaX residues by the protease RCEl, followed by SAM-dependent methylation of the resulting cysteine carboxylate by Icmt. These two membrane-bound enzymes recognize and modify both farnesylated and geranylgeranylated proteins. The overall result of these three post-translational steps is to convert a hydrophillic protein into a more hydrophobic, membrane-associated one.

The intense interest in this pathway, and specifically in FTase, was initially derived from the fact that mutant Ras proteins, the products of ras oncogenes, are key causative agents in -30% of human cancer. The development of selective inhibitors of FTase is a key area of current cancer chemotherapeutic research, and a large number of potent inhibitors of FTase have been developed, with two compounds in advanced trials for treatment of several carcinomas. Despite the promise demonstrated in the pre-clinical and clinical evaluation of these agents, they also exhibited a significant and surprising drawback: many human tumors driven by the mutant form of K-Ras are quite resistant to FTIs. It was expected that these tumors would be particularly sensitive to FTI treatment, since FTIs were designed to act as anti-Ras agents. However, it has been confirmed by several groups that, in the presence of FTIs, the crucial oncoprotein target K-Ras is geranylgeranylated by GGTase I, and this alternative modification apparently allows mutant K-Ras to continue its growth-promoting actions. Thus, there has been interest in developing other methods for the inactivation of Ras proteins.

There was significant early interest in these two steps catalyzed by RCEl and Icmt, but progress in this area was stymied by an inability to isolate and purify these membrane- bound proteins. Moreover, it was felt that these steps were of secondary importance, as farnesylation itself seemed to be sufficient for activity of mutant Ras proteins. However, it has been recently demonstrated that genetic disruption of the mouse RCEl or Icmt gene leads to a profound mislocalization of K-Ras, and thus presumably a blockage of its ability to promote cell growth. Taken together, these data may suggest that a) inhibition of Icmt might lead to mislocalization of both farnesylated and geranylgeranylated K-Ras; b) this

mislocalization may well interefere with the biological activity of K-Ras, and thus c) Icmt inhibitors may be intriguing potential anticancer agents. The present application is thus directed to the examination of the substrate specificity of Icmt with a view toward the development of substrate-based inhibitors of the enzyme. In the present invention, active compounds are disclosed as anti-cancer/anti-tumor agents as well as agents to treat disease states or conditions which are modulated through isoprenyl cysteine methyltransferase enzyme, including hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others.

Members of the Ras family, implicated in many human cancers, are modified post- translationally, targeting them to the appropriate intracellular location. Ras and other - CaaX proteins undergo three sequential reactions: isoprenylation of the cysteine, in particular farnesylation by farnesyltransferase (FTase); proteolysis of the three terminal amino acids (- aaX); and a-carboxyl methylation of the isoprenylated cysteine. This process is crucial for membrane localization and thus activity of the key Ras oncoproteins. FTase inhibitors are being evaluated in clinical trials as cancer chemotherapeutic agents. Unfortunately, these compounds have surprisingly little effect on many Ras-transformed tumors. There is now growing interest in the subsequent enzymatic step, a-carboxyl methylation by

isoprenylcysteine methyltransferase (Icmt), as an alternative target for the inhibition of Ras protein action. Carboxyl methylation is critical for the proper localization of Ras proteins in yeast and mouse cells. Given this finding, we believe that Icmt represents an excellent target for chemotherapeutic intervention. Potent cell permeable inhibitors will be evaluated for their ability to mislocalize Ras, interfere with Ras-mediated signaling, block anchorage-independent growth of pancreatic ductal carcinoma, and block tumor growth in vivo.

OBJECTS OF THE INVENTION

In one aspect of the invention, an object of the present invention is to provide compounds and methods for the treatment of tumors and/or cancer in mammals. In another aspect of the present invention, an object of the present invention is to provide pharmaceutical compositions useful for the treatment of tumors and/or cancer, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others.

In still other aspects of the invention, objects of the present invention provide compounds and methods for the treatment of neoplasia, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others.

In still other aspects of the present invention, objects of the invention provide methods of inhibiting isoprenylcysteine methyltransferase, an enzyme which is believed to modulate a number of disease states or conditions including neoplasia, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others.

Any one or more of these and/or other objects of the present invention may be readily gleaned from the description of the present invention which follows.

SUMMARY OF THE INVENTION

The present invention relates to a series of novel FTP-triazole compounds as potent inhibitors of Icmt, through a focus on thioether and prenyl mimetics. These mimetics were coupled utilizing a copper-assisted cycloaddition to assemble the potential inhibitors. Using the resulting triazole from the coupling as a prenyl mimetic resulted in a biphenyl substituted FTP triazole termed "TAB-Me", which is a submicromolar inhibitor of Icmt (IC50 = 0.8 ± 0.1 μΜ; calculated Kj = 0.4 μΜ). Subsequent work has led to a more potent inhibitor, "STAB-Me", with a calculated Kj value of 140 nM for Icmt. Both of these compounds block the membrane localization of K-Ras and the methylation of famesylated proteins in cellular model systems. Moreover, they exhibit selective cytostatic effects toward Icmt+/+ MEFs, and block the growth of the pancreatic tumor cell line PaTu (STAB-ME in PaTu - 7.3 μΜ +/- 0.7; TAB-Me in PaTu - 8.15 μΜ +/- 3).

In one embodiment, the present invention is directed to compounds of the chemical formula (I):

where A is -(CH ₂ )„-, -C(O)-, -(CH ₂ )iC(R ^A ) ₂ -, -C(R ^A ) ₂ (CH ₂ )i-, C _1-6 alkyl optionally substituted with 1-3 halogens,

B is S, S(O), S(O) ₂ , or -C(O)- ;

Each R ^A is independently H, a C ₁ -C ₃ alkyl optionally substituted with one or two hydroxyl groups and up to three halogen groups, C=O, or a halogen group;

R ^Sa and R ^Sb are absent, H, OH, C ₁ -C ₃ alkyl, NR ¹ R ¹ , -(C=O)(OH), -(NH)(C=OR ¹ ,

-(NH)(C=O)-NH-R ¹ , together with the carbon to which they are bound form -C=O, and where R ^Sa is CH ₃ it may provide a chiral center, in which case R ^Sa is i is 0, 1, 2, or 3;

n is an integer from 0-12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12); R ^s is H, N ₃ , CN, N0 ₂ , halogen, C ₁ -C ₆ alkyl optionally substituted with one or two hydroxyl groups or up to three halogen groups (often F), OR ¹ , SR ¹ ,

-C(O)R ¹ , -OC(O)R ¹ , -C(O)OR ¹ , -N(R ^N )C(O)OR ¹ , -C(O)N(H)OH, NR ¹ R ¹ , NC(O)R ¹ , -C(O)NR ¹ R ¹ , -P(O)(OR ¹ ) ₂ , or together R ^s and R ^Sa form a five or 6-membered carbocyclic or heterocyclic group (e.g. a 1,3 dioxolane group, etc.);

R ^N is H or a C _1- C ₃ alkyl group;

Each R ¹ is independently H, C ₁ -C ₆ alkyl which is optionally substituted with one or two hydroxyl groups or up to 3 halogen groups, an optionally substituted C ₄ -C ₂₀ hydrocarbyl group, preferably an alkyl or alkene group (which may include multiple unsaturations, preferably a C5-C ₁₀ alkyl group (including butyl, sec-butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, isononyl, decyl), an optionally substituted aryl group (e.g., phenyl including benzyl, ethylphenyl, methoxyphenyl, ethoxyphenyl, C ₁ -C ₄ alkyl phenyl, heteroarylphenyl or heterocyclylphenyl or an optionally substituted l-,2- or 3- naphthyl group), an optionally substituted heterocycle or heteroaryl group (especially including, 1- or 2-furanyl, 1-, 2-, or 3-oxanyl, 2- or 3-thiophenyl, 2- or 3-pyrrolyl, 2-, 3- or 4- pyridinyl, 2-, or 3-pyrollidinyl or as otherwise disclosed herein), an optionally substituted biphenyl (preferably a biphenyl group containing from 1 to 4, preferably 3 halogen substitutes (e.g., F, CI, Br, I, often F) or a triphenyl group, substituted or unsubstituted C ₁ -C ₄ alkene-biphenyl (where the biphenyl is optionally substituted with from 1 to 4, preferably 3, halogen substitutes; and

R ² is G-D, where G is an optionally substituted C ₁ -C ₂₀ alkyl, alkenyl, alkynyl or aryl group and D is absent, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl-optionally substituted aryl, (optionally substituted C ₁ -C ₁₀ alkyl, alkenyl or alkynyl)-optionally substituted aryl, (optionally substituted C ₁ -C ₁₀ alkyl, alkenyl or alkynyl)- (optionally substituted aryl)- (optionally substituted aryl (optionally substituted C ₁ -C ₁₀ alkyl or alkenyl)-0-(optionally substituted aryl-optionally substituted aryl);

or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof.

In certain embodiments, compounds of the invention have the chemical formula (II):

where x is 1-6, preferably 1-4, more preferably 2 or 3; y is 1-3, preferably 1-2, more preferably 1; and z is 1-3, preferably 1-2, more preferably 2, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof.

In alternative preferred embodiments, the present invention relates to a compound according to the chemical formula (II):

where R is H or a C ₁ -C ₄ alkyl group, preferably H or CH ₃ , more preferably CH ₃ ; and

R ₁ is an optionally substituted C ₄ -C ₂₀ hydrocarbyl group, preferably an alkyl or alkene group (which may include multiple unsaturations, preferably a C ₅ -C ₁₀ alkyl group (including butyl, sec-butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, isononyl, decyl), an optionally substituted phenyl (including benzyl, ethylphenyl,

methoxyphenyl, ethoxyphenyl, C1-C4 alkyl phenyl, optionally substituted aryl-aryl including optionally substituted bi-phenyl, C _1- C4 alkyl, alkenyl or alkynyl-optionally substituted aryl- aryl including C ₁ -C ₄ alkyl, alkenyl or alkynyl- optionally substituted bi-phenyl,

heteroarylphenyl or heterocyclylphenyl), an optionally substituted l-,2- or 3-naphthyl, an optionally substituted heterocycle or heteroaryl group (especially including, 1- or 2-furanyl, 1-, 2-, or 3-oxanyl, 2- or 3-thiophenyl, 2- or 3-pyrrolyl, 2-, 3- or 4-pyridinyl, 2-, or 3- pyrollidinyl), or an optionally substituted biphenyl or triphenyl group, or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof. In certain embodiments, R ₁ of formula (II) is a group, where z is 1-3, preferably 2.

One or more additional compounds according to the present invention are disclosed in further detail herein, and include such exemplary compounds as the following:

Pharmaceutical compositions according to the present invention comprise an effective amount of one or more of the above-depicted compounds or as otherwise described in the attached appendices hereof, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient and further optionally in combination with an additional anticancer agent.

The method of the present invention involves the use of compounds to treat neoplasia and other diseases and conditions such as hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others of animals, especially mammals, including humans, encompassed by a compound as otherwise described above or in the attached appendices hereof. In this method an effective amount of one or more compounds according to the present invention, optionally in combination with at least one additional anticancer agent is administered to a patient in need.

The compounds of the present invention are used to treat benign and malignant neoplasia, including various cancers such as, stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, renal, brain/ens, head and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acute mylogenous leukemia, Ewings Sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms Tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus, larynx, melanoma, kidney, lymphoma, among others. Compounds according to the present invention are particularly useful in the treatment of breast cancer, including breast cancer which is of a multiple drug resistant phenotype.

A method of treating hyperproliferative cell growth, restenosis following

cardiovascular surgery, hyperplasia, including renal hyperplasia, among others using one or more of the disclosed compositions are other inventive aspects of the present invention.

Further inventive aspects of the present invention relate to the use of the present compositions in the treatment of arthritis and chronic inflammatory diseases, including rheumatoid arthritis and osteoarthritis, among others.

The present invention also relates to methods for inhibiting the growth of neoplasia, including a malignant tumor or cancer comprising exposing the neoplasia to an inhibitory or therapeutically effective amount or concentration of at least one of the disclosed compounds. This method may be used therapeutically, in the treatment of neoplasia, including cancer or in comparison tests such as assays for determining the activities of related analogs as well as for determining the susceptibility of a patient's cancer to one or more of the compounds according to the present invention.

Methods for treating abnormal cell proliferation or growth of non-transformed cells, including the treatment of psoriasis, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, among others, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others, comprising administering a therapeutically effective amount of one or more of the disclosed compounds for treating the condition or disease are also contemplated within the scope of the present invention.

The present invention also relates to a method for inhibiting isoprenylcysteine methyltransferase comprising exposing said enzyme to an effective amount of any one or more of the compounds which are set forth hereinabove. Others aspects according to the present invention relate to a method of inhibiting isoprenyl cysteine methyltransferase enzyme in a patient in order to treat a disease or condition modulated by said enzyme comprising administering to said patient an effective amount of any one or more of the compounds compound which are set forth hereinabove. Disease states or conditions which are believed to be modulated by this enzyme include for example, neoplasia, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others.

Brief Description of the Figures

Figure 1: Simplified schematic depicting the post-translational modifications of the Ras family of proteins. Farnesylation (FTase), endoproteolysis (Rce-1) of the C-terminal -AAX residues and carboxylmethylation (Icmt) are necessary for biological activation of Ras proteins.

Figure 2: Structures of Icmt substrates (a,b) and inhibitors (c,d).

Figure 3: Plausible mechanistic pathway of analog 16 under assay conditions to yield 12.

Figure SI: Possible intra-molecular hydrogen bonding in compound 29 could result in lack of activity vis-a-vis compound 12.

Figure S2: Compound 12 (Farnol) is a mixed competitive Icmt inhibitor with a predominant competitive component (alpha value greater than 1). Procedure: ¾ determinations were completed using a modified in vitro vapor diffusion assay. Briefly, 5 μg of protein was incubated with 10 mM Tris-HCl (pH 7.5), 0-200 μΜ AFC and 20 uM SAM (50-60 mCi/mmol). Farn-OL was incubated with protein at a final concentration of 0.5, 1.5 and 3 μΜ. The reaction was continued as previously described. Lineweaver-Burk plots were used to illustrate the mode of inhibition (mixed-competitive) with an a- value greater than 1, describing predominantly competitive properties. Figure S3: Compound 12 alters the subcellular localization of GFP K-Ras in Jurkat T cells. Jurkat T cells were transiently transfected with GFP K-Ras and treated with DMSO as a control, simvastatin (45 μΜ) or compound 12 at the indicated concentration for 24 hours. The histogram depicts the differences in GFP K-Ras localization after indicated treatment.

Differences were defined as partial or complete loss of normal localization. Representative images for Jurkat cells transiently transfected with GFP-K-Ras, exhibiting normal

localization, partial mislocalization, and full mislocalization, are shown in: Majmudar et. al Bioorg Med Chem 2012 20, 283-295.

Figure 1A. Cell growth inhibition of mouse embryonic fibroblasts by 12n. ^a MEFs are plated in 96 well plates at a density of 1000 cell/well in DMEM supplemented with 10% FBS. After incubation for 24 hrs, the media is replaced with DMEM supplemented with 5% FBS and 12n (various cone.) or DMSO (0.1%). The cell viability is determined after five days using MTT. Twenty microliters of MTT (5 mg/ml) is added to each well and incubated for 4 hrs at 37°C. Afterwards, the media is removed and 150 μΐ of DMSO is added to each well. The absorbance is determined at 590 nm using a Molecular Devices VERSAmax microplate reader. ^b The IC ₅₀ of Icmt ^{+ +} was determined to be 33 μΜ ± 1 and Icmt^ of > ΙΟΟμΜ. IC ₅₀ determinations were made using Graphpad Prism V4.

Figure 2A. This figure represents a generic illustration to library building that led to the identified lead structures.

Figure 4. Table 1 A shows the enzymatic inhibition of a number of compounds according to the present invention. Enzymatic inhibitory determinations of each compound were establish by incubating His-hlcmt crude membrane extracts (5 μg), AFC (10 μΜ) and ¹⁴ C-SAM (20 μΜ) in Tris-HCl (pH 7.5). These reaction mixtures were incubated for 30 min (30 °C) before the reaction was stopped using NaOH and the base labile ^l4 C-methyl groups transferred were determined using a Packard 1600A Liquid Scintillation Analyzer. Competitive and uncompetitive inhibition constants were determined using the Cheng-Prusoff method (Y.C. Cheng and W.H. Prusoff; Biochemical Pharmacology; 22: 3099-3108, 1973). Figure 5. Table 2 A shows growth inhibition determinations were found by seeding each cell line (1 - 1.2 xlO ³ cells per well) in a 96-well plate with complete media containing 10% FBS. After 24 h incubation (37 °C, 5% G0 ₂ ), the media was removed and replaced with drugged complete media supplemented with 5% FBS. The cells are incubated for 5 days before the addition of MTT (4 mg/ml). The plates were incubated for an additional 4 h before the media was removed and DMSO (100 μΐ) was used to solubilize the formazan crystals. The absorbance of each well was measured (Abs= 590 nm) using Molecular Devices VersaMax plate reader. Each drug concentration was tested three times in triplicate.

Detailed Description of the Invention

The following terms shall be used throughout the specification to describe the present invention.

The term "compound", as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. Within its use or description in context, the term generally refers to a single compound, but in certain instances may also refer to stereoisomers (cis and/or trans, etc.), anomers, epimers and/or optical isomers (including racemic mixtures), as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds as well as pharmaceutically acceptable salts, solvates and polymorphs thereof.

The term "patient" is used throughout the specification to describe a subject animal, such as a mammal, preferably a human, to whom treatment, including prophylactic treatment, with the compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal.

The term "effective amount" is used throughout the specification to describe concentrations or amounts of compounds according to the present invention which may be used to produce an effect within context, whether that effect relates to a favorable change in the disease or condition treated, or the change is a remission, a decrease in growth or size of cancer or a tumor, a favorable physiological result, a reduction in the growth or elaboration of a microbe, or the like, depending upon the disease or condition treated.

The term "independently" is used herein to indicate that the variable, which is independently applied, varies independently from application to application.

The term "non-existent" or "absent" refers to the fact that a substituent is absent and the group to which such substituent is attached forms an additional bond with an adjacent atom or group.

The term "optionally substituted" means optional substitution with the specified groups, radicals or moieties. It should be noted that any atom with unsatisfied valences in the text, schemes, examples and tables herein is assumed to have the hydrogen atom(s) to satisfy the valences.

The term "alkyl" is used throughout the specification to describe a hydrocarbon radical containing between one and twenty carbon units, one and eight, four and twelve, five and ten, five and fifteen or one and four carbon units. Alkyl groups for use in the present invention include linear, branched-chain groups or cyclic groups (cycloalkyl groups). The term "hydrocarbyl" refers to a substituent or group having carbon and hydrogen groups and may be saturated or unsaturated. Alkyl groups are subsumed under the term hydrocarbyl in describing the present invention.

"Aryl" refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., benzene or phenyl) or multiple condensed rings (e.g., naphthyl) and can be bound to a compound according to the present invention at any position on the ring(s). Other examples of aryl groups include heterocyclic aromatic ring systems "heteroaryl" having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as imidazole, furyl, pyrrole, pyri- dyl, indole and fused ring systems, among numerous others, which may be substituted or unsubstituted. Biphenyl and triphenyl groups also represent aryl groups according to the present invention. "Alkoxy" as used herein refers to an alkyl group bound through an ether linkage; that is, an "alkoxy" group may be represented as ~0~alkyl where alkyl is as defined above. "Acyl" as used herein referes to an alkyl or other hydrocarbyl group bound through a keto linkage. "Ester" refers to a carboxy ester (where the carbonyl of the ester is attached to the basic pharmacophore) or ester (where the oxygen is attached to the basic pharmacophore). "Amide" refers to an amide group where the nitrogen is attached to the basic pharmacophore and "carboxamide" refers to an amide group where the carbonyl is attached to the basic pharmacophore.

The term "cyclic" shall refer to a carbocyclic or heterocyclic group, preferably a 5- or 6-membered ring, but may include 4 and 7-membered rings or fused rings. "Bicyclic" or "bicyclo" refers to bicyclic

The term "heterocycle" or "heterocyclic" shall mean an optionally substituted moiety which is cyclic and contains at least one atom other than a carbon atom, such as a nitrogen, sulfur, oxygen or other atom. A heterocyclic ring shall contain up to four atoms other than carbon selected from nitrogen, sulfur and oxygen. These rings may be saturated or have unsaturated bonds. Fused rings are also contemplated by the present invention. Bicyclo groups are also contemplated for use herein. A heterocycle according to the present invention is an optionally substituted imidazole, a piperazine (including piperazinone), piperidine, furan, pyrrole, imidazole, thiazole, oxazole or isoxazole group, among numerous others. Depending upon its use in context, a heterocyclic ring may be saturated and/or unsaturated. In instances where a heterocyclic ring is fully unsaturated, there is overlap with the term "heteroaryl".

Exemplary heterocyclic groups (which term subsumes exemplary heteroaryl groups within context) which may be used in the present invention include for example, pyrrole, imidazole, diazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, azepine, diazepine, furan, dihydrofuran, tetrahydrofuran, pyran, oxanyl, oxepine, thiophene, thiopyran, thiepine, oxazole, isoxazole, thiazole, isothiazole, furazan, oxadiazole, oxazine, oxadiazine, oxazepine, oxadiazepine, thiadiazole, thiazine, thiadiazine, thiazepine, thiadiazepine, indole, isoindole, indolizine, benzofuran, isobenzofiiran, benzothiophene, isobenzothiophene, dithianaphthalene, indazole, quinoline, isoquinoline, quinolizine, purine, phthalazine, ptridine, naphthyridine, quinoxaline, quinazoline, cinnoline, pyrrolopyridine, benzoxazole, benzothiazole, benzimidazole, chromene, benzoxepine, benzoxazepine, benzoxadiazepine, benzothiepine, benzothiazepine, benzothiadiazepine, benzazepine, benzodiazepine, benzofurazan, benzothiadiazole, benzotriazole, carbazole, (-)carboline, acridine, phenazine, dibenzofuran, xanthene, dibenzothiophene, phenothiazine, phenoxazine, phenoxathiin, thianthrene, phenanthridine, phenanthroline, perimidine, pyridonaphthyridine, pyrazoloisoquinoline, pyrazolonaphthyridine, pyrimidoindole, indolizinoindole, aziridine, azetidine, pyrroline, pyrrolidine, imidazoline, imidazolidine, triazoline, triazolidine, tetrazoline, tetrazolidine, pyrazoline, pyrazolidine, dihydropyridine, tetrahydropyridine, piperidine, dihydropyrazine, tetrahydropyrazine, piperazine, dihydropyrimidine,

tetrahydropyrimidine, perhydropyrimidine, dihydropyridazine, tetrahydropyridazine, perhydropyridazine, dihydroazepine, tetrahydroaepine, perhydroazepine, dihydrodiazepine, tetrahydrodiazepine, perhydrodiazepine, oxirane, oxetane, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, dihydrooxepine, tetrahydrooxepine, perhydrooxepine, thiirane, thietane, dihydrothiophene, tetrahydrothiophene, dihydrothiopyran,

tetrahydrothiopyran, dihydrothiepine, tetrahydrothiepme, perhydrothiepine, dihydrooxazole, tetrahydrooxazole(oxazolidine), dihydroisoxazole, tetrahydroisoxazole(isoxazolidine), dihydrothiazole, tetrahydrothiazole(thiazolidine), dihydroisothiazole,

tetrahydroisothiazole(isothiazolidine), dihydrofurazan, tetrahydrofurazan, dihydrooxadiazole, tetrahydrooxadiazole(oxadiazolidine), dihydrooxazone, tetrahydrooxazine,

dihydrooxadiazine, tetrahydrooxadiazine, dihydrooxazepine, tetrahydrooxazepine, perhydrooxazepine, dihydrooxadiazepine, tetrahydrooxadiazepine, perhydrooxadiazepine, dihydrothiadiazole, tetrahydrothiadiazole(thiadiazolidine), dihydrothiazine,

tetrahydrothiazine, dihydrothiadiazine, tetrahydrothiadiazine, dihydrothiazepine,

tetrahydrothiazepine, perhydrothiazepine, dihydrothiadiazepine, tetrahydrothiadiazepine, perhydrothiadiazepine, morpholine, thiomorpholine, oxathiane, indoline, isoindoline, dmydrobenzofuran, perhydrobenzofuran, dmydroisobenzofuran, perhydroisobenzofuran, dihydrobenzothiophene, perhydrobenzothiophene, dihydroisobenzothiophene,

perhydroisobenzothiophene, dihydroindazole, perhydroindazole, dihydroquinoline, tetrahydroquinoline, perhydroquinoline, dihydroisoquinoline, tetrahydroisoquinoline, perhydroisoquinoline, dihydrophthalazine, tetrahydrophthalazine, perhydrophthalazine, dihydronaphthyridine, tetrahydronaphthyridine, perhydronaphthyridine, dihydroquinoxaline, tetrahydroquinoxaline, perhydroquinoxaline, dihydroquinazoline, tetrahydroquinazoline, perhydroquinazoline, tetrahydropyrrolopyridine, dihydrocinnoline, tetrahydrocinnoline, perhydrocinnoline, benzoxathiane, dihydrobenzoxazine, dihydrobenzothiazine,

pyrazinomorpholine, dihydrobenzoxazole, perhydrobenzoxazole, dihydrobenzothiazole, perhydrobenzohiazole, dihydrobenzimidazole, perhydrobenzimidazole, dihydrobenzazepine, tetrahydrobenzazepine, dihydrobenzodiazepine, tetrahydrobenzodiazepine, benzodioxepane, dihydrobenzoxazepine, tetrahydrobenzoxazepine, dihydrocarbazole, tetrahydrocarbazole, perhydrocarbazole, dihydroacridine, tetrahydroacridine, perhydroacridine,

dihydrodibenzofuran, dihydrodibenzothiophene, tetrahydrodibenzofuran, tetrahy- drodibenzothiophene, perhydrodibenzofuran, perhydrodibenzothiophene, tetrapyri donaphthyri dine, tetrahydro-pcarboline, dihydroazepinoindole, hexahydroazepinoindole, tetrahydropyrazoloisoquinoline, tetrabydropyrazolonaphthyridine, dihydroazepinoindazole, hexahydroazepinoindazole, dihvdropyrazolopyridoazepine,

hexahydropyrazolopyridoazepine, tetrahydropyrimidoindole, dihydrothiazinoindole, tetrahydrothiazinoindole, dihydrooxazinoindole, tetrahydrooxazinoindole, hexahydroin- dolizinoindole, dihydroindolobenzdiazepine, octahydroindoloquinolizine,

hexahydroimidazopyridoindole, perhydrodibenzothiophene, tetrapyridonaphthyridine, tetrahydrocarboline, dihydroazepinoindole, hexahydroazepineindole,

tetrahydropyrazoloisoquinoline, tetrabydropyrazolonaphthyridine, dihydroazepinoindazole, hexahydroazepinoindazole, dihydropyrazolopyridoazepine,

hexahydropyrazolopyridoazepine, tetrahydropyrimidoindole, dihydrothiazinoindole, tetrahydrothiazinoindole, dihydrooxazinoindole, tetrahydrooxazinoindole,

hexahydroindolizinoindole, dihydroindolobenzdiazepine,

octahydroindoloquinolizine, hexahydroimidazopyridoindole,

hexahydropyrrolothiazepinoindole, dioxolane, dioxane, dithiolane, dithiane, dioxaindan, benzodioxane, chroman, benzodithiolane, benzodithiane, azaspiro[4.4]nonane,

oxazaspiro[4.4]nonane, oxazaspiro[2.5]octane, dioxaspiro[4.4] nonane, azaspiro[4.5]decane, thiaspiro[4.5]decane, dithiaspiro[4.5]decane, dioxaspiro[4.5]decane, oxazaspiro[4.5] decane, azaspiro[5.5]undecane, oxaspiro[5.5]undecane, dioxaspiro[5.5]undecane, 2,3,4,9- tetrahydrospiro[P-carboline-l,P-cyclopentane], azabicyclo[2.2.1]heptane, oxabicy- clo[2.2.1]heptane, azabicyclo[3.1.1]heptane, azabicyclo [3.2.1]octane,

oxabicyclo[3.2.1]octane, azabicyclo[2.2.2] octane, diazabicyclo[2.2.2]octane, among others.

The term "unsubstituted" shall mean substituted only with hydrogen atoms. The term "substituted" shall mean, within the chemical context of the compound defined, a substituent (each of which substituents may itself be substituted) selected from a hydrocarbyl (which may be substituted itself, preferably with an optionally substituted alkyl or halogen (fluoro) group, among others), preferably an alkyl (preferably, about 1-8 carbon units in length, up to about 12 or more carbon units), an optionally substituted aryl (which also may be heteroaryl and may include an alkylenearyl or alkyleneheteroaryl), an optionally substituted heterocycle (especially including an alkyleneheterocycle), CF ₃ , halogen (especially fluoro), thiol, hydroxyl, carboxyl (carboxylic acid), oxygen (to form a keto group), C ₁ -C ₈ alkoxy, CN, nitro, an optionally substituted amine (e.g.. an alkyleneamine or a C ₁ -C ₆ monoalkyl or dialkyl amine, which may be optionally hydroxyl substituted), C ₁ -C ₈ acyl, C ₁ -C ₈ alkylester, C ₁ -C ₈ alkyleneacyl (keto), C ₁ -C ₈ alkylene ester, carboxylic acid, alkylene carboxylic acid, C ₁ -C ₈ thioester, C ₂ -C ₈ ether, C ₁ -C ₈ thioether, amide (amido or carboxamido), substituted amide (especially mono- or di-alkylamide) or alkyleneamide, an optionally substituted carbamate or urethane group, wherein an alkylene group or other carbon group not otherwise specified contains from 1 to 8 carbon units long (alternatively, about 2-6 carbon units long) and the alkyl group on an ester group is from 1 to 8 carbon units long, preferably up to 4 carbon units long. Various substituents may themselves be substituted with substituents as otherwise described herein. Various optionally substituted moieties may be substituted with 5 or more substituents, preferably no more than 3 substituents and preferably from 1 to 3 substituents. The term substituted may include, within context, substituents such as alkylene groups (represented as a -(CH ₂ ) _n or-(CH ₂ ) _y group where n is 0, 1, 2, 3, 4, or 5, preferably from 1 to 3 and y is 1, 2, 3, 4 or 5, preferably 1 to 3) which can bridge one moiety to a ring or other group on a pharmacophore or other moiety or substituent. In preferred embodiments, preferred substitutents include (within an appropriate context) one or more halogen groups (F, CI, Br or I) or a C ₂ -C ₁₀ alkyl, acyl, ester, amido or carboxamido group, preferably no more than three halogen groups, preferably two halogen groups, which are most preferably F

The term "neoplasia" is used to describe the pathological process that results in the formation and growth of a neoplasm, i.e., an abnormal tissue that grows by cellular proliferation more rapidly than normal tissue and continues to grow after the stimuli that initated the new growth cease. Neoplasia exhibits partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue which may be benign (benign tumor) or malignant (carcinoma). The term "cancer" is used as a general term to describe any of various types of malignant neoplasms, most of which invade surrounding tissues, may metastasize to several sites and are likely to recur after attempted removal and to cause death of the patient unless adequately treated. As used herein, the term cancer is subsumed under the term neoplasia. Representative cancers include, for example, squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, and renal cell carcinomas, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias, including non-acute and acute leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, T-lineage acute lymphoblastic leukemia (T-ALL), adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas;

myeloproliferative diseases; sarcomas, including Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor and teratocarcinomas, among others, which may be treated by one or more compounds according to the present invention. A more complete list of cancers which may be treated using compounds according to the present invention may be found at the website:

cancer.gov/cancertopics/alphalist, relevant portions of which are incorporated by reference herein.

The term "additional anticancer agent" is used to describe a compound which may be combined with one or more compounds according to the present invention in the treatment of cancer and include such compounds/agents as everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY- 142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR T inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint- 1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111 , 131-I-TM-601 , ALT- 110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, I O 1001, IPdRi KRX- 0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS- 100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, liposomal doxorubicin, 5'-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901 , AZD-6244, capecitabine, L-Glutamic acid, N -[4-[2-(2-amino-4,7-dihydro-4-oxo-lH- pyrrolo[2,3- d ]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,

DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258,); 3-[5-(methylsulfonylpiperadinemethyl)- indolylj-quinolone, vatalanib, AG- 013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6 ,Azgly 10 ] (pyro-Glu-His-Trp-Ser- Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH ₂ acetate [C ₅₉ H ₈₄ N ₁₈ Oi ₄ -(C ₂ H ₄ 0 ₂ )x where x = 1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS- 214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951 , aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate,

cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,

mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291 , squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox,gefitinib, bortezimib, paclitaxel, cremophor-free paclitaxel, docetaxel, epithilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA- 923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR- 3339, ZKl 86619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2- hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001 , ABT-578, BC-210,

LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L- 779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab,

hydrocortisone, interleukin-11 , dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard,

methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, , diphenhydramine, hydroxyzine,

metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, darbepoetin alfa and mixtures thereof.

The term "hyperproliferative cell growth" is used to describe conditions of abnormal cell growth of a non-transformed cell often, of the skin, distinguishable from cancer.

Examples of such conditions include, for example, skin disorders such as hyperkeratosis (including ichthyosis), keratoderma, lichen, planus and psoriasis, warts (including genital warts), blisters and any abnormal or undesired cellular proliferation. The term "restenosis" is used to describe the recurrence of stenosis after corrective surgery on the heart, including the heart valve, or the narrowing of a structure (usually a coronary artery) following the removal or reduction of a previous narrowing of such structure. It is usually a result of a form of hyperplasia-neointimal hyperplasia.

The term "hyperplasia", "hypertrophy" or "numerical hypertrophy" is used to describe an increase in the number of cells in a tissue or organ, excluding tumor formation, and refers to all types of hyperplasia, including cystic hyperplasia, cystic hyperplasia of the breast, nodular hyperplasia of the prostate and renal hyperplasia, neointimal hyperplasia, among numerous others.

A preferred therapeutic aspect according to the present invention relates to methods for treating neoplasia, including benign and malignant tumors and cancer in animal, especially mammalian, including human patients, comprising administering effective amounts or concentrations of one or more of the compounds according to the present invention to inhibit the growth or spread of or to actually shrink the neoplasia in the animal or human patient being treated.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in an effective amount for the treatment of a condition or disease state such as neoplasia, including cancer, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others or a related condition or disease as otherwise described, optionally in combination with a

pharmaceutically acceptable additive, carrier or excipient.

Certain of the compounds, in pharmaceutical dosage form, may be used as prophylactic agents for preventing a disease or condition from manifesting itself. In certain pharmaceutical dosage forms, the pro-drug form of the compounds according to the present invention may be preferred.

The present compounds or their derivatives, including prodrug forms of these agents, can be provided in the form of pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salts or complexes refers to appropriate salts or scomplexes of the active compounds according to the present invention which retain the desired biological activity of the parent compound and exhibit limited toxicological effects to normal cells. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid, among others; (b) base addition salts formed with metal cations such as zinc, calcium, sodium, potassium, and the like, among numerous others, which are formed at the carboxylic acid position of compounds according to the present invention.

Modifications of the active compound can affect the solubility, bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species. Further, the modifications can affect the anticancer activity of the compound, in some cases increasing.the activity over the parent compound. This can easily be assessed by preparing the derivative and testing its anticancer activity according to known methods well within the routineer's skill in the art.

The compounds of this invention may be incorporated into formulations for all routes of administration including for example, oral, topical and parenteral including intravenous, intramuscular, intraperitoneal, intrabuccal, transdermal and in suppository form, among numerous others.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in an effective amount for treating neoplasia, cancer and other diseases and conditions which have been described herein, including psoriasis, hyperproliferative cell growth, restenosis following cardiovascular surgery, hyperplasia, including renal hyperplasia, chronic inflammatory diseases including rheumatoid and osteoarthritis, among others, optionally in combination with a pharmaceutically acceptable additive, carrier and/or excipient. One of ordinary skill in the art will recognize that a therapeutically effective amount of one of more compounds according to the present invention will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient (animal or human) treated.

In the pharmaceutical aspect according to the present invention, the compound according to the present invention is formulated preferably in admixture with a

pharmaceutically acceptable carrier. In general, it is preferable to administer the

pharmaceutical composition in orally-administrable form, but a number of formulations may be administered via a parenteral, intravenous, intramuscular, transdermal, buccal,

subcutaneous, suppository or other route. Intravenous and intramuscular formulations are preferably administered in sterile saline. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity. In particular, the modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.) which are well within the ordinary skill in the art. It is also well within the routineer's skill to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect to the patient.

In certain pharmaceceutical dosage forms, the pro-drug form of the compounds may be preferred. One of ordinary skill in the art will recognize how to readily modify the present compounds to pro-drug forms to facilitate delivery of active compounds to a targeted site within the host organism or patient. The routineer also will take advantage of favorable pharmacokinetic parameters of the pro-drug forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound.

The amount of compound included within therapeutically active formulations according to the present invention is an effective amount for treating the infection or condition. In general, a therapeutically effective amount of the present preferred compound in dosage form usually ranges from slightly less than about 0.025mg./kg. to about 2.5 g./kg., about 0.1-50 mg/kg, about 1-25 mg/kg, about 2.5-5 mg/kg to about 100 mg/kg of the patient or about 10-50 mg/kg, depending upon the compound used, the condition or infection treated and the route of administration, although exceptions to this dosage range may be contemplated by the present invention.

Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal and suppository administration, among other routes of administration.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients including those which aid dispersion may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

The present compounds may be used to treat animals, and in particular, mammals, including humans, as patients. Thus, humans, equines, canines, bovines and other animals, and in particular, mammals, suffering from tumors, and in particular, cancer, or other diseases as disclosed herein, can be treated by administering to the patient an effective amount of one or more of the compounds according to the present invention or its derivative or a

pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier, additive or excipient, either alone, or in combination with other known pharmaceutical agents, depending upon the disease to be treated. This treatment can also be administered in conjunction with other conventional cancer therapies, such as radiation treatment or surgery.

The active compound is included in the pharmaceutically acceptable carrier, additive or excipient in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated.

The compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing from less than 1 mg to a gram or more, from about 1 to 3000 mg, about 5 to 500 mg of active ingredient per unit dosage form. An oral dose of about 0.5 to 750 mg, about 1-500 mg, about 5-500mg, about 10-500 mg, about 15 to about 350 mg, about 25-250 mg is usually convenient.

The concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the

administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed

composition. The active ingredient may be administered at once, or may be divided into a .number of smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material-of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable salt thereof may also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as other anticancer agents, and in certain instances depending upon the desired therapy or target, other antiprolierative agents, antirestenosis agents, antinflammatories, or other related compounds which may be used to treat disease states or conditions according to the present invention.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include.the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as

ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers include, for example, physiological saline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared by dissolving appropriate lipid(s) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. Other methods of preparation well known by those of ordinary skill may also be used in this aspect of the present invention.

A wide variety of biological assays have been used and are accepted by those skilled in the art to assess anti-cancer activity of compounds. Any of these methods can be used to evaluate the activity of the compounds disclosed herein.

One common method of assessing activity is through the use of test panels of cancer cell lines. These tests evaluate the in vitro anti-cancer activity of particular compounds in cancer cell lines, and provide predictive data with respect to the use of tested compounds in vivo. Other assays include in vivo evaluations of the compound's effect on human or in an appropriate animal model, for example, using mouse tumor cells implanted into or grafted onto mice or in other appropriate animal models.

Representative Compounds Representative compounds of the invention include, but are not limited to, those described in this section.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is -(CH ₂ ) _n -, -(CH ₂ ) _i C(R ^A ) ₂ -, or -C(R ^A ) ₂ (CH ₂ ) _i - ;

B is S;

R ^Sa and R ^Sb are independently H, C ₁ -C ₆ alkyl, or together with the carbon to which they are bound form -C=O;

R ^s is OH, halogen, O-(C ₁ -C ₆ alkyl), -C(O)OR ¹ or (CH) _n -OH, where n is 1-6;

R ¹ is H or C ₁ -C ₆ alkyl which is optionally substituted with one or two hydroxyl groups or up to 3 halogen groups;

G is an optionally substituted C ₁ -C ₁₀ alkyl or optionally substituted C ₁ -C ₁₀ alkenyl; and D is absent or is a five or six membered heteroaryl which contains 1 , 2 or 3 nitrogens and which is substituted by a C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkenyl or C ₁ -C ₁₂ alkoxy, said C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkenyl or C ₁ -C ₁₂ alkoxy being optionally substituted by (1) an optionally-substituted phenyl (2) an optionally-substituted biphenyl (3) an optionally-substituted triphenyl (4) an optionally-substituted phenyl which itself is optionally substituted by a five or six membered heteroaryl, or (5) a five or six-membered partially or completely saturated heterocyclic group containing a N, S or O atom.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is CH ₂ ;

R ^Sa and R ^Sb are H;

R ^s is OH or halogen;

B is S;

G is an optionally substituted C _1- C20 alkyl or alkenyl group; and

D is absent.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is CH ₂ ; R ^Sa is H and R ^sb is -OH or d-C ₆ alkyl;

R ^s is (CH)n-OH, where n is 1 -3 ;

B is S;

G is an optionally substituted C]-C ₂₀ alkyl or alkenyl group; and

D is absent.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is C _1-6 alkyl optionally substituted with one or two halogens;

R ^Sa is absent and R ^Sb together with the carbon to which it is bound forms -C=O;

R ^s is (CH) _n -OH, where n is 1-3;

B is S;

G is an optionally substituted C1-C20 alkenyl group; and

D is absent.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is C ₁ -6 alkyl optionally substituted with one or two halogens;

R ^Sa and R ^Sb are H;

R ^s is halogen;

B is S;

G is an optionally substituted C ₁ -C ₂ o alkenyl group; and

D is absent.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is CH ₂ ;

R ^Sa and R ^sb are H;

R ^s is OH or halogen;

B is S;

G is an optionally substituted C _! -C ₂₀ alkyl or alkenyl group; and

D is absent. A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein the ompound is:

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is C ₁ -3 alkyl;

R ^Sa is absent and R ^Sb together with the carbon to which it is bound forms -C=O;

R ^s is OH;

B is S;

G is an optionally substituted C ₁ -C ₆ alkenyl group; and

D is triazole which is substituted by a C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkenyl or C _1- C12 alkoxy, said C _1- C ₁ 2 alkyl, C ₁ -C ₁₂ alkenyl or C ₁ -C ₁₂ alkoxy being optionally substituted by (1) an optionally- substituted phenyl (2) an optionally-substituted biphenyl (3) an optionally-substituted triphenyl (4) an optionally-substituted phenyl which itself is optionally substituted by a five or six membered heteroaryl, or (5) a five or six-membered partially or completely saturated heterocyclic group containing a N, S or O atom.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is C1.3 alkyl;

R ^Sa is absent and R ^Sb together with the carbon to which it is bound forms -C=O;

R is C _1-3 alkoxy;

B is S;

G is an optionally substituted C ₁ -C ₆ alkenyl group; and

D is triazole which is substituted by a C ₁ -C ₁₂ alkyl, said C ₁ -C ₁₂ alkyl being substituted by (1) an optionally-substituted phenyl (2) an optionally-substituted biphenyl (3) an optionally- substituted triphenyl (4) an optionally-substituted phenyl which itself is optionally substituted by a five or six membered heteroaryl, or (5) a five or six-membered partially or completely saturated heterocyclic group containing a N, S or O atom. A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein:

A is C _1-3 alkyl;

R ^Sa is absent and R ^Sb together with the carbon to which it is bound forms -C=O;

R is C _1-3 alkoxy;

B is S;

G is an optionally substituted C ₁ -C ₆ alkenyl group; and

D is triazole which is substituted by a C ₁ -C ₁₂ alkyl, said C ₁ -C ₁₂ alkyl being substituted by an optionally-substituted biphenyl.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, in the compound has the formula:

A compound of formula (II):

or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein R is H or a CH ₃ and R ₁ is selected from the group consisting of:

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein R is H or a CH ₃ and ¾ is selected from the group consisting of:

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein the compound has the formula:

where x is an integer from 1 to 3, L is -OH or a C ₁ -C ₃ alkoxy, and M is selected from the group consisting of:

wherein any phenyl of a M group may be optionally substituted with 1-3 substituents selected from the group consisting of C _1-6 alkyl, alkenyl or alkynyl, C _1-6 alkoxy, or halogen.

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein the compound has the formula:

where x is independently an integer from 0 to 3, preferably 1 to 3, R _z1 and are

independently H, C _1-6 alkyl, alkenyl or alkynyl, C _1-6 alkoxy, or halogen (often F), g for either R _z1 or R _z2 can be the same or different and is independently 0, 1, 2 or 3, R ^sa and R ^sb are the same or different and are the same as above or preferably selected from the group consisting of H, -OH, -(CH ₂ ) _n -OH, C _1-6 alkyl, alkenyl or alkynyl, C _1-6 alkoxy, and halogen, and R ^s is the same as above or preferably -(CH ₂ ) _n -OH, and M is selected from the group consisting of:

A compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein the compound has the formula:

where x is an integer from 1 to 3, L is -OH or a C _1- C3 alkoxy, R _z1 , R _z2 R _z3, and R _z4 are independently H, C _1-6 alkyl, alkenyl or alkynyl, C _1-6 alkoxy, or halogen, g for any of R _z1 , R _z2 R _z3 , and R _z4 can be the same or different and is independently 0, 1, 2 or 3, and M is selected from the group consisting of:

A com ound of the formula:

where x is 1, 2 or 3, y is 0 or 1, R ^q and R ^q are independently H or halogen, R ^sa together with the carbon to which it bound forms -C=O and R ^sb is absent, and R ^s is selected from the group consisting of H, -OH, -(NH)OH, halogen, N ₃ , C _1-6 alkyl, alkenyl or alkynyl, C _1-6 alkoxy, a 5 or 6-membered saturated, partially unsaturated or aromatic heterocyclic ring containing one or two heteroatoms selected from the group consisting of N and O,

where k is 0, 1, 2 or 3; or where x is 1, 2 or 3, y is 0 or l, R ^l and R ^q2 are independently H or halogen, R ^sa and R ^sb are each H, and R is selected from the group consisting of H, -OH, -(NH)OH, halogen, N ₃ , -(CH ₂ ) _n -OH, where n is 0, 1, 2, or 3

where k is 0, 1, 2 or 3; or pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof.

A com ound of the formula:

where x is 1, 2 or 3, R _z1 and R _z2 are independently H, C _1- alkyl, alkenyl or alkynyl, C _1-6 alkoxy, or halogen, g for R _z1 and R _z2 can be the same or different and is independently 0, 1, 2 or 3, R ^sa and R ^sb are the same or different and are selected from the group consisting of H, - OH, C _1-6 alkyl, alkenyl or alkynyl, C _1- alkoxy, and halogen, and R ^SA is

-(CH ₂ )n-OH, where n is 0, 1, 2, or 3; or pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof. A com ound of the formula:

or pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof, wherein x is 1, R _z1 and are F, g for R _z1 is 1, g for R _z1 is 2, R ^sa is H, R ^sb is H or C _1-6 alkyl, and R ^SA is-(CH ₂ ) _n -OH, where n is 0 or 1.

A compound selected from the group consisting of:

aceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof. A compound selected from the group consisting of:

or an enantiomer or racemate thereof;

or a pharmaceutically acceptable salt, stereoisomer, enantiomer, solvate or polymorph thereof.

Representative Chemical Synthesis and Biological Activity

1. Background.

The compounds according to the present invention are synthesized by methods which are well known in the art. Compounds which contain the aryl, including naphthyl, or biphenyl groups as depicted above, may be readily synthesized by analogy following the well-described methods set forth in the attached manuscript and thesis which are attached as appendices.

Early studies on the synthesis of prenylated peptides focused on the solid-phase synthesis of non-prenylated peptides followed by the selective prenylation of a free cysteine residue in solution. Naider and Becker, J. M. Biopoly. 1997, 43, 3. Fragment condensation routes have also been employed. Xie, et al., J. Org. Chem. 2000, 65, 8552. A number of groups have developed methods for the solid-phase synthesis of prenylated peptides on a variety of linkers such as oxime, PTMSEL and sulfonamide. See, Dolence, et al., Bioconj. Chem., 2001, 12, 35; Lumbierres, et al., Tet. Lett. 2006, 47, 2671 and Palomo, et al., Angew. Chem. Int. Ed, 2006, 45, 477. However, most of these methods involve coupling an already prenylated cystein resiude to resin-bound peptide. A method that would allow the on-resin attachment of prenyl groups is desirable in order to synthesize more diverse libraries that incorporate famesyl analogs and fluorescent and cross-linking lipid agents into prenylcysteine derivatives.

The solution-phase synthesis of a 23 -member library of amide-modified

farnesylcysteine derivatives (AMFCs). See Donelson, et al., Bioorg.Med. Chem. Lett. , 2006, 16, 4420. This initial library was screened against Icmt, and six inhibitors possessing bulky aromatic and aliphatic groups attached to the a-nitrogen of the cysteine were identified. A farnesylcysteine analog containing a N-l-adamantylcarbonyl group, 4j was identified as the most potent inhibitor of Icmt with an IC ₅₀ of 12 μΜ. Encouraged by these findings, a method was developed for the solid-phase synthesis of prenylcysteine analogs to facilitate the synthesis of a more comprehensive library of AMFCs. Icmt is a membrane bound protein for which no crystal structure is available to aid in the rational design of inhibitors. Therefore it was necessary to synthesize a library of AMFCs to better understand the structure-activity relationships for this class of inhibitors. To validate this technique, a set of AMFCs based on the initial adamantyl lead were prepared.

The solid-phase synthesis of farnesylcysteine derivatives was complicated by the acid lability of the thioether bond connecting cysteine to the prenyl group. It has been shown that the solid-phase synthesis of lipidated peptides requires mild, non-acidic cleavage conditions. In 2002, the Waldmann group reported an elegant solid-phase method for the synthesis of prenylated peptides terminating in a methyl ester. See Ludolph, et al., J Am. Chem. Soc, Unfortunately, this method did not work particularly for the generation of peptides that end in a free carboxylate, an important feature for Icmt inhibitors. Palomo, et al, Ibid.

Subsequently, Waldmann reported problems as well, due to radical side-reactions that occurred during cleavage from the hydrazine resin. Lumbierres, et al., Ibid. Thus, for the purposes of synthesizing the desired prenylcysteine library, a new approach to the preparation of prenylcysteine derivatives on solid-phase was developed.

The 2-chlorotrityl chloride resin has proven to be a versatile and useful tool for the synthesis of a variety of peptides. Sohma, et al., J Pep. Sci, 2005, 1 1, 441 and Kitagawa, et al., Tet. Lett., 1997, 38, 599. The 2-chlorotritylchloride resin has certain advantages over other solid supports. First, due to the SNI mechanism of the loading step, attaching the cysteine to the chlorotrityl resin does not require activation of the cysteine carboxyl group, which decreases the likelihood of racemization. Second, the cleavage conditions are very mild and result in the rapid, quantitative release of the modified cysteine from the bead.

Third, the resin is commercially available and relatively inexpensive. Lastly, amino acid residues can be anchored to the chlorotrityl linker through not only the carboxyl group, but also the a-amino group and certain side chains, which allow for synthesis of peptides in the N to C direction (See, Thieriet, and Albericicio, Org. Lett., 2000, 2, 1815) and selective functionalization of the carboxyl group.

Prior to beginning the synthesis of the AMFC set, several steps of the proposed solid- phase synthetic route to prenylcysteine analogs needed optimization, including the loading of the protected cysteine derivative, the deprotection of the cysteine sulfur, and in particular the cleavage of the analog from resin. We found that using dithiothreitol as a reducing agent for cleavage of the cysteine protecting group, with diisopropylethylamine as a base, resulted in fewer side products than using tributylphosphine and water. A number of methods have been reported for the cleavage of compounds from 2-chlorotrityl resin. See Barlos, et al., Int. J. Pep. Prot. Res. 1991, 37, 513 and Bollhagen, et al, J Chem. Soc. Chem. Comm., 1994, 22 2559. We compared a variety of cleavage cocktails including: 1 :4

hexafluoroisopropanol/dichloromethane, 1 :2:7 acetic acid/trifluoroethanol/dichloromethane, 0.5:99.5 trifluoroacetic acid/dichloromethane, 1 :99 trifluoroacetic acid/dichloromethane, and 3:97 trifluoroacetic acid/dichloromethane. While none of these conditions resulted in significant loss of the farnesyl group from the cysteine, the 0.5:99.5 mixture of trifluoroacetic acid/dichloromethane was chosen for subsequent use due to its speed and quantitative release of the compounds from the resin. The 1 :4 hexafluoroisopropanol/dichloromethane conditions were attractive, due to the essentially neutral nature of this mixture, but it was not always found to result in efficient release of the analog from the bead. Additionally, the higher cost of HFIP combined with the fact that it is used in a higher proportion to TFA made this option less desirable. While the 1 :2:7 acetic acid/trifluoroethanol/dichloromethane solution worked well, it was much slower than the TFA solution, which required only 2-3 minutes for complete product release.

The solid-phase synthetic route to prenylcysteine analogs was outlined in Figure 3, Scheme 1 of PCT/US08/11506, filed October 6, 2008 (WO2009/048541). The commercial 2-chlorotritylchloride resin is coupled to Fmoc-Cys(SStBu)-OH, using 1% collidine in DCM. Any unreacted trityl moieties were capped by addition of a 1% collidine/MeOH solution to the reaction mixture. Reductive removal of the dithiotertbutyl protecting group from 1 with dithiothreitol was followed by coupling of the desired farnesyl side chain to the free thiol, using farnesyl chloride and collidine as the base to afford intermediate 2. Next, the Fmoc protecting group was removed with 20% piperidine/DMF. Coupling of the selected carboxylic acid with the resulting free amine was accomplished using HBTU/HOBt. See, Dourtoglou, et al., Synth., 1984, 7, 572. The polymer-bound prenylcysteine analog 3 was then released from the resin using the optimized cleavage conditions. The crude

prenylcysteine was obtained in good purity (70-90%, by HPLC). Purification by C ₁₈ sep pak cartridge, (Fisher) afforded prenylcysteine analogs suitable in purity for characterization and biochemical evaluation in 45-65% yield based on resin loading.

2. S-Farnesyl-Thiopropionic Acid (FTP) Triazoles; Synthesis and Rationale.

The post-translational processing of members of the Ras protein superfamily is under active investigation because approximately 20% of human cancers result from mutated Ras proteins. ¹ These proteins contain a -CaaX motif that is first isoprenylated on the cysteine thiol with a farnesyl group by farnesyltransferase (FTase) or a geranylgeranyl group by geranyl geranyl transferase- 1 (GGTase-1). Following lipidation, two critical modifications occur sequentially at the endoplasmic recticulum (ER) by membrane-associated enzymes. First, the endoprotease Ras converting enzyme- 1 (Rce-1) cleaves the terminal -aaX residues, resulting in a newly exposed prenylcysteine at the C-terminus of the protein. Second, isoprenylcysteine carboxylmethyltransferase (Icmt) methyl esterifies the carboxylate using S- adenosyl-methionine (SAM) as the methyl donor. The increased hydrophobicity imparted by these modifications is thought to allow for proper localization of the Ras proteins to the plasma membrane, and thus their function. ²

Since blocking Ras localization should prevent its functioning in tumor progression, this posttranslation pathway has been the target for the development of therapeutics against mutant Ras-based tumors. To this end, potent FTase inhibitors were developed that showed promise against some tumors, but proved ineffective against K-Ras driven tumors, ³ due in part to alternative geranylgeranylation. We chose to focus on developing inhibitors of Icmt, the only known enzyme that catalyzes the methyl esterification of the terminal prenylcysteine. ⁴ Genetic knockout studies have provide support for the importance of Icmt- mediated methylation in Ras signaling. ⁵

Substrate specificity studies revealed several key elements necessary for recognition by Icmt, including a 15-20-carbon unit isoprene chain and a requirement for a thioether bond bridging the prenyl and cysteine motifs. ⁶ ' ⁷ Early work by the Rando group identified the minimal substrate for Icmt as farnesyl thiopropionic acid (FTP, l). ⁸ Using this information as a starting point, inhibitors of Icmt have been developed from studies of rotationally restricted prenylcysteines, ⁹ library screening efforts, ¹⁰ amide-, ¹¹ ' ¹² and prenyl-modified ¹³

prenylcysteines. In recent studies, we have established the prenylcysteine analog POP-3MB as a low micromolar inhibitor of human Icmt. ¹²

We have now utilized FTP as a starting point for non-amino acid Icmt inhibitor scaffolds, where either the sulfur was replaced with a triazole or a triazole was incorporated within the isoprene chain. This approach allowed us to utilize the synthetic advantages of copper-assisted dipolar cycloaddition for rapid and facile analog assembly. ¹⁴ ' ¹⁵ Herein, we report the synthesis and evaluation of FTP analogs assembled utilizing click chemistry. ¹⁴ Triazole 12n represents our most potent Icmt inhibitor to date, with an in vitro IC50 of 0.8 ± 0.1 uM.

In the initial design, a 1,4-disubstituted 1,2,3-triazole was positioned as the thioether replacement and cysteine backbone modifier that would join the lipid and carboxylate motifs of the FTP analogs (Scheme 1). This approach had two key advantages: a) it replaced the potentially labile allylic thioether, and b) the building blocks needed for the click reaction were readily available. Sodium azide displacement of bromo-esters 3a-d afforded the requisite azido-esters 4a-d. Activation of farnesol to the mesylate followed by organocuprate addition afforded the prenyl alkyne 6a. ¹⁶ In a similar manner, alkylation of either farnesyl chloride or geranyl bromide afforded alkynes 6b-c. Copper (I) mediated dipolar

cycloaddition ¹⁴ of the azide and alkyne coupling partners was followed by saponification to afford the triazolylprenyl carboxylates 7a-g. This synthetic sequence generated analogs with varying attachments between the carboxylate-triazole and prenyl-triazole moieties, allowing for an initial investigation of the triazole motif as a cysteine backbone replacement.

Scheme l ^{a b} : Triazole for Sulfur Analogs

"Reagents and Conditions: (a) NaN ₃ , DMF, 80 °C; (b) i. TMS-propyne, nBuLi, THF, -78-0 °C ii. TBAF, THF, rt (c) i. nBuLi, THF, 0 °C ii. Ethynyl-MgCl, CuCN -10 °C; (d) sodium ascorbate, cupric sulfate, tBuOH/H ₂ 0, rt. ^b Number in parenthesis indicates Icmt specific activity as percent of AFC control in the presence of 10 μΜ inhibitor.

These analogs (Scheme 1) were evaluated for activity in an established Icmt vapor

1 7

diffusion assay. None of the compounds were substrates for Icmt; instead, 7a-g were modest inhibitors of Icmt. By adjusting the length of the carboxylate-triazole linker, an optimal two methylene bridge was determined (7b). Modulating the spacing between the prenyl-triazole motif was also investigated and found to be important to achieve inhibitory potency. With 7b as the parent molecule, we investigated both increasing the length of the linker between the triazole and prenyl moiety and altering the prenyl chain length in 7e-g. Increasing the linker reduced inhibition, and reducing the lipid tail was also detrimental to inhibitory activity, despite retaining similar overall length to FTP. It is possible that these shorter analogs do not possess enough appropriate hydrophobic character to effectively bind to the enzyme. Alternatively, the triazole may be positioned within the prenyl-binding site, in a manner that prevents the proper alignment of the carboxylate.

While FTP analogs bearing a triazole for sulfur substitution present a potential new approach for the design of Icmt inhibitors, these results also suggest the importance of maintaining the sulfur heteroatom (or an isostere thereof) in the structure of inhibitor compound. The positioning of the triazole was important for inhibitory activity, and these analogs also appear to require extensive isoprene character. Nevertheless, triazole-containing analogs based on the minimal FTP structural motif warranted further exploration as this facile chemical approach could quickly generate diverse compounds.

Previous efforts examined certain characteristics for prenyl-modified AFC analogs. ¹³ These data demonstrated that the correct (E)-geometry of the first isoprene must be maintained as well as inclusion of the sulfur heteroatom. ⁷ Further, simple saturated lipids are not suitable for creating a high-affinity interaction, ¹³ suggesting the possibility for specific "drug-like" structural motifs that will bind to the active site of Icmt.

Compounds developed for other prenyl or lipid binding pockets were taken into consideration in our design. Squalene synthase inhibitor studies revealed that heterocycles could be inserted as isoprene mimics, but geometrical mimicry of the isoprene unit was key. ¹⁸ Aryl substitutions can also be incorporated into the central and terminal isoprene positions of FPP analogs, leading to nanomolar FTase ligands. ^19-21 Collectively, these ideas point to an examination of aryl and heteroaryl modifications to the isoprenoid in our design of Icmt inhibitors.

This focused investigation would again utilize the synthetic advantage of copper- mediated dipolar cycloaddition. A library generated by shifting the triazole ring into the prenyl moiety while maintaining the allylic thioether was investigated. This design allowed for retention of the important first isoprene group and the specific "cysteine-like" moiety in FTP, both of which we hypothesized as key factors in Icmt recognition and binding.

Synthesis of the FTP-triazoles began from iodide 8, which underwent Zr-assisted carboalumination, ²² affording the desired trisubstituted olefin 9 (Scheme 2). Corey-Kim chlorination provided the activated intermediate for alkylation with methyl 3- mercaptopropionate, resulting in ester 10. Sodium azide displaced the primary iodide, affording the desired cycloaddition partner 11. Cu (I)-mediated cycloaddition with a variety of terminal alkynes provided the FTP-triazole analogs. These were evaluated as both the methyl esters (12a-m), and as the carboxylates (13a-m) following saponification. (77%) 12c (67%) 12d (59%)

72%)

) 121 (89%) 12m (80%)

) 131 (89%) 13m (90%)

^a Reagents and Conditions: (a) Me ₃ Al, Cp ₂ ZrCl ₂ , (HCO) _n , 0 °C-rt; (b) i. N-chlorosuccinimide, Me ₂ S, DCM, -40 °C-rt ii. methyl thiopropionate, DIEA, DCM, 0 °C-rt; (c) NaN ₃ , DMF, 80 °C; (d) sodium ascorbate, cupric sulfate, tBuOH/H ₂ 0, rt; (e) LiOH, MeOH rt. ^b Number in parenthesis indicates Icmt specific activity as percent of AFC control in the presence of 10 μΜ inhibitor.

These FTP-triazole analogs were evaluated by the in vitro vapor diffusion assay as previously described. ²⁴ The carboxylates 13a-m lacked measurable substrate activity.

Furthermore, and not surprisingly, the methyl esters (12a-m) also were not substrates, since a free carboxylate is required for methyl transfer.

Inhibition studies using AFC as the substrate demonstrated that all of the analogs exhibited at least weak Icmt inhibition. The alkyl analogs 12a-d and 13a-d were modest inhibitors, and surprisingly the esters showed inhibitory activity comparable to the acids. Modestly increased inhibition of Icmt was seen with 12d, bearing a terminal prenyl group.

In order to achieve both a higher-affinity interaction and more drug-like properties, a more suitable prenyl mimetic was utilized. We utilized a series of aryl alkynes that were incorporated using the facile cycloaddition strategy. Aryl rings were introduced that offered a variety of steric and ring electronic characteristics attached to the triazole (12e-i and 13e-i). Neither the acid nor ester compounds were substrates or inhibitors of Icmt. These data suggest that neither saturated lipids nor rigid aryl groups directly joined to the triazole satisfied the structural requirements for prenyl substitutions.

Natural prenylcysteine substrates have a high degree of flexibility and the

aforementioned aryl analogs possess an inflexible motif. The next series of compounds was designed to investigate the effects of increasing the flexibility between the triazole and the terminal isoprene mimetic (12j-m and 13j-m). Increased inhibition of Icmt was afforded with increased spacing between the triazole and aryl motifs. Analog 12k was the most potent analog in the non-cysteine series, with an IC ₅₀ of 41 ± 5DM. With the incorporation of aryl and heteroaryl groups within the prenyl region a parallel introduction of flexibility appears necessary for effective binding.

A significant advantage of this cycloaddition-based approach is that extensive libraries of terminal alkynes are readily available or easily prepared and compound diversity can be rapidly generated. To this end, alkynes derived from alkylating commercially available phenols with propargyl bromide were synthesized (121 and 13I). ²⁵ Unfortunately, presence of the ether linkage led to a significant decrease in inhibitory activity. In this study, an all carbon linker has been found to be most favorable for imparting inhibitory activity. Incorporation of the polar tetrahydropyranyl moiety resulted in a poor Icmt inhibitor (IC5 ₀ > 100 DM for 13m).

Our results to this point suggested strongly that flexibility between the triazole and isoprene mimetic must be maintained; moreover, heteroatom tolerances were apparently limited. Because Icmt can effectively turnover C ₂₀ -geranylgeranylated substrates, ⁶ we hypothesized that further extension into the region of the terminal isoprene could be utilized for tighter binding of our FTP-triazole analogs. To examine this hypothesis, we initially utilized a series of biphenyl alkynes as we have previously shown that the biphenyl group is an effective isoprene mimetic with FTase and Icmt. ^{12, 13, 19}

Scheme 3 ^a ' : Triazole substitutions in FTP: Use of Biphenyl Alkynes

in parenthesis indicates Icmt specific activity as percent AFC control with 10 μΜ inhibitor.

The design considerations presented above led to FTP triazole analog 12n.

Compound 12n, which contains a methylated carboxyl, was a very potent inhibitor if Icmt in vitro. Under our single point screening assay, the specific activity of Icmt for AFC in the presence of 10 uM 12n decreased to 14% that of the no inhibitor control (Scheme 3). We experimentally determined an in vitro IC ₅₀ of 0.8 ± 0.1 DM for 12n and a calculated Ki of 0.4 DM (Cheng and Prusoff method ²⁶ ). To the best of our knowledge, 12n represents the first substrate-based compound to exhibit sub-micromolar inhibition against Icmt. Following the trend of the other FTP-triazole analogs, the methyl ester analog 12n was much more potent than the corresponding carboxylate 13n (Scheme 3).

With the success of the 4-biphenyl motif in increasing inhibitory potency, we next altered the orientation of the biphenyl to explore the SAR of this new scaffold. As the biphenyl motif was moved towards the ortho position, activity substantially decreased (12o-12p). Ester 12o was nearly 30 times less potent than 12n, with an IC ₅₀ of 29 ± 5 DM. As observed with the shorter analogs, the presence of an oxygen linker was detrimental to inhibition despite the presence of the biphenyl group.

To further SAR efforts, several other methyl esters and acids with altered aromatic ring electronics were synthesized (12r-13u) using the 4-biphenyl FTP-triazole scaffold of 12n. Again, the methyl esters demonstrated greater enzyme inhibition compared to the acids. A trend was observed, with compounds containing electron-withdrawing groups in the terminal aromatic ring (12s, 12t) being more potent than the electron rich 12u. Again, note the increased potency of the esters over their carboxylate counterparts, in contrast to the majority of Icmt inhibitors previously prepared that have a free carboxylate. ²⁷ See Figure 1 A.

A key indicator of inhibitor specificity is the dependence of its cellular effects on the presence of the enzymatic target. A model system utilizing either Icmt ^{+ +} or Icmt ^{" "} MEF cells was available for use in determining the cellular selectivity for 12n. ⁴ These cell lines were derived from wild-type and knockout Icmt mice, as described previously. ⁴ Both cell lines were incubated with increasing amounts of 12n for 24 h and cell viability was measured by a standard MTT assay. Treatment of MEF Icmt ^7" cells with 12n had little effect on cell viability (Figure 1) demonstrating an IC50 above the highest concentration tested (>100uM). On the other hand, wild-type MEF ^{+ +} cell viability decreased upon 12n treatment, with an observed IC50 of 33 ± 0.9 μΜ. These data suggest that 12n is inhibiting cell growth through specific inhibition of Icmt.

FTP-triazole 12n is a potent inhibitor of Icmt both in a biochemical assay and in a model MEF cell system. Removal of the amide motif of the prenylcysteine analogs, and insertion of the biphenyl prenyl-mimetic has led to a paradigm shift in the design of substrate-based inhibitors of Icmt. The lead triazole analog 12n not only possesses higher potency than our best previous Icmt inhibitor (POP-3MB: IC50 = 2.5 μΜ) ¹² but it also possesses a lower molecular weight (450 versus 606), a lower CLogP (5.5 versus 8.8), and is much more easily assembled. It exhibits significantly improved ligand efficiency, and is thus a better starting point for a drug development effort. References for S-Farnesyl-Thiopropionic Acid (FTP) Triazoles: Synthesis and Rationale

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3. Additional Representative Compounds and Related Syntheses.

Additional representative compounds of the invention and related syntheses are described below.

(a) Phenoxy Phenyl and Related Analogs.

The general synthetic design for the synthesis of the phenoxy phenyl and related analogs begins with treating L-cysteine methylester hydrochloride 2.1 with trans-trans- farnesylchloride 2.2 under basic conditions of 7N ammonia in methanol. The resulting farnesylcysteine methylester 2.3 was coupled with the appropriate carboxylic acid using HOBT and HBTU to yield the methylester derivatives (2.4a-h) of the compound of interest. Saponification using lithium hydroxide yielded the final compounds of choice (2.5a-h).

Scheme 2.1 details this general synthesis. In cases where the carboxylic acid was not commercially available (2.4e-h and 2.5e-h), the appropriate aromatic carboxylic acid was synthesized and coupled to farnesylcysteine methylester.

Scheme 2.1 General synthesis of phenoxy-pher.yl analogs

(a) 7N NH ₃ in Methanol.0 "C, 3h, 80% (b) RCOOH, HOBT, HBTU, OIEA, DMF, 0 ^* C to rt, 6

lOh, 48-93% (c) LiOH in Methanol, 2fi, 60-85%.

Carboxylic acids needed for the synthesis for compounds 2.5a-d were commercially available. For compounds 2.5e-h, each carboxylic acid was synthesized as shown in Scheme 2.2A-D. The general method involved using an alkoxide anion as the nucleophile to displace a benzylic halide to obtain the respective phenoxy-phenyl analog. The aromatic bromide was then converted into a Grignard reagent using magnesium turnings in THF and quenching the formed Grignard with dry ice. Carboxylic acid 2.8 used to prepare analog 2.5e was synthesized using the Ullmann-type ether coupling.

Scheme 2.2A-D Synthesis of various carboxylic acids

fa) Potassium hydroxide, 5 equiv; Cu powder, 0.1 equiv; water, reflux, 10 hours, 86% (b)

NCS, dimethylsulfide, DCM, -50 °C to rt, 2 hours, 73% (c) NaH, OMF, 0 °C to rt, 4 hours,

62-77% (d) Mg turnings, 1,2 dibromoethane, rt, 3 hours, then CO ₂ quench, then 10%

HCl, 45 - 78%.

We also designed and synthesized molecules in which the phenoxy phenyl amide scaffold was maintained while modifying the nature or length of the prenyl chain. To this end, we synthesized analogs 2.25a-b shown in Scheme 2.3. Analog 2.25a is the enantiomer of POP-FC, while analog 2.25b bears a longer aliphatic chain. Analog 2.25c with the undecyl moiety was designed to probe the importance of the prenyl chain in achieving hicmt inhibition. We reported that a similar change from prenyl to straight chain alkyl is detrimental to hicmt inhibition when the amide linkage was replaced with a sulfonamide bond.

Scheme 2.3 Synthesis of prenyt-modified phenoxy-phenyl farnesytcysteines

fa} 7N NH ₃ in Methanol, 0 "C, 3-12 hours, 66 - 80% (b) 2-phenoxybenzoIc acid, HOBT,

HBTU, DIEA _> DMF, CTC to rt, 6 hours, 48-93% (c) LiOH in Methanol, 2 hours, 60-85%.

In order to investigate the importance of the amide proton in hlcmt inhibition, we envisioned an N-methyl analog 2.32. The N-methyl analog 2.32 was synthesized using a solid-phase methodology using the advanced intermediate 2.28, which was synthesized as reported previously. Compound 2.28 was loaded on the 2-chlorotrityl-chloride

resin, which is essential to this synthesis due to the acid sensitivity of the prenyl chain.

After loading, the disulfide-protecting group was removed using DTT and the farnesyl group was attached to the free thiol using farnesyl chloride. The 2-phenoxybenzoic acid was then coupled to the secondary amine using standard coupling procedures

previously used in our laboratory. Compound 2.32 was obtained upon cleavage from the resin using 0.2% TFA in DCM. The synthesis of the N-methyl analog is shown in Scheme 2.4.

Additional phenoxy phenyl analogs and related compounds nclude, but are not limited to, those described below.

Compound 2.3: (/?)-methyl 2-amino-3-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l- yl)thio)propanoate: L-cysteine methylester hydrochloride (1.1 eq., 5 mmol, 858.25 mg) was charged to a dry round bottom flask and the atmosphere inside the flask was replaced with argon. The flask was maintained at 0 °C and 5 mL of 7N ammonia in methanol was added to methylester hydrochloride. The contents were allowed to stir for 15 minutes

following which, trans-trans -faraesylchloride (1.0 eq., 4.54 mmol, 1.2 mL) was syringed into the flask. The reactants were allowed to stir together for 3 hours at 0 °C. The reaction was monitored by TLC analysis. Upon completion of the reaction, the contents of the round bottom flask were evaporated to dryness under reduced pressure. The

resulting slurry was impregnated onto a silica gel column and purified by column

chromatography under the influence of gravity via isocratic elution using a mixture of methanol (10%) and dichloromethane (90%). This yielded farnesylcysteine methylester as a viscous yellow oil (1.39 g, 82% yield). 1H NMR (300 MHz, CDC13) δ 5.12 (dt, J= 10.7, 6.2 Hz, 1H), 3.72 (s, 3H), 3.62 (dd, J= 7.8, 4.6 Hz, 1H), 3.34 - 3.10 (m, 2H), 2.85 (td, J= 14.6, 14.0, 5.9 Hz, 2H), 2.01 (ddd, J= 32.0, 10.1, 6.5 Hz, 8H), 1.67 (s, 6H), 1.59 (s, 6H). 13C NMR (75 MHz, CDC13) δ 174.48, 148.59, 135.12, 131.26, 124.26, 124.07, 118.13, 54.18, 52.11, 39.63, 36.61, 30.55, 29.19, 27.31, 26.64, 25.65, 21.23, 17.64, 16.02.

Amide-modified farnesylcysteine methylester intermediates (2.4a-h):

A dry round bottom flask was charged with the appropriate carboxylic acid (1.0 eq., 0.5 mmol) and the atmosphere in the flask was replaced with argon. Dimethylformamide

(DMF, 4 mL/mmol of carboxylic acid) was syringed into the flask under a steady stream of argon. Diisopropylethylamine (DIEA, 1.1 eq., 0.55 mmol, 96 μΚ), HOBT (1.1 eq., 0.55 mmol, 74.3 mg) and HBTU (1.1 eq., 0.55 mmol, 208.6 mg) were added to the reaction vessel and the contents were allowed to react at 0 °C for 30 min. This was followed by addition of a solution of farnesylcysteine methylester, 2.3 (1.1 eq., 0.55 mmol, 186.7 mg) in DMF (1 mL/mmol). The contents were allowed to react for two hours. The

reaction was monitored by TLC analysis. On completion of the reaction, 10% aqueous citric acid (15 mL) was added to the reaction mixture and the product was extracted using ethylacetate (3 x 20 mL) as the solvent. The combined organic extracts were pooled together, dried over anhydrous sodium sulfate (1 g) and filtered. The organic solvent was removed under vacuum utilizing a rotary evaporator and the residue was loaded on a short (2-3 inches) gravity silica gel plug. The silica gel plug was flushed with 3% methanol in dichloromethane to obtain the crude coupling product.

Synthesis of the amide-modified farnesylcysteine (free acid) Compounds

2.5a-h:

Solvent from the crude product obtained from the previous step was removed under reduced pressure and the resulting residue was dissolved in methanol (2 mL/mmol) and was transferred to a round-bottom flask. The atmosphere in the flask was replaced with argon and the contents were allowed to cool to 0 °C. Lithium hydroxide (1.1 eq., 0.55 mmol, 21 mg) was added to the mixture and the contents were allowed to react for two hours. The reaction was monitored by TLC analysis (2% methanol in methylene

chloride). Following the completion of the reaction, as envisioned by consumption of the starting material, the solvent was removed under reduced pressure. The resulting residue was re-suspended in acetone (5 mL) and silica gel (700 mg). The mixture was concentrated under reduced pressure to afford the adsorbed silica gel. This adsorbed silica gel was loaded on a silica gel column (7-9 inches, 1 inch in diameter).

Chromatographic separation was performed under the influence of gravity using

isocratic elution with mixture of methanol (10%) and methylene chloride (90%) to afford compounds 2.5a-h in 60-85%) yields as waxy solids.

Compound 2.5a: (i?)-2-(4-phenoxybenzamido)-3-(((2E,6E)-3,7,l 1-trimethyldodeca- 2,6,10-trien-l-yl)thio)propanoic acid: 1H NMR (300 MHz, CDC13) δ 7.80 (m, 3H), 7.33 (dd, J= 18.3, 10.6 Hz, 2H), 7.06 (m, 4H), 5.41 - 4.74 (m, 4H), 3.13 (m, 4H), 2.10 - 1.87 (m, 8H), 1.67 (d, J= 6.5 Hz, 3H), 1.59 (d, J= 4.5 Hz, 3H), 1.56 (s, 3H). 13C NMR (75 MHz, CDC13) 6176.81, 135.09, 134.77, 131.17, 131.06, 129.86, 124.31, 124.03, 123.81, 119.74, 117.51, 39.66, 39.56, 26.69, 26.48, 25.70, 25.66, 25.63, 17.65, 17.59, 16.08, 15.95, 15.79.

Compound 2.5b: (i?)-2-(3-phenoxybenzamido)-3-(((2E,6E)-3,7,l 1-trimethyldodeca- 2,6,10-trien-l-yl)thio)propanoic acid: IH NMR (300 MHz, CDC13) δ 8.03 - 7.68 (m, 4H), 7.67 - 7.46 (m, 3H), 7.37 (t, J= 7.2 Hz, 1H), 7.20 (d, J= 18.4 Hz, 1H), 5.38 (dd, J= 13.7, 7.3 Hz, 3H), 4.98 (d, J= 29.8 Hz, 1H), 3.61 - 2.94 (m, 4H), 2.48 - 2.08 (m, 8H), 1.94 (d, J= 14.1 Hz, 3H), 1.88 (s, 3H), 1.85 (s, 6H). 13C NMR (75 MHz, CDC13) δ 176.38, 167.17, 157.37, 156.40, 139.73, 135.21, 135.05, 131.12, 129.73, 124.26, 124.22, 123.76, 123.57, 121.85, 121.50, 119.32, 119.07, 117.94, 53.49, 39.61, 39.51, 29.98, 26.64, 26.43, 25.62, 25.59, 17.61, 16.01, 15.91.

Compound 2.5c: (i?)-2-(2-benzylbenzamido)-3-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10- trien-l-yl)thio)propanoic acid: 1H NMR (300 MHz, CD30D) δ 7.32 (d, J= 6.7 Hz, 1H), 7.18 - 6.89 (m, 8H), 5.05 (t, J= 7.4 Hz, 1H), 4.92 (t, J= 6.3 Hz, 2H), 4.51 (s, 1H), 4.13 - 3.93 (m, 2H), 3.11 - 2.99 (m, 2H), 2.78 (ddd, J= 22.1, 13.6, 5.6 Hz, 2H), 1.98 - 1.72 (m, 8H), 1.50 (d, J= 4.1 Hz, 6H), 1.42 (s, 6H). 13C NMR (75 MHz, CD30D) δ 143.06, 141.43, 141.16, 138.46, 136.99, 132.89, 132.31 , 131.87, 131.14, 130.22, 129.32, 127.96, 127.87, 126.29, 125.97, 122.37, 41.69, 41.57, 40.32, 35.24, 31.26, 28.63, 28.28, 26.84, 18.71, 17.21,

17.07.

Compound 2.5d: (i?)-2-(dibenzo[6,i/]furan-4-carboxamido)-3-(((2E,6E)-3,7,l 1- trimethyldodeca-2,6,10-trien-l-yl)thio)propanoic acid: IH NMR (300 MHz, CDC13) δ 8.56 (s, 1H), 8.09 (d, J= 17.2 Hz, 1H), 7.85 - 7.42 (m, 3H), 7.35 (s, 1H), 7.22 (s, 2H), 5.15 (s, 1H), 5.09 - 4.81 (m, 3H), 3.38 - 3.04 (m, 4H), 1.94 (dd, J= 19.0, 7.0 Hz, 8H), 1.64 (s, 3H), 1.56 (s, 3H), 1.47 (d, J= 12.5 Hz, 5H). 13C NMR (75 MHz, CDC13) δ 171.57, 168.75, 156.31,

152.11, 140.64, 136.04, 132.16, 129.75, 128.50, 125.29, 124.77, 124.21, 123.82, 121.35, 120.57, 112.87, 61.28, 40.60, 40.49, 34.29, 31.15, 27.65, 27.39, 26.64, 18.63, 17.00,

16.89, 12.33.

Compound 2.5e: (i?)-2-(2-(phenylthio)benzamido)-3-(((2E,6E)-3,7,l 1-trimethyldodeca- 2,6,10-trien-l-yl)thio)propanoic acid: 1H NMR (300 MHz, CDC13) δ 7.61 (d, J= 32.1 Hz, 1H), 7.37 (s, 3H), 7.28 (d, J= 3.6 Hz, 3H), 7.14 (s, 2H), 7.01 (s, 1H), 5.23 - 4.93 (m, 3H), 4.80 (s, 1H), 3.12 (s, 2H), 2.93 (t, J= 28.2 Hz, 2H), 1.99 (dd, J= 17.7, 6.9 Hz, 8H), 1.67 (s, 3H), 1.59 (s, 5H), 1.55 (s, 3H). 13C NMR (75 MHz, CDC13) δ 171.57, 168.04, 139.63, 136.60, 135.04, 133.98, 133.09, 131.15, 130.88, 129.32, 128.84, 127.92, 126.13, 124.27, 123.81, 119.51, 77.42, 77.00, 76.57, 58.58, 39.62, 39.53, 33.17, 30.02, 26.65, 26.46,

25.65, 17.64, 16.13, 15.95.

Compound 2.5f: (i?)-2-(2-(phenoxymethyl)benzamido)-3-(((2E,6E)-3,7,l 1- trimethyldodeca-2,6,10-trien-l-yl)thio)propanoic acid: IH NMR (300 MHz, CDC13) δ 7.61 (s, 1H), 7.47 (d, J- 6.6 Hz, 2H), 7.30 (d, J= 17.7 Hz, 1H), 7.21 (t, J= 7.0 Hz, 3H), 6.91 (dd, J= 20.5, 7.4 Hz, 3H), 5.34 - 4.92 (m, 5H), 4.61 (s, 1H), 2.97 (s, 3H), 2.62 (s, 1H), 2.10 - 1.79 (m, 8H), 1.68 (s, 3H), 1.60 (s, 3H), 1.55 (s, 3H), 1.51 (s, 3H). 13C NMR (75 MHz, CDC13) δ 174.55, 158.04, 139.77, 135.35, 135.15, 131.23, 130.82, 129.42, 129.01, 128.37, 128.03, 124.31, 123.79, 121.11, 119.20, 114.95, 67.76, 53.84, 39.67, 39.52, 29.78, 26.70, 26.47, 17.67, 16.06, 15.96. Compound 2.5g (i?)-2-(2-(benzyloxy)benzamido)-3-(((2E,6E)-3,7,l 1-trimethyldodeca- 2,6,10-trien-l-yl)thio)propanoic acid: IH NMR (300 MHz, CDC13) δ 8.72 (d, J= 7.2 Hz, 1H),8.22 (dd, J= 7.8, 1.6 Hz, IH), 7.50 (d, J= 6.5 Hz, 2H), 7.46 - 7.29 (m, 4H), 7.13 - 6.98 (m,2H), 5.26 (d, J= 10.5 Hz, 2H), 5.10 (d, J= 18.0 Hz, 3H), 4.94 (dd, J= 12.1, 6.7 Hz, IH), 3.08(ddd, J- 20.4, 13.2, 7.8 Hz, 3H), 2.79 (ddd, J= 20.5, 13.9, 7.8 Hz, 2H), 2.05 - 1.90 (m, 8H),1.67 (s, 3H), 1.62 - 1.57 (m, 9H). 13C NMR (75 MHz, CDC13) δ 175.33, 164.65, 156.96, 139.67, 135.43, 135.19, 133.04, 132.37, 131.20, 129.17, 128.49, 127.99, 124.33, 123.68, 121.39, 120.94, 119.57, 112.68, 77.43, 77.00, 76.58, 71.22, 52.44, 52.29, 39.61, 39.53, 32.90, 29.68, 26.63, 26.34, 25.62, 17.62, 16.01, 15.93.

Compound 2.5h: (i?)-2-(2-((benzyloxy)methyl)benzamido)-3-(((2E,6E)-3,7,l 1- trimethyldodeca-2,6,10-trien-l-yl)thio)propanoic acid: IH NMR (300 MHz, CDC13) δ 8.10 (d, J= 6.8 Hz, IH), 7.71 (t, J= 8.2 Hz, IH), 7.36 (dd, J= 1 1.4, 4.5 Hz, 2H), 7.33 - 7.29 (m, 4H), 7.27 (d, J= 3.7 Hz, IH), 5.19 - 4.99 (m, 3H), 4.66 (d, J= 11.3 Hz, IH), 3.36 (s, 4H), 3.07 (dd, J= 12.5, 7.4 Hz, 2H), 2.87 - 2.71 (m, 2H), 2.11 - 1.88 (m, 8H), 1.65 (d, J= 12.1 Hz, 3H), 1.58 (s, 6H), 1.56 (s, 3H). 13C NMR (75 MHz, CDC13) δ 175.72, 170.17, 140.64, 138.58, 136.17, 136.09, 132.14, 131.54, 131.21 , 130.05, 129.28, 129.22, 128.65, 128.59, 125.21, 124.69, 120.37, 73.07, 71.12, 51.07, 40.57, 40.47, 34.25, 30.78, 27.60, 27.36, 26.58, 18.57, 16.97, 16.87.

Compound 2.8 2-(phenylthio)benzoic acid:

A round bottom flask was charged with 2-iodobenzoic acid 2.6 (1.0 eq., 10 mmol, 2.5 g), potassium hydroxide (5.0 eq., 50 mmol, 2.8 g) and distilled water (40 mL). Thiophenol (1.0 eq., 10 mmol, 1.02 mL) and Copper powder (0.1 eq., 1.0 mmol, 63.5 mg) were then added to the round bottom flask. The contents were refluxed for 12 hours, cooled to room temperature and acidified with 4 N hydrochloric acid. Acidification resulted in precipitation of product, which was filtered and recrystallized with hot methanol to yield white crystals in 86% overall yield. Melting point: 167-170 °C. IH NMR (300 MHz, CDC13) δ 7.85 (dd, J= 7.8, 1.4 Hz, IH), 7.37 - 7.27 (m, 2H), 7.24 - 7.12 (m, 3H), 7.02 - 6.95 (m, IH), 6.87 (dd, J= 10.9, 4.2 Hz, IH), 6.52 (d, J= 8.1 Hz, IH). 13C NMR (75 MHz, CDC13) δ 172.50, 145.62, 136.84, 134.13, 133.11, 133.00, 130.81, 130.30, 128.12, 126.19,

125.24.

General Synthetic methodology for bromides 2.12, 2.16 and 2.20

In general, phenol (or the corresponding benzyl alcohol) (1.3 eq., 1.3 mmol) was charged to a dry round-bottom flask followed by the addition of anhydrous DMF (3 mL/mmol).

The atmosphere in the flask was replaced with argon. Upon complete dissolution of the phenol (or the benzyl alcohol), the contents were cooled to 0 °C and allowed to stir for 15 min. This was followed by addition of sodium hydride (1.3 eq., 1.3 mmol, 31 mg) and the compounds were allowed to react for one hour. Following this time period, a

solution of the appropriate benzyl bromide (1.0 eq., 1.0 mmol) was added as a solution in anhydrous DMF (1 mL/mmol) over a period of 10 min. The contents were allowed to react for 3 hours at 0°C. The reaction was quenched with ice and the product was

extracted with ether (3 x 20 mL), followed by washing with 10% citric acid (3 x 20 mL), 2M NaOH (to remove the unreacted phenol) and brine (2 x 10 mL). The organic fractions were pooled, dried over anhydrous sodium sulfate (1 g), filtered and concentrated

under vacuum to yield a waxy-solid product in each case. A chromatographic separation was performed using ethylacetate (20%) and hexanes (80%) if it was necessary.

Compound 2.12: l-bromo-2-(phenoxymethyl)benzene Yield: 82%, melting point 33 - 35 °C. IH NMR (300 MHz, CDC13) δ 7.71 (d, J= 8.0 Hz, 2H), 7.45 (dt, J= 7.6, 6.4 Hz, 3H), 7.29(dd, J= 12.1, 4.4 Hz, IH), 7.14 (t, J= 8.1 Hz, 3H), 5.27 (s, 2H). 13C NMR (75 MHz, CDC13) 8159.27, 137.20, 133.40, 130.41, 130.01, 129.69, 128.38, 123.11, 122.05, 115.71 , 70.09.

Compound 2.16: l-(benzyloxy)-2-bromobenzene: Yield: 67% IH NMR (300 MHz, CDC13) 57.42 (dd, J= 7.8, 1.6 Hz, IH), 7.33 (d, J= 7.3 Hz, 2H), 7.27 - 7.10 (m, 3H), 7.02 (dd, J= 7.8,1.1 Hz, IH), 6.76 - 6.61 (m, 2H), 4.90 (s, 2H). 13C NMR (75 MHz, CDC13) δ 155.71 , 137.26,134.12, 129.29, 129.20, 128.63, 127.73, 127.71, 122.85, 114.49, 71.29.

Compound 2.20: l-((benzyloxy)methyl)-2-bromobenzene: Yield: 56% IH NMR (300 MHz, CDC13) δ 7.62 - 7.49 (m, 2H), 7.49 - 7.27 (m, 6H), 7.23 - 7.08 (m, IH), 4.69 (s, 4H). 13C NMR (75 MHz, CDC13) δ 138.97, 138.57, 133.43, 130.06, 129.84, 129.41, 129.37, 128.67, 128.33, 123.68, 73.67, 72.50.

General Procedure for synthesis of carboxylic acids 2.13, 2.17 and 2.21:

A dry, flame dried round bottom flask was charged with magnesium turnings (4.0 eq. ₅ 7.53 mmol, 183.2 mg) and was further flame-dried under a steady stream of argon for five minutes. Upon cooling to ambient temperature, anhydrous THF (4 mL/mmol) and one drop of 1 ,2-dibromoethane were added to the flask. The contents were allowed to react for 5 min, followed by addition of the appropriate bromide (1.0 eq., 1.88 mmol, as a solution in anhydrous THF, 3 mL) over a period of 5 min. The Grignard reagent was allowed to form for 2.5 hours and was quenched with dry ice (about 2 grams). This was followed by addition of 10% aqueous hydrochloric acid (10 mL) to the quenched

mixture. The contents from the reaction quench were transferred to a separatory

funnel, followed by addition of ethylacetate (20 mL). The aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were treated with 5% sodium hydroxide (3 x 5 mL). Concentrated hydrochloric acid was added to the

combined aqueous phase until the pH of the solution was 2.5, which resulted in the precipitation of the product. Filtration of the precipitate yielded the desired carboxylic acid in 45 - 78% yields as white solids.

Compound 2.13: 2-(phenoxymethyl)benzoic acid: Melting point 123 - 125 °C. IH NMR (300 MHz, CDC13) δ 11.72 (s, IH), 8.23 (d, J= 7.8 Hz, IH), 7.87 (d, J= 7.8 Hz, IH), 7.65 (s,lH), 7.45 (s, IH), 7.38 - 7.25 (m, 2H), 7.11 - 6.93 (m, 3H), 5.61 (s, 2H). 13C NMR (75 MHz,CDC13) 6 173.87, 159.53, 141.79, 134.68, 132.73, 130.52, 130.48, 128.25, 127.24, 122.00,115.93, 115.90, 69.02.

Compound 2.17: 2-(benzyloxy)benzoic acid: Melting point 69 - 71 °C. IH NMR (300 MHz, CDC13) δ 10.52 (s, IH), 7.77 (dt, J= 27.4, 13.7 Hz, 2H), 7.36 (d, J= 4.1 Hz, IH), 7.32 (s, 2H),7.00 (t, J= 7.5 Hz, IH), 6.79 (ddd, J= 19.3, 17.3, 8.3 Hz, 3H), 5.15 (s, 2H).

Compound 2.21: 2-((benzyloxy)methyl)benzoic acid: Melting point 135 - 138 °C. IH NMR (300 MHz, CDC13) δ 7.72 - 7.49 (m, 3H), 7.49 - 7.31 (m, 5H), 7.25 - 7.11 (m, IH), 4.73 (s, 4H). 13C NMR (75 MHz, CDC13) δ 171.87, 137.97, 135.77, 133.43, 130.06, 129.84, 129.41, 129.37, 128.67, 127.45, 123.68, 72.58, 71.65. Synthesis of compounds 2.23a and 2.23b

The general synthetic strategy followed the procedure mentioned for synthesis of compound 2.3.

Compound 2.23a (S)-methyl 2-amino-3-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l- yl)thio)propanoate: 1H NMR (300 MHz, CDC13) δ 5.14 (dt, J= 10.7, 6.2 Hz, 1H), 3.72 (s, 3H), 3.62 (dd, J= 7.8, 4.6 Hz, 1H), 3.34 - 3.12 (m, 2H), 2.85 (td, J= 14.6, 14.0, 5.9 Hz, 2H), 2.01 (ddd, J= 32.0, 10.1, 6.5 Hz, 8H), 1.67 (s, 6H), 1.59 (s, 6H). 13C NMR (75 MHz, CDC13) δ 173.92, 148.59, 135.10, 131.26, 124.26, 124.07, 118.13, 54.18, 52.11, 39.63, 36.61,30.53, 29.19, 27.31, 26.64, 25.63, 21.23, 17.61, 15.98.

Compound 2.23b (i?)-methyl 2-amino-3-(undecylthio)propanoate: 1H NMR (300 MHz, CDC13) δ 3.71 (s, 3H), 3.63 (dd, J= 7.4, 4.7 Hz, 1H), 2.97 - 2.66 (m, 2H), 2.50 (dd, J= 9.4, 5.3 Hz, 2H), 1.62 - 1.44 (m, 2H), 1.23 (m, 16H), 0.90 - 0.76 (m, 3H). 13C NMR (75 MHz, CDC13) δ 175.48, 65.47, 55.06, 53.13, 38.21, 34.50, 33.56, 32.83, 30.57, 30.44, 30.26, 29.74, 23.61, 15.05, 8.27.

Synthesis of compounds 2.24a and 2.24b

The synthetic strategy followed was identical to the one described for the synthesis of compounds 2.4a-h.

Compound 2.24a (5)-methyl 2-(2-phenoxybenzamido)-3-(((2E,6E)-3,7,l 1- trimethyldodeca-2,6,10-trien-l-yl)thio)propanoate: IH NMR (300 MHz, CDC13) δ 8.60 (d, J= 7.2 Hz, 1H), 8.19 (dd, J= 8.1, 2.1 Hz, 1H), 7.33 (d, J= 7.9 Hz, 2H), 7.16 - 7.03 (m, 4H), 6.81 (d, J= 8.2 Hz, 1H), 5.05 (q, J= 8.1, 7.7 Hz, 3H), 4.95 (dt, J= 6.8, 5.3 Hz, 1H), 3.63 (s, 3H), 3.16 - 2.85 (m, 4H), 2.08 - 1.88 (m, 8H), 1.62 (s, 3H), 1.54 (s, 9H). 13C NMR (75 MHz, CDC13) 8 171.86, 164.99, 156.35, 156.09, 140.30, 135.80, 133.63, 132.87, 131.76, 130.69, 125.21, 124.99, 124.43, 124.18, 123.89, 120.36, 120.22, 118.94, 53.32, 53.05, 40.34, 40.21, 33.74, 30.48, 27.35, 27.02, 26.36, 18.34, 16.66. Compound 2.24b (i?)-methyl 2-(2-phenoxybenzamido)-3-(undecylthio)propanoate 1H NMR (300 MHz, CDC13) 6 8.65 (d, J= 7.2 Hz, 1H), 8.29 - 8.18 (m, 1H), 7.66 - 7.48 (m, 2H), 7.43 - 7.31 (m, 4H), 6.88 (s, 1H), 5.09 - 4.92 (m, 1H), 3.70 (s, 3H), 3.04 (dd, J= 5.2, 3.0 Hz, 2H), 2.42 (t, J= 7.4 Hz, 2H), 1.31 - 1.21 (m, 16H), 0.87 (t, J= 6.3 Hz, 3H). 13C NMR (75 MHz, CDC13) 5 172.17, 165.38, 156.66, 156.34, 133.91, 133.11, 130.93, 125.47, 124.43, 120.52, 120.08, 119.17, 81.93, 53.68, 53.40, 34.89, 33.53, 32.80, 30.49, 30.35, 30.23, 30.15, 29.62, 28.97, 26.51, 23.59, 15.04.

Synthesis of compounds 2.25a and 2.25b

The synthetic strategy was similar to the one described for synthesis of compounds

2.5a-h.

Compound 2.25a (S)-2-(2-phenoxybenzamido)-3-(((2E,6£)-3,7,l 1-trimethyldodeca- 2,6, 10-trien- 1 -yl)thio)propanoic acid

1H NMR (300 MHz, CDC13) δ 10.02 (bs, 1H), 8.77 (d, J= 6.7 Hz, 1H), 8.22 (d, J= 7.1 Hz, 1H), 7.35 (t, J= 7.9 Hz, 3H), 7.23 - 7.04 (m, 4H), 6.83 (d, J= 8.2 Hz, 1H), 5.07 (dd, J= 8.1 3.9 Hz, 3H), 4.95 (d, J= 5.5 Hz, 1H), 3.18 - 2.86 (m, 4H), 1.99 (tt, J- 27.6, 13.8 Hz, 8H),

I .64 (d, J= 14.5 Hz, 3H), 1.58 (d, J= 4.3 Hz, 6H), 1.55 (s, 3H). 13C NMR (75 MHz, CDC13) δ

174.65, 165.04, 155.79, 155.01, 139.55, 134.99, 132.96, 132.15, 131.03, 129.85, 124.49, 124.17, 123.67, 123.26, 122.50, 119.66, 119.41, 117.77, 52.82, 39.52, 39.40, 32.71,

29.74, 26.54, 26.27, 25.55, 17.54, 15.85.

Compound 2.25b (i?,E)-3-((3,7-dimethylocta-2,6-dien-l-yl)thio)-2-(2- phenoxybenzamido)propanoic acid: 1H NMR (300 MHz, CD30D) δ 10.28 (s, 1H), 8.69 (d, J = 6.6 Hz, 1H), 8.20 (dd, J= 7.8, 1.7 Hz, 1H), 7.36 (dd, J= 10.7, 4.8 Hz, 3H), 7.16 (t, J= 7.4 Hz, 2H), 7.12 - 7.05 (m, 2H), 6.84 (d, J= 8.2 Hz, 1H), 5.10 - 4.98 (m, 2H), 4.91 (dd, J=

I I .4, 6.0 Hz, 1H), 3.17 - 2.85 (m, 4H), 2.07 - 1.88 (m, 4H), 1.64 (s, 3H), 1.54 (d, J= 4.7 Hz, 6H). 13C NMR (75 MHz, CD30D) δ 166.37, 157.14, 134.02, 133.69, 132.52, 130.86, 125.56, 124.19, 121.04, 119.56, 37.67, 35.60, 33.33, 33.05, 30.97, 30.81, 30.74, 30.71, 30.49, 23.49, 14.39.

Compound 2.27 (R)-(9H-fluoren-9-yl)methyl 4-((tert-butyldisulfanyl)methyl)-5- oxooxazolidine-3-carboxylate:

To a room temperature solution of 600 mg (1.39 mmol) Fmoc-Cys(StBu)-OH, 2.26 in 10 mL benzene under natural atmosphere was added 215.5 mg (155.1 mg/1 mmol)

paraformaldehyde and 7 mol % para-toluene sulfonic acid (0.097mmol, 18.5 mg). The reaction was heated to reflux in a round bottom flask and stirred for 14 h. The reaction was concentrated to provide clear oil. The contents were loaded on a flash silica gel column and the oxazolidinone 2.27 was purified via isocratic column chromatography (40% ethyl acetate in hexanes) to provide 256 mg (42%) 2.27 as a viscous semi-solid. The NMR peaks matched the data reported earlier.99 1H NMR (300 MHz, CDC13) δ 7.77 (d, 7= 7.5 Hz, 2H), 7.56 (d, J= 7.2 Hz, 2H), 7.41 (t, J= 7.2 Hz, 2H), 7.33 (t, J= 7.6 Hz, 2H), 5.38 (d, J= 29.3 Hz, 2H), 4.75 - 4.50 (m, 2H), 4.25 (t, J= 5.8 Hz, 1H), 4.15 - 3.95 (m, 1H), 3.55 (d, J= 13.7 Hz, 0.5H), 3.21 (ddt, J= 11.1, 8.2, 3.6 Hz, .5H), 2.98 (dd, J= 17.1, 8.9 Hz, 0.5H), 2.65 (d, J= 19.3 Hz, 0.0H), 1.26 (s, 9H). 13C NMR (75 MHz, CDC13) δ 171.93, 152.80, 144.24, 142.14, 128.80, 128.08, 125.63, 120.94, 79.39, 69.91, 66.71, 56.31, 49.24, 47.85, 21.90. Compound 2.28 (i?)-2-((((9H-fluoren-9-yl)metlioxy)carbonyl)(n etliyl)an ino)-3- (tertbutyldisulfanyl) propanoic acid:

To a room temperature stirring solution of 256 mg (0.57 mmol) 2.27 in 3 mL chloroform under natural atmosphere was added 1.18 mL (5.7 mmol) triisopropyl silane followed by the rapid addition of 1.4 mL (2.5 mL/mmol) trifluoroacetic acid. The reaction was stirred at room temperature for 16 h and then concentrated. The oil was dissolved in 20 mL

DCM and concentrated. This was repeated three consecutive times. After impregnation onto a gravity silica gel column, 2.28 was purified via isocratic column chromatography using ethyl acetate (40%) and hexanes (60%) to provide 320 mg (72%) 2.28 as a white solid in a 1.5: 1.0 conformer ratio as reported previously.99

1H NMR (300 MHz, CDC13) - 7.78 (d, J= 7.2 Hz, 2H), 7.60 (dd, J= 17.1, 7.7 Hz, 2H), 7.46 27.38 (m, 1H), 7.33 (td, J= 7.3, 4.9 Hz, 2H), 5.49 - 5.18 (m, 1H), 4.76 - 4.36 (m, 1H), 4.28 (m 1H), 3.85 - 3.70 (m, 0.5H), 3.58 (m, 0.5H), 3.40 - 3.10 (m, 0.5H), 3.03 (s, 3H), 1.27 (s, 9H). ESI-LRMS m/z = 468 [M+Na]+.

Compound 2.32 (i?)-2-(iV-methyl-2-phenoxybenzamido)-3-(((2E,6E)-3,7,l 1- trimefhyldodeca-2,6, 10-trien- 1 -yl)thio)propanoic acid:

2-Chlorotrityl chloride resin (750 mg, 1.01 mmol/g loading capacity) was placed in a 25 mL fritted peptide vessel under argon. To the vessel was added 15 mL of anhydrous methylene chloride and the resin was allowed to swell by allowing the contents to gently rock on a peptide shaker for 30 minutes. Meanwhile, in a separate vial, 2.28 (1.68 g, 3.78 mmol) was dissolved in 5 mL of anhydrous methylene chloride to which was added 2,4,6- collidine (501 μί, 3.78 mmol). After the resin was swollen, the solvent was removed from the resin, the solution of 2.28 was transferred to the resin. The mixture was gently agitated for 6 h on a peptide shaker under argon. Then, 5 mL of a 1% solution of 2,4,6- collidine in MeOH was then added, and the mixture was agitated for an additional 10 min. The mixture was drained, and the resin was washed 3 times each

with DMF, methylene chloride, and DMF. Based on previous protocols developed in the laboratory, 80% loading efficiency was assumed.

General Procedure for Reduction of -StBu Group. Dithiothreitol (DTT, 770 mg, 3.78 mmol) was dissolved in a 2% diisopropylethylamine/DMF solution (10 mL) and added to the resin-bound cysteine, the atmosphere in the vessel was replaced with argon, and the reaction vessel was gently agitated overnight. The solvent was drained, and the resin washed 3 times each with DMF, methylene chloride, and DMF. The deprotection step was repeated again with a fresh solution of DTT.

General Procedure for Farnesylation of Resin-Bound Fmoc- Cysteine. DIEA (1.3 mL, 7.5 mmol) was added to a solution of farnesyl chloride (995 μί, 3.78 mmol) in 4 mL of methylene chloride. This solution was added to the resin-bound cysteine under argon, and the reaction vessel was gently agitated for 4 h at ambient temperature. The solvent was then drained, and the resin was washed as before. Cleavage of N-Fmoc Group from loaded Cys derivative. A 20% solution of piperidine in DMF (8 mL) was added to the resin- bound famesylcysteine, and the vessel was agitated for 25 min. The solvent was drained and rinsed once with DMF, and the process was repeated. The solvent was drained, and the resin was rinsed as before.

Procedure for Coupling of 2-phenoxy benzoic acid. 2-phenoxybenzoic acid (3.0 eq., 2.27 mmol, 487 nig), HOBt (3.0 eq., 2.27 mmol, 306 mg), HBTU (3.0 eq., 2.27 mmol, 860 mg) were dissolved in 4 mL of DMF, and DIEA (3.0 eq., 2.27 mmol, 395 μί) was then added. After 5 min, this solution was then added to the resin set on a peptide rocker for 12 h.

The solvent was drained, and the resin was rinsed as before.

Procedure for Cleavage of AMFC from Resin. The resin-bound compound 2.31 was treated once with a 0.5% solution of trifluoroacetic acid in methylene chloride for 1 min. The solution was drained into a round-bottom flask, and the resin was washed three times with 10 mL anhydrous methylene chloride.

The solvent was removed in- vacuo, and the crude material was loaded directly onto a silica column and purified using isocratic elution using methanol (0.5%) and methylene chloride (99.5%) and purified to give 52 mg of 2.32, a 16% yield based on starting cysteine derivative loaded onto the resin. 1H NMR (300 MHz, CDC13) δ 7.33 - 7.11 (m, 4H), 7.10 - 6.93 (m, 3H), 6.92 - 6.81 (m, 2H), 6.80 - 6.56 (m, 1H), 4.94 (d, J= 1.3 Hz, 3H), 4.30 - 4.13 (m, 1H), 3.12 - 2.98 (m, 1H), 2.94 (d, J= 7.6 Hz, 1H), 2.87 (d, J= 5.8 Hz, 1H), 2.81 (s, 2H), 2.72 (dd, J= 13.9, 4.2 Hz, 1H), 2.03 - 1.97 (m, 2H), 1.97 - 1.75 (m, 8H), 1.51 (s, 4H), 1.44 (s, 6H). 13C NMR (75 MHz, CDC13) δ 171.94, 157.82, 136.14, 132.05, 131.93, 130.91, 130.87, 125.44, 125.38, 125.13, 124.55, 121.60, 121.09, 120.98, 120.36, 119.12, 62.48, 40.84, 40.70, 30.69, 29.84, 27.78, 27.46, 25.97, 17.84, 16.31, 16.24, 16.20. ESILRMS (pos) m/z 536 [M+H]+, 558 [M+Na]+.

(b) Carbamates.

The carbamate library (3.8a-g) was afforded through S-alkylation of cysteine with farnesylchloride, followed by reaction of farnesylcysteine 3.7 with various commercially available chloroformates in presence of potassium carbonate and acetone (Scheme 3.2).

We also wanted to investigate the effect of a thiocarbamate linker, and to this end we synthesized compound 3.10. The synthesis of this analog is depicted in Scheme 3.3.

(c) Alcohol Analog.

An alcohol-bearing analog 4.11 (which we termed "Farnol") is a potent

Hlcmt inhibitor, has a submicromolar IC50.

Alcohol analog and related compounds can be synthesized as shown in Schemes 4.1 and 4.2 below.

Methylthiopropionate 4.1 was S-alkylated with all-traw-farnesyl chloride 4.2 to yield thioether 4.3. Compound 4.3 was either saponified to yield FTP A 4.4, or reduced using diisobutylaluminium hydride to yield the alcohol analog 4.5. Coupling of the carboxylic acid 4.4 with hydroxylamine hydrochloride afforded hydroxamic acid 4.8. The alcohol analog 4.5 was modified via bromination to analog 4.6, and then converted to the phosphonate ethyl ester 4.7.

Additional Synthesis Details:

Compound 4.5 3-(((2E, 6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)propan-l-ol: The atmosphere in a dry, flame-dried round bottom flask was replaced with argon.

Freshly distilled (over calcium hydride) methylene chloride (5 mL/mmol) was added to the flask under a steady stream of argon. Compound 4.3 (1.0 eq., 3.2 mmol, 1.03 g) was then added (as a solution in 2 mL methylene chloride) to the round bottom flask. The contents were cooled to -78 °C and all times, the atmosphere in the flask was kept under argon. Once the contents in the flask reached a temperature of -78 °C, a solution of 1M diisobutylaluminium hydride in methylene chloride (DIBAL-H, 2.5 eq., 8.0 mmol, 8.0 mL) was added slowly to the reaction vessel. The contents were allowed to react at - 78 °C for 2 hours and then the temperature of the flask was allowed to slowly increase to 0 °C. The contents were allowed to react for a further 2 hours. Upon completion of the reaction, the reaction mixture was added slowly to a cooled solution of 10% citric acid. The contents were transferred to a separatory funnel and the aqueous layer was extracted with methylene chloride (3 x 15 mL). The organic extracts were combined, washed with brine and concentrated under reduced pressure. The residue was

impregnated onto a silica gel column. Purification through isocratic elution using ethyl acetate (30%) and hexanes (70%) yielded compound 4.5 in 65% yield as a viscous oil. IH NMR (300 MHz, CDC13) δ 5.20 (tt, J= 13.4, 6.7 Hz, IH), 5.13 - 4.93 (m, 2H), 3.68 (q, J= 5.8Hz, 2H), 3.13 (d, J= 7.8 Hz, 2H), 2.54 (t, J= 7.0 Hz, 2H), 2.31 (s, IH), 2.15 - 1.89 (m, 8H),1.80 (dq, J= 13.1, 6.6 Hz, 2H), 1.66 - 1.63 (s, 3H), 1.63 (s, 3H), 1.56 (s, 6H). 13C NMR (75 MHz, CDC13) δ 138.75, 135.12, 131.12, 124.18, 123.64, 120.21, 61.62, 39.56, 39.47, 31.80, 29.15, 27.61, 26.57, 26.27, 25.57, 17.55, 15.95, 15.89.

Compound 4.11 2-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)ethanol:

The synthetic method used to synthesize 4.11 was similar to the one described for the preparation of compound 4.5. Column chromatographic purification (30% ethyl acetate and 70% hexanes) yielded 69% of 4.11 as a viscous oil. IH NMR (300 MHz, CDC13) δ 5.18 (tt, J- 11.1, 5.6 Hz, IH), 5.13 - 4.94 (m, 2H), 3.65 (t, J= 6.1 Hz, 2H), 3.13 (t, J= 7.5 Hz, 2H), 2.64 (s, IH), 2.61 (d, J= 6.0 Hz, 2H), 2.15 - 1.84 (m, 8H), 1.63 (d, J= 0.7 Hz, 3H), 1.61 (s, 3H), 1.54 (d, J= 10.4 Hz, 6H). 13C NMR (75 MHz, CDC13) δ 139.04, 135.12, 131.05, 124.13, 123.54, 120.08, 60.27, 39.52, 39.41, 33.81, 28.66, 26.52, 26.18, 25.53, 17.51, 15.89, 15.85. ESI-LRMS (pos) m/z = 283 [M]+, 321 [M+K]+.

Scheme 4.2 Synthesis of FTA-based carboxylate replaced compounds

(a) 7N NH3/MeOH, 0 °C, 3 hours, 70-75% (b) DIBAL-H, anhydrous DCM, 0 °C - rt, 4 hours, 69% (c) LiOH, MeOH, rt, 4 hours, 79% (d) 7N NH3/MeOH, rt, 3 hours, 74% (e) Dess-Martin periodinane, 0 °C, DCM, then NaHC03, 4 hours, 64% (f) acetic anhydride 0 °C, 2hours, 88% (g) NH20H HCl, DIEA, HOBT, HBTU, 65% (h) PPh3, CBr4, imidazole, DCM, 0 °C- rt, 2 hours, 77% (i) Cs2C03, diethylphosphite, 8 days, 43% (j) NaN3, DMF, 50 °C, 10 hours, 61%.

(d) Alcohol and Diol-Triflurophenyl Analogs.

Compound 6.53 was synthesized based on its predicted potency from the predictive QSAR model. This compound matched its prediction, and has provided us with the first sub- 100 nanomolar substrate-based hlcmt inhibitor described till date. Compounds

4.21e and 4.21f were earlier synthesized as carboxylate modifications and have an

estimated IC50 of 1.5 μΜ. We hypothesized that combining the diol for ester

replacement and the trifluoro-biphenyl motifs would be beneficial for hlcmt inhibitors because the alcohol-based modifications would replace the ester metabolic soft spot in our molecules. With that in mind, we synthesized analogs 6.60 - 6.62 (Scheme 6.7).

Both compounds 6.60 and 6.61 display superior hlcmt inhibition and are equipotent to their ester counterparts. Compound 6.60 has an IC50 of 250 ± 0.02 nM, while the IC50 for compound 6.61 was experimentally determined to be 93 ± 0.003 nM. Compound 6.62 inhibits hlcmt by 97% at 10 μΜ in vitro.

Additional Synthesis Details:

Compound 6.40 (E)-methyl 2-((3-methyl-5-(4-(2-(4-phenoxyphenoxy)ethyl)-lH-l,2,3- triazol-l-yl)pent-2-en-l-yl)thio)acetate: 1H NMR (300 MHz, CDC13) δ 7.43 (d, J= 2.7 Hz, 1H), 7.35 - 7.23 (m, 2H), 7.03 (dd, J- 8.7, 6.2 Hz, 1H), 6.99 - 6.88 (m, 5H), 5.18 (t, J= 7.9 Hz, 1H), 4.44 (td, 7= 7.1, 2.7 Hz, 2H), 4.22 (td, J= 6.6, 2.8 Hz, 2H), 3.71 (s, 3H), 3.19 (dt, J = 6.5, 2.8 Hz, 4H), 3.06 (d, J= 2.7 Hz, 2H), 2.61 (t, J= 7.0 Hz, 2H), 1.67 (s, 3H). 13C NMR (75 MHz, CDC13) δ 170.92, 158.26, 154.80, 150.23, 144.45, 135.17, 129.52, 123.02, 122.40, 121.74, 120.69, 117.53, 115.46, 67.21, 52.28, 48.39, 40.06, 32.01, 29.54, 26.12, 15.69.

Synthesis of compound 6.56 (E)-3-((5-bromo-3-methylpent-2-en-l-yl)thio)propane-l,2- diol:

A dry round bottom flask was charged with a-thioglycerol (0.9 eq., 4.3 mmol, 374 μί,) and the atmosphere in the flask was replaced with argon. The flask was placed in an ice bath and 7N ammonia in methanol (3 mL) was syringed into the flask and the contents were allowed to react with each other for not more than one minute. Compound 6.3

(obtained from the reaction as a solution in methylene chloride) was then slowly

syringed into the reaction flask. The contents were allowed to reach ambient

temperature slowly and were allowed to react for 15 hours. Upon completion of

reaction (on certain occasions, the reaction proceeded very sluggishly and complete reaction, as evidenced by disappearance of starting material, was only observed after 96 hours), the contents of the reaction mixture were evaporated under reduced pressure.

The resulting slurry was then adsorbed on gravity silica gel and isocratic elution with ethyl acetate (15%) and hexanes (85%) yielded compound 6.56 as colorless oil in 79% yield. IH NMR (300 MHz, CDC13) δ 5.29 (t, J= 7.8 Hz, IH), 3.83 - 3.47 (m, 5H), 3.43 (t, J = 7.1 Hz, 2H), 3.25 - 3.07 (m, 2H), 2.62 - 2.46 (m, 4H), 1.64 (s, 3H). 13C NMR (75 MHz, CDC13) δ 135.67, 123.31, 70.38, 65.28, 42.22, 34.15, 31.08, 29.23, 15.48.

Compound 6.57 (£)- 1 -((5-bromo-3-methylpent-2-en- 1 -yl)thio)propan-2-ol:

Compound 6.57 was synthesized using a similar synthetic strategy ad described in the synthesis of compound 6.56 above, l-mercaptopropan-2-ol 6.55 (0.9 eq., 2.25 mmol, 217 μΐ,) was used instead of a-thioglycerol. Compound 6.57 was isolated in 74% yield. IH NMR (300 MHz, CDC13) δ 5.33 (t, J= 7.5 Hz IH), 3.77 (dpd, J= 12.4, 6.5, 3.0 Hz, IH), 3.39 (t, J= 7.2 Hz, 2H), 3.11 (qd, J= 13.5, 7.6 Hz, 2H), 2.74 (s, IH), 2.61 (dd, J= 13.7, 3.9 Hz, IH), 2.52 (t, J- 7.2 Hz, 2H), 2.35 (dd, J= 13.7, 8.5 Hz, IH), 1.60 (s, 3H), 1.16 (d, J= 6.2 Hz, 3H). 13C NMR (75 MHz, CDC13) δ 135.35, 123.43, 65.42, 42.15, 40.05, 30.93, 28.90, 21.88, 15.35.

Compound 6.58 (E)-3-((5-azido-3-methylpent-2-en- 1 -yl)thio)propane- 1 ,2-diol:

A dry scintillation vial was charged with (E)-3-((5-bromo-3-methylpent-2-en-l- yl)thio)propane-l,2-diol 6.56 (1.0 eq., 2.0 mmol, 536 mg) as a solution in anhydrous DMF (3 mL) and sodium azide (2.5 eq., 5.0 mmol, 324 mg). The atmosphere in the flask replaced with argon and the contents were allowed to react with each other at 40 °C for 8 hours. Upon completion of reaction, the contents were allowed to cool and

transferred to a separatory funnel containing 10 mL of 10% aqueous citric acid. Ethyl acetate (15 mL) was added to the separatory funnel and the aqueous phase was

extracted with ethyl acetate. Further ethyl acetate extractions (2 x 15 mL) were carried out and the combined organic extracts were pooled together, washed with brine (15 mL) and dried over sodium sulfate (800 mg). Evaporation of the solvent under reduced pressure yielded a brown oil that was impregnated onto a silica gel column. Isocratic elution with methanol (5%) and methylene chloride (95%) yielded compound 6.58 as yellow oil in 65% yield. IH NMR (300 MHz, CDC13) 5 5.18 (ddd, J= 9.3, 7.1, 1.3 Hz, IH), 3.61 (qd, J= 6.2, 3.3 Hz, IH), 3.55 - 3.46 (m, IH), 3.36 (dd, J= 11.3, 6.5 Hz, IH), 3.18 (t, J = 7.0 Hz, 2H), 3.02 (d, J= 7.7 Hz, 2H), 2.50 - 2.32 (m, 2H), 2.15 (t, J- 7.0 Hz, 2H), 1.52 (d, J= 1.2 Hz, 3H). 13C NMR (75 MHz, CDC13) δ 134.46, 122.80, 70.50, 64.82, 48.86, 37.97, 30.98, 29.06, 15.39.

Compound 6.59 (E)-l-((5-azido-3-methylpent-2-en-l -yl)thio)propan-2-ol:

A dry scintillation vial was charged with (E)-l-((5-bromo-3-methylpent-2-en-l- yl)thio)propan-2-ol 6.57 (1.0 eq., 0.96 mmol, 244 mg) as a solution in anhydrous DMF (3 mL) and sodium azide (2.5 eq., 1.92 mmol, 125 mg). The atmosphere in the flask

replaced with argon and the contents were allowed to react with each other at 40 °C for 8 hours. Upon completion of reaction, the contents were allowed to cool and

transferred to a separatory funnel containing 10 mL of 10% aqueous citric acid. Ethyl acetate (15 mL) was added to the separatory funnel and the aqueous phase was

extracted with ethyl acetate. Further ethyl acetate extractions (2 x 15 mL) were carried out and the combined organic extracts were pooled together, washed with brine (15 mL) and dried over sodium sulfate (800 mg) and filtered. Evaporation of the solvent under reduced pressure yielded a brown oil that was impregnated onto a silica gel

column. Isocratic elution with ethyl acetate (15%) and hexanes (85%) yielded compound 6.59 as yellow oil in 71% yield. IH NMR (300 MHz, CDC13) δ 5.35 - 5.21 (m, IH), 3.84 - 3.70 (m, IH), 3.27 (t, J= 7.0 Hz, 2H), 3.21 - 3.00 (m, 2H), 2.80 (d, J= 2.2 Hz, IH), 2.60 - 2.51 (m, IH), 2.34 (dd, J= 13.6, 8.4 Hz, IH), 2.24 (t, J- 7.0 Hz, 2H), 1.15 (d, J= 6.2 Hz, 3H), 1.65 (s, 3H). 13C NMR (75 MHz, CDC13) δ 134.71, 123.07, 65.45, 49.09, 40.05, 38.20, 28.96, 21.78, 15.60.

Compound 6.60 (E)-3-((5-(4-(2-([l,l'-biphenyl]-4-yl)ethyl)-lH-l,2,3-triazo l-l-yl)-3- methylpent-2-en-l-yl)thio)propane-l,2-diol: The alkyne 6.44 (1.2 eq., 0.6 mmol, 124 mg) was charged to a scintillation vial and suspended in a 1 : 1 mixture of tert-butanol and water (3 mL total). Sodium ascorbate (0.5 eq., 0.25 mmol, 50 mg) was added to the reaction mixture, followed by compound 6.58 (1.0 eq., 0.5 mmol, 120 mg) and copper sulfate pentahydrate (0.25 eq., 0.13 mmol, 32 mg). The contents were allowed to stir at ambient temperature under an atmosphere of argon for 5 hours. Upon completion of reaction (as evidenced by

disappearance of starting material), the contents were transferred to a separatory

funnel, followed by addition of ethyl acetate (10 mL) and brine (10 mL). The aqueous layer was further extracted with ethyl acetate (3 10 mL). The organic fractions were

pooled together, washed with brine (10 mL) and dried over sodium sulfate (500 mg).

The organic solvent was removed under reduced pressure to yield a pale to dark

yellow/brown oil. This was impregnated on a silica gel column and purified through isocratic elution with methanol (1%) and methylene chloride (99%) to yield compound 6.60 as yellow oil in 58% yield. 1H NMR (300 MHz, CDC13) δ 7.62 - 7.56 (m, 2H), 7.56 - 7.50 (m, 2H), 7.43 (t, J= 7.5 Hz, 2H), 7.33 (t, J= 7.0 Hz, 1H), 7.29 - 7.23 (m, 2H), 7.13 (s, 1H), 5.10 (t, J= 7.8 Hz, 1H), 4.41 (dd, J= 7.5, 5.9 Hz, 2H), 3.78 - 3.67 (m, 2H), 3.58 (q, J= 6.4 Hz, 1H), 3.12 - 2.97 (m, 5H), 2.51 (q, J= 5.8, 5.0 Hz, 2H), 2.47 - 2.32 (m, 2H), 1.68 (s, 3H). 13C NMR (75 MHz, CDC13) δ 147.30, 140.79, 140.02, 138.96, 133.81, 128.89, 128.70, 127.07, 127.02, 126.87, 124.56, 120.87, 70.46, 65.26, 48.18, 40.34, 34.95, 34.01, 29.18, 27.20, 15.56. ESI-LRMS (pos) m/z = 438 [M+H]+, 460 [M+Na]+, ESI-HRMS (pos) calculated mass = 438.2215, actual mass = 438.2213.

Compound 6.61 (E)-3-((3-methyl-5-(4-(2-(3,3',4'-trifiuoro-[l,l ^, -biphenyl]-4-yl)ethyl)-lH- 1 ,2,3-triazol- 1 -yl)pent-2-en- 1 -yl)thio)propane- 1 ,2-diol:

The alkyne 6.52 (1.2 eq., 07 mmol, 172 mg) was charged to a scintillation vial and suspended in a 1 :1 mixture of tert-butanol and water (3 mL total). Sodium ascorbate

(0.5 eq., 0.28 mmol, 54 mg) was added to the reaction mixture, followed by compound 6.58 (1.0 eq., 0.55 mmol, 127 mg) and copper sulfate pentahydrate (0.25 eq., 0.14

mmol, 34 mg). The contents were allowed to stir at ambient temperature under an

atmosphere of argon for 5 hours. Upon completion of reaction (as evidenced by

disappearance of starting material), the contents were transferred to a separatory

funnel, followed by addition of ethyl acetate (10 mL) and brine (10 mL). The aqueous layer was further extracted with ethyl acetate (3 x 10 mL). The organic fractions were pooled together, washed with brine (10 mL) and dried over sodium sulfate (500 mg).

The organic solvent was removed under reduced pressure to yield a pale to dark

yellow/brown oil. This was impregnated on a silica gel column and purified through isocratic elution with methanol (5%) and methylene chloride (95%) to yield compound 6.61 as yellow oil in 74% yield. IH NMR (300 MHz, CDC13) 5 IH NMR (300 MHz, CDC13) 57.37 - 7.26 (m, IH), 7.26 - 7.12 (m, 5H), 5.09 (t, J= 7.8 Hz, IH), 4.42 (t, J= 6.7 Hz, 2H), 3.91 - 3.43 (m, 5H), 3.07 (d, J= 7.8 Hz, 2H), 3.03 (s, 4H), 2.52 (t, J= 6.7 Hz, 2H), 2.48 - 2.33 (m, 2H), 1.72 (s, 3H). 13C NMR (75 MHz, CDC13) δ 162.87, 159.62, 151.81 (dd, JCF = 31.5, 12.9 Hz, 1C), 148.51 (dd, JCF = 32.5, 12.8 Hz, 1C), 146.91, 139.04 (d, JCF = 8.3 Hz, 1C),136.64 (d, JCF = 5.5 Hz, 1C), 133.75 , 131.20 (d, JCF = 5.6 Hz, 1C), 127.22 (d, JCF = 16.2 Hz,lC), 124.45, 122.46 (d, JCF = 29.1 Hz, 1C), 120.86, 117.55 (d, JCF - 17.4 Hz, 1C), 115.68 (d,JCF = 18.0 Hz, 1C), 113.56 (d, JCF = 23.6 Hz, 1C), 70.57, 65.18, 48.23, 40.24, 38.20, 33.95,29.17, 28.47, 25.78, 15.53. ESI-LRMS (pos) m/z = 492 [M+H]+, 514 [ +Na]+, 530 [M+K]+, ESI-HRMS (pos) calculated mass = 492.1933, actual mass = 492.1930.

Compound 6.62 (E)-l-((3-methyl-5-(4-(2-(3,3',4 ^, -trifluoro-[l,l'-biphenyl]-4-yl)ethyl)-lH- 1 ,2,3-triazol- 1 -yl)pent-2-en- 1 -yl)thio)propan-2-ol:

Compound 6.62 was prepared similarly as described for the synthesis of compound

6.61. Azide 6.59 (1.0 eq., 0.23 mmol, 50 mg) was used instead of azide 6.58. IH NMR (300 MHz, CDC13) δ 7.36 - 7.25 (m, 2H), 7.17 (d, J= 6.8 Hz, IH), 5.13 (t, J= 7.8 Hz, 3H), 7.13 (s, IH), 5.13 (t, J= 6 Hz, IH), 4.40 (t, J= 7.2 Hz, 2H), 3.84 - 3.71 (m, IH), 3.15 - 3.06 (m, 2H), 3.02 (s, 6H), 2.53 (t, J= 7.0 Hz, 3H), 2.49 - 2.26 (m, 2H), 1.66 (s, 3H), 1.21 (d, J= 6.2 Hz, 3H). 13C NMR (75 MHz, CDC13) 5 162.86, 159.60, 151.77 (dd, JCF = 32.0, 12.5 Hz, 1C), 148.48 (dd, JCF = 32.9, 12.8 Hz, 1C), 146.78, 138.97 (d, JCF = 7.5 Hz, 1C), 137.24 -136.14 (m), 133.89, 131.19, 127.43, 124.13, 122.49 (d, JCF = 41.0 Hz), 120.77, 117.52 (d, JCF = 17.4 Hz, 1C), 115.65 (d, JCF = 18.0 Hz, 1C), 113.52 (d, JCF = 23.6 Hz, 1C) 77.43, 65.70,48.26, 40.10, 39.89, 28.98, 28.48, 25.82, 21.88, 15.60. ESI-LRMS (pos) m/z = 492 [M+H]+,514 [M+Na]+, ESI-HRMS (pos) calculated mass = 492.1933, actual mass =

492.1930.

Synthesis of compound 6.64 (E)-(5-bromo-3-methylpent-2-en-l-yl)(2- methylbutyl)sulfane :

A dry round bottom flask was charged with 2-methylbutane-l -thiol (1.0 eq., 2.14 mmol, 263 μΐ) and the atmosphere in the flask was replaced with argon. The flask was placed in an ice bath and 7N ammonia in methanol (3 mL) was syringed into the flask and the contents were allowed to react with each other for not more than one minute.

Compound 6.3 (obtained from the reaction as a solution in methylene chloride) was then slowly syringed into the reaction flask. The contents were allowed to reach ambient temperature slowly and were allowed to react for 20 hours. Upon completion.

of reaction, the contents of the reaction mixture were evaporated under reduced

pressure. The resulting slurry was then adsorbed on gravity silica gel and isocratic

elution with ethyl acetate (15%) and hexanes (85%) yielded compound 6.64 as colorless oil in 82% yield. 1H NMR (300 MHz, CDC13) δ 5.41 - 5.27 (m, 1H), 3.45 (t, J= 7.3 Hz, 2H),

3.13 (d, J= 7.8 Hz, 2H), 2.58 (t, J= 7.3 Hz, 2H), 2.52 - 2.25 (m, 2H), 1.67 (d, J= 1.3 Hz,

3H), 1.63 - 1.40 (m, 2H), 1.19 (dt, J= 13.3, 7.3 Hz, 1H), 0.96 (d, J= 6.6 Hz, 3H), 0.88 (t, J=

7.4 Hz, 3H). 13C NMR (75 MHz, CDC13) δ 134.78, 124.22, 38.38, 34.79, 31.09, 29.62,

28.82, 18.95, 15.59, 11.33.

Synthesis of compound 6.65 (E)-l-(3-methyl-5-((2-methylbutyl)thio)pent-3-en-l-yl)-4-(2- (3,3',4'-trifluoro-[l ,1 '-biphenyl]-4-yl)ethyl)-lH-l ,2,3-triazole:

Compound 6.64 (1.0 eq., 0.20 mmol, 54 mg) was charged to a round bottom flask. DMF (4 mL) and sodium azide (2.0 eq., 0.40 mmol, 26 mg) were added to the flask, followed by sodium ascorbate (0.5 eq., 0.27 eq., 34 mg), alkyne 6.52 (1.2 eq., 0.40 mmol, 104 mg) and copper sulfate pentahydrate (0.25 eq., 0.08 mmol, 21.2 mg). The contents were allowed react with each other at 40 °C for 8 hours. Upon completion of the reaction, the contents were allowed to cool to ambient temperature and were transferred to a

separatory funnel. 10% aqueous citric acid (10 mL) followed by ethyl acetate (15 mL) were added to the separatory funnel. The aqueous phase was extracted further with fresh ethyl acetate (2 15 mL). The combined organic extracts were washed further with 10% citric acid (2 x 10 mL) and brine (20 mL). The ethyl acetate fractions were then dried over sodium sulfate (1 g), filtered and concentrated under reduced pressure. The resulting oil was impregnated onto a silica gel column and isocratic elution with ethyl acetate (50%) and hexanes (50%) was used to isolate compound 6.65 as a white waxy solid in 83% yield. 1H NMR (300 MHz, CDC13) δ 7.34 (ddd, J= 1 1.6, 7.5, 2.1 Hz, 1H), 7.30 -7.17 (m, 5H), 7.15 (s, 1H), 5.22 (t, J= 7.9 Hz, 1H), 4.40 (t, J= 7.2 Hz, 2H), 3.05 (s, 6H), 2.56(t, J= 7.2 Hz, 2H), 2.46 - 2.18 (m, 2H), 1.60 - 1.37 (m, 2H), 1.69 (s, 3H), 1.54 - 1.42 (m, 2H), 1.19 (td, J= 13.2, 6.7 Hz, 1H), 0.94 (d, J= 6.7 Hz, 3H), 0.87 (t, J= 7.3 Hz, 3H). 13C NMR (75 MHz, CDC13) δ 162.95, 159.70, 151.87 (dd, JCF = 32.6, 12.4 Hz, 1C), 148.94 - 148.14 (m, 1C), 146.75, 139.03 (d, JCF = 7.7 Hz, 1C), 133.33, 131.21 (d, JCF = 5.7 Hz, 1C), 127.50 (d, JCF = 16.3 Hz, 1C), 124.43, 122.75 (dd, JCF = 6.0, 3.4 Hz, 1C), 122.28 (d, JCF = 2.9Hz, 1C), 120.80, 117.58 (d, JCF = 17.4 Hz, 1C), 115.74 (d, JCF = 17.8 Hz, 1C), 1 13.61 (d, JCF =23.7 Hz, 1C), 48.53 , 40.06 , 38.67 , 34.79 , 29.68 , 28.73 , 25.96 , 18.86 , 15.83 , 11.30.

The invention is illustrated further in the following non-limiting examples. Example 1

Identification of a Novel Nanomolar Inhibitor of hlcmt via

a Carboxylate Replacement Approach

1. Background.

Pancreatic cancer has one of the poorest prognoses amongst all cancers. ¹ Over 85% of all pancreatic cancers are caused by mutations in the K-Ras oncogene that permanently activate the enzyme. K-Ras, and other members of the Ras protein superfamily, possess a CaaX motif at the C-terminus, where C is a cysteine, "a" is an aliphatic amino acid and "X" can be one of many different amino acids. K-Ras undergoes three sequential post- translational modifications ³ (Figure 1) beginning with famesylation of the cysteine residue sulfur by farnesyltransferase (FTase). This lipidation reaction is followed by endoproteolysis of the -aaX residues by Rce-1 and finally, the free carboxylate of the cysteine residue is methyl esterified by the integral membrane enzyme, isoprenylcysteine carboxyl

methyltransferase (Icmt).

Clinical failure of potent FTase inhibitors (FTIs) ⁴ ' ⁵ and cardiomyopathy caused by Rce-1 ablation, ⁶ has shifted the focus for inhibition of K-Ras downstream to Icmt. Like prenylation, carboxylmethylation is important for proper localization and functioning of K- Ras; ^7"9 Furthermore, K-Ras that is prenylated and proteolyzed but not methylated does not promote cellular transformation. ¹⁰ ' ¹¹ Although Icmt methylates both farnesylated and geranylgeranylated proteins, methylation is not necessary for the localization of some geranylgeranylated proteins, ¹² suggesting that Icmt inhibition will have a more profound inhibitory effect on the activity of farnesylated proteins, such as K-Ras, than

geranylgeranylated proteins. Thus, Icmt is a promising drug target for K-Ras driven cancers. Small molecule inhibitors of human Icmt have been identified both from high throughput screens of synthetic and natural product libraries ^13"15 and from substrate-based design approaches. ^{16, 17} Our previous substrate-based inhibitors used N- Acetyl -S- farnesylcysteine (AFC, Figure 2a), the minimal peptidic Icmt substrate, as the scaffold molecule for further modifications. Modifications to the AFC scaffold at both the amide and the prenyl regions yielded several low micromolar inhibitors, the most potent being POP- 3MB-FC, which demonstrated an in vitro IC ₅₀ of 2.5 μΜ (Figure 2d). ^16"20 Previous attempts to modify the carboxylate of the substrate famesylthiopropionic acid (FTPA) resulted in weak inhibitors of bovine Icmt. ²¹ Herein, we have synthesized and tested a larger set of carboxylate replacements based on FTPA and have identified a novel inhibitor, 12, in which the carboxyl group was replaced with an alcohol moiety. This inhibitor was not a substrate for human Icmt (hlcmt) but inhibited the enzyme with an IC ₅ o of 860 nM. In cells, incubation with 12 resulted in GFP K-Ras mislocalization in Jurkat T cells.

2. Chemistry.

Our compound set was initially focused on carboxylate variants of the FTPA molecule (Scheme 1). Methylthiopropionate (1) was S-alkylated ²² with all-trara-farnesyl chloride (2) to yield thioether 3. Compound 3 was either saponified to yield FTPA (4), or reduced using diisobutyl aluminium hydride to yield the alcohol analog 5. Coupling of carboxylic acid 4 with hydroxylamine hydrochloride afforded hydroxamic acid 8. The alcohol analog 5 was modified via bromination to analog 6, and then converted to the phosphonate ethyl ester 7.

Scheme 1

(a) 7N NH ₃ /MeOH, 0°C, 3h, 70-75% (b) LiOH, MeOH, rt, 4h, 71% (c) DIBAL-H, anhydrous DCM, 0°C - rt, 4h, 65% (d) PPh ₃ , CBr ₄ , Imidazole, DCM, 0°C - rt, 2h, 80% (e) Cs ₂ C0 ₃ , Diethylphosphite, 8days, 50% (f)NH ₂ OH HC1, DIEA, HOBT, HBTU, 62%.

Scheme 2

Our second sets of analogs were farnesyl thioacetic acid (FTA) derivatives where the carboxylate motif was replaced with various substituents. We synthesized analogs 10 to 18 as shown in Scheme 2. Aldehyde analog 14, the protected alcohol 15 and the azido analog 18 were prepared as described below. The aldehyde 14 was afforded by treating alcohol 12 with Dess-Martin periodinane in dichloromethane. Analog 15 was synthesized via acetylation of alcohol 12. Displacement of the bromide in 16 with sodium azide yielded the azido analog, 18. Bromide 16 and azide 18 were intermediates toward the synthesis of the phosphonate and the tetrazole respectively. Unfortunately, attempts to synthesize the tetrazole were unsuccessful in our hands.

Scheme 3

To efficiently achieve chemical diversity, we coupled various commercially available thiols to farnesyl chloride (2; Scheme 3). Utilizing either 7N ammonia in methanol or diisopropylethyl amine (DIEA) in dichloromethane, we synthesized compounds 20a-h from thiols 19a-h. For the synthesis of compound 20c, ethanedithiol was used 10 times in excess to the electrophile, to ensure mono-alkylation. To evaluate the effect of a urethane functional group, we synthesized analog 21 by treating the free amino analog 20g with

ethylchloroformate in the presence of potassium carbonate and acetone as the solvent. The acid and the alcohol derivatives of the S-alpha-gem-dimethyl analog 20h were prepared by treating 20h with lithium hydroxide or diisobutylaluminium hydride to yield compounds 22 ad 23 respectively. Compounds 26a and 26b were then synthesized to evaluate the effects of the ketone and the 1,3-dioxolane functional groups on hlcmt inhibition. The free thiol intermediate 26 was used to alkylate chloroacetone or 2-(bromomethyl)-l,3-dioxolane to yield analogs 26a and 26b respectively.

Scheme 4

To evaluate the effects of fluorine substituents alpha to the allylic thioether, we designed and synthesized compounds 28 and 29 (Scheme 4). Methyl 2-bromo-2,2- difluoroacetate 27 was treated with famesylmercaptan ²³ to yield the methyl ester 28. DIBAL- H reduction of ester 28 yielded alcohol 29. Our previous work has highlighted the importance of the prenyl moiety in the substrate-based analogs for Icmt inhibition. ¹⁸ ' ²⁰ To evaluate this key feature, we synthesized analogs 31a-c, 32b and 33a-c (Scheme 5). These analogs were generated by displacing various prenyl (or homoprenyl) halides with methylthioglycolate 9. Homofarnesyl iodide 30a was generated from homofarnesyl alcohol, which was prepared using previously established protocols. ²⁴ Alkylation of

methylthioglycolate 9 with prenyl electrophiles was accomplished utilizing

diisopropylethylamine as the base and dichloromethane as the solvent. Alkylation yielded esters 31a-c in good yields. We then converted ester 31b to the corresponding carboxylic acid 32b using lithium hydroxide mediated saponification. The alcohols 33a-c were derived from the esters 31a-c using DIBAL-H as the reducing agent.

cheme 5

3. Biochemical and Cellular Evaluation.

All analogs synthesized were screened as both substrates and inhibitors of hlcmt using a robust and quantitative vapor diffusion assay (VDA). ^25"27 None of the compounds (except compound 2, 50% substrate activity of AFC) were substrates for hlcmt (data not shown) and were subsequently screened as inhibitors in a single point assay (10 μΜ) in the presence of 25 μΜ AFC as the substrate (Table 1). Analog 12 was the most potent inhibitor evaluated and was determined to have an IC ₅₀ of 0.86 ± 0.02 μΜ. The mode of inhibition was mixed- competitive with an a- value greater than 1, indicating that 12 exhibits predominantly competitive properties (see Figure S2), with a K _ic = 0.44 uM +/- 0.01 and a K _iu = 0.43 uM +/- 0.01.

Compound 12 was then further evaluated for its cellular activity. As the lack of methylation leads to -Ras mislocalization in cells, we examined the effect of treatment of 12 on Jurkat T cells transiently expressing GFP-K-Ras. These cells were treated with DMSO as a negative control, simvastatin (45 μΜ) as a positive control, or 12 (10 μΜ) for 24 h. We observed that treatment with 12 resulted in partial mislocalization of GFP-K-Ras in Jurkat T cells at 10 μΜ. At 45 μΜ, the simvastatin control treatment results in almost complete GFP K-Ras mislocalization in Jurkat T cells (see Figure S3).

4. Discussion.

A full determination of the structure-activity relationships around a ligand for a biological target is a key step in the development of a therapeutically useful agent based on the ligand. We have used the minimal Icmt substrate AFC as a template for the development of low micromolar inhibitors, and have examined a number of structural aspects of this chemotype. However, we have not examined the carboxylate, due in part to a lack of success in Rando's early attempts to replace this moiety. ²¹ In addition to the basic SAR knowledge gained from an examination of carboxylate isosteres, ³⁷ there are particular reasons to develop replacements for the carboxylate moiety. The general structure of our AFC analogs, with a lipid mimic on one end and a carboxylate on the other end, resembles a surfactant, and may lead to unfavorable physical properties (such as micelle formation). AFC analogs and methylated derivatives thereof have also exhibited a variety of biological effects believed to result from binding to proteins other than Icmt, and removal of the carboxylate may decrease the risk of these off-target effects. Thus, we initiated an examination of carboxylate replacements in the simpler farnesylthiopropionate skeleton as a model for AFC.

In our efforts to obtain a suitable surrogate for the carboxylate group, we chose to initially focus our attention on the FTPA backbone. The phosphonate and hydroxamic acid analogs 7 and 8 were designed to evaluate the effect of acidity of the phosphonate and the chelation potential of the hydroxamic acid. Alcohol analog 5 was also designed to evaluate the effect of modulating the effect of change in pKa of the terminal functionality. In the next set of analogs synthesized, we focused on the shorter FTA backbone as FTA is a known Icmt inhibitor. This series was evaluated more extensively to develop SAR information. In addition to modifications explored previously, we also included the aldehyde, the acetate (a reverse ester) and the amide analogs. Intermediates were also evaluated for their hlcmt inhibition potential. Upon identification of compound 12, we designed a series to explore SAR around the hydroxy motif (Scheme 3). Effects of steric bulk and electron-withdrawing groups alpha to the sulfur atom were also explored. Finally, we chose to evaluate the importance of the length and nature of the prenyl chain for hlcmt inhibition. We have previously reported the significance of the prenyl chain for hlcmt inhibition and wanted to evaluate the effects of this particular modification in the carboxylate replacement series. In the FTPA analog series 3-8, which was the initial set synthesized, we observed that the alcohol analog 5 was the most potent hicmt inhibitor. The methyl ester analog 3 and the bromo variant analog 6 were very poor hicmt inhibitors. The modest inhibitory potency of 3 was surprising in view of the potency of ester analogs in one of our previous hicmt inhibitor series. ²⁸ Although the bromide is not a carboxylic acid isostere, we chose to evaluate this synthetic intermediate in our screen. Icmt is a target with no structural information, and through the use of such chemical probes, we intend to garner as much information as possible about the enzyme binding pocket. The phosphonate ester 7 was synthesized to evaluate the bioisosteric compatibility of the phosphonate ester in our compounds. The poor potency of analog 7 suggests that phosphonate replacements are not a promising approach for hicmt inhibition. Furthermore, Rando et. al. had earlier synthesized a phosphonic acid variant and also found it to be a weak Icmt inhibitor. ²¹ Hydroxamic acid analogs 8 and 13 were synthesized with the hypothesis that they would bind to a divalent metal, possibly zinc, if it is present near the hicmt active site. ²⁷ ' ²⁹ Both of these analogs displayed poor hicmt inhibition, suggesting that the metal, if present, may not be in proximity to the carboxylate.

The amide derivative 10b inhibited hicmt with similar potency as the FTA analog 11. The equipotency of these analogs adds evidence to the hypothesis that the carboxylate motif may be replaceable for substrate-based hicmt inhibitors. In contrast, analogs that bore an aldehyde, phosphonate or azide (14, 17 and 18) were poor hicmt inhibitors. The azide 18 was an intermediate for the synthesis of a terminal tetrazole and we chose to evaluate this intermediate as well; unfortunately, synthesis of the tetrazole scaffold did not provide us enough pure material for biochemical evaluation.

The alcohol-bearing analog 12 is the most potent inhibitor in the series. The nature of the interactions this analog may have with the hicmt binding pocket is not known, but some conclusions can be drawn from the biochemical activities of the analogs synthesized based on alcohol 12. Esterification of the alcohol to the acetate (analog 15) diminished the activity significantly. Replacement of the hydroxyethyl moiety with a propyl or isopropyl substitutent (analogs 20a-b), an aminoethyl motif (compound 20g) or the ethanethiol (analog 20c), all resulted in significant loss of inhibitory activity. The most striking observation was the complete loss of inhibitory activities of the methyl ether analog (compound 20d) and the dioxolane analog 26b. These data suggest that the hydroxy group of 12 is part of a critical hydrogen bond donor interaction with a functional group in the hlcmt binding pocket. The significant inhibitory activity of analog 16, which contains a bromide may be attributed to its spontaneous conversion to the alcohol derivative 12, as shown in Figure 3. Under the assay conditions, the bromide analog 16 may undergo an intra-molecular cyclization to a charged thiirane intermediate that collapses under aqueous conditions to the alcohol 12. The loss of this putative interaction in the methyl ether analog 20d results in complete loss of inhibition. Strikingly, inhibitory activity is restored to a significant extent when the hydroxy

functionality in added back in the molecular framework of the small molecule (analog 20e). Addition of an extra hydroxy functionality is not beneficial to inhibition, as evidenced from the reduced activity of the (i?)-thioglycerol analog 20f.

We also investigated the effects of substituents alpha to the allylic thioether. Analogs 20h, 22 and 23 are all poor inhibitors of hlcmt. Particularly striking is the observation that analog 23, which bears the hydroxy group, was a poor hlcmt inhibitor. The presence of the gem-dimethyl group appears to lend steric impedance to analog binding. The ketone bearing analog 26a and the 1,3-dioxolane analog 26b were also synthesized to evaluate the effects of these molecular modifications on hlcmt inhibition. Both these analogs were poor hlcmt inhibitors at the test concentration. To evaluate the effects of smaller, electron-withdrawing substituents alpha to the allylic thioether, we replaced the gem-dimethyl group with a gem- difluoro functionality (analogs 28 and 29). Intriguingly, the inhibition of the ester analog 28 increased by 8-fold as compared to the gem-dimethyl substituted analog 20h and 5-fold versus the unsubstituted analog 3. The gem-difluoro analog that possesses the hydroxy group (analog 29), displays approximately 50% lower inhibition vis-a-vis analog 12. ³⁰

Next, to evaluate the importance of the prenyl region for these carboxylate-replaced analogs, we synthesized analogs 31-33. Analogs where the farnesyl group was replaced by the one-carbon longer homofarnesyl chain (analogs 31a and 33a) were two-fold and four-fold less potent compared to their counterparts (analogs 10 and 12) with the farnesyl chain, respectively. We have earlier shown that the presence of sulfur is critical to hlcmt inhibition in AFC derived analogs, ³¹ and these data suggest that the entire allylic thioether motif may be important for binding to hlcmt. Other analogs bearing the homofarnesyl chain can be synthesized and evaluated to test this hypothesis. Analogs that bear the shorter geranyl moiety in place of the farnesyl chain are completely devoid of any inhibitory activity (analogs 31b, 32b and 33b). The 20-carbon geranylgeranyl scaffold in analog 33c exhibited intermediate behavior, with 40% of the activity of analog 12. We have demonstrated twice before with AFC-based analogs that the nature, length and character of the prenyl chain or mimetic are highly critical to hicmt inhibition. ¹⁸ ' ²⁰ This focused prenyl replaced study with the seven analogs 31-33 highlights the importance of the isoprene region of the molecule. All these data are evidences that support our hypothesis that the prenyl chain may be the most important pharmacophoric requirement for hicmt inhibition. Our cellular studies with 12 have shown that this novel alcohol bearing small farnesylated molecule inhibits hicmt in cells and that it also causes GFP-K-Ras mislocalization in cells.

5. Conclusions.

Herein, we report for the first time, a substrate-based sub-micromolar inhibitor of hicmt that does not contain the carboxylate functionality, which is methylesterified in hicmt substrates. We have synthesized a small molecule, 12 that not only inhibited hicmt in vitro with sub-micromolar potency, but also resulted in GFP-K-Ras mislocalization in Jurkat T cells. This compound also appears to have critical interactions in the hicmt binding pocket, which do not appear to be present in the analogs where the alcohol is masked as an ester or ether. We have again shown that the prenyl region is a crucial molecular motif required for hicmt inhibition. We have also demonstrated that the amide bond is dispensable for hicmt inhibitor design. Removal of the amide simplifies the molecules, makes the molecules smaller with higher ligand efficiency. Our data serve as a proof of principle and hold promise for substrate-based hicmt inhibitors that do not bear the carboxylate motif. Taking cues from Rando's earlier work, ²¹ we have furthered the understanding of inhibition requirements for hicmt. Our non-canonical approach has provided us significant dividends in terms of identifying novel scaffolds for hicmt inhibition, a target of increasing interest in anti-cancer research. Additionally, our research group has recently reported triazole-based sub-micromolar hicmt inhibitors that bear a metabolically labile ester motif. ²⁸ The results obtained through this carboxylate replacement investigation are guiding efforts to replace the ester motif in triazole-based hicmt inhibitors. 6. Experimental Section,

Compound 3: methyl 3-(((2E, 6E)-3,7,H-trimethyldodeca-2,6,10-trien-l- yl)thio)propanoate: The atmosphere in a dry round bottom flask was replaced with argon and 7N ammonia/methanol (3 mL) was added to the flask. The contents of the flask were cooled to 0 °C. This was followed by the addition of methylthiopropionate (1.0 eq.) (1) to 7N ammonia/methanol (Sigma-Aldrich) at 0 °C and the contents were allowed to stir together for three minutes. This was followed by the slow; drop wise addition of farnesyl chloride (1.1 eq.). The contents were allowed to react with each other for 3 h at 0 °C. Following the completion of the reaction, the solvent from the flask was removed under reduced pressure using a rotary evaporator. The residue was adsorbed on silica gel (dry loading). The product was isolated using column chromatography by isocratic elution with 5% ethyl acetate in hexanes. The isolated yield regularly ranges from 70-75%. ¹ H NMR (300 MHz, CDC1 ₃ ) δ 5.15 (dd, J= 23.4, 15.9 Hz, 1H), 5.03 (s, 2H), 3.63 (s, 3H), 3.11 (d, J= 7.7 Hz, 2H), 2.74 - 2.60 (m, 1H), 2.51 (s, 2H), 2.10 - 1.72 (m, 8H), 1.61 (s, 6H), 1.54 (s, 6H). 13C NMR (75 MHz, CDC13) δ 172.12, 138.82, 135.03, 130.95, 124.13, 123.56, 120.08, 51.46, 39.49, 39.40, 34.50, 29.13, 26.50, 26.17, 25.70, 25.49, 17.46, 15.85, 15.79.

Compound 4: 3-(((2E, 6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl)thio)propanoic acid was synthesized upon saponification of compound 3. Compound 3 (1.0 eq.) was dissolved in methanol followed by addition of lithium hydroxide (2 eq.). The contents were allowed to stir in the reaction vessel at room temperature for 4 h under an atmosphere of argon. On completion of the reaction, methanol was removed from the vessel under reduced pressure using a rotary evaporator. The residue was re-suspended in 2% methanol-methylene chloride mixture and was purified by isocratic elution using 2% methanol in methylene chloride. The isolated yield was 71%. 1H NMR (300 MHz, CDC1 ₃ ) δ 10.09 (s, 1H), 5.30 - 5.18 (m, 1H), 5.08 (td, J- 5.3, 3.1 Hz, 2H), 3.18 (d, J= 7.7 Hz, 2H), 2.78 - 2.68 (m, 2H), 2.68 - 2.58 (m, 2H), 2.03 (qd, J= 14.3, 6.4 Hz, 8H), 1.67 (d, J= 2.2 Hz, 3H), 1.66 (d, J- 0.6 Hz, 3H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 178.28, 139.29, 135.32, 131.27, 124.27, 123.69, 120.03, 39.66, 39.57, 34.73, 29.34, 26.67, 26.34, 25.68, 25.49, 17.66, 16.06, 15.99. Compound 5: 3-(((2E, 6E)-3,7,H-trimethyIdodeca-2,6,10-trien-l-yl)thio)propan-l-ol :

The atmosphere in a dry, flame-dried round bottom flask was replaced with argon. Freshly distilled (over calcium hydride) methylene chloride (5 mL/mmole) was added to the flask under a steady stream of argon. Compound 3 was then added (as a solution in 2 mL methylene chloride) to the round bottom flask. The contents were cooled to -78 °C and all times, the atmosphere in the flask was kept under argon. Once the contents in the flask reached a temperature of -78 °C, a solution of diisobutylaluminium hydride (DIBAL-H, 3.0 eq.) was added slowly to the reaction vessel. The contents were allowed to react at -78 °C for 2 h and then the temperature of the flask was allowed to slowly increase to 0 °C. The contents were allowed to react for a further 2h. Upon completion of the reaction, the reaction mixture was added slowly to a cooled solution of 10% citric acid. The contents were transferred to a separatory funnel and the aqueous layer was extracted with methylene chloride (3 x 15 mL). The organic extracts were combined, washed with brine and concentrated under reduced pressure. The residue was subjected to column chromatography using isocratic elution using 30% ethyl acetate in hexanes, which yielded compound 5 in 65% yield as a viscous oil. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.20 (tt, J= 13.4, 6.7 Hz, 1H), 5.13 - 4.93 (m, 2H), 3.68 (q, J= 5.8 Hz, 2H), 3.13 (d, J= 7.8 Hz, 2H), 2.54 (t, J= 7.0 Hz, 2H), 2.31 (s, 1H), 2.15 - 1.89 (m, 8H), 1.80 (dq, J= 13.1, 6.6 Hz, 2H), 1.66 - 1.63 (s, 3H), 1.63 (s, 3H), 1.56 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) 6 138.75, 135.12, 131.12, 124.18, 123.64, 120.21, 61.62, 39.56, 39.47, 31.80, 29.15, 27.61, 26.57, 26.27, 25.57, 17.55, 15.95, 15.89.

Compound 6: (3-bromopropyl)((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien- l- yl)sulfane: To a dry round bottom flask was added freshly distilled methylene chloride and the atmosphere in the flask was replaced with argon. Triphenylphosphine (1.5 eq) and imidazole (1.5 eq.) were added to the flask and were allowed to stir at 0 °C for 10 min. This was followed by slow addition of alcohol 5. The contents were allowed to react for another 5 min, following which carbon tetrabromide (1.5 eq.) was added in portions. The atmosphere in the flask was again replaced with argon. The contents were allowed to react for 2h, and the temperature of the reaction mixture slowly increased to room temperature. Upon completion of reaction, the reaction mixture was transferred to a separatory funnel and was washed with brine. The organic layer was collected and the brine was re-extracted with methylene chloride (2 x 15 mL). The combined organic extracts were pooled and concentrated under reduced pressure to yield a yellowish-brown residue, which on purification with column chromatography (isocratic elution, 2% ethyl acetate in hexanes) yielded 80% of compound 6. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.22 (td, J= 7.7, 1.1 Hz, 1H), 5.08 (ddd, J= 6.8, 5.2, 2.7 Hz, 2H), 3.61 - 3.36 (m, 2H), 3.14 (d, J= 7.7 Hz, 2H), 2.77 - 2.45 (m, 2H), 2.06 (tt, J= 16.5, 8.1 Hz, 8H), 1.67 (s, 3H), 1.64 (d, J= 10.0 Hz, 3H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 138.96, 135.24, 131.23, 124.25, 123.70, 120.25, 39.65, 39.55, 32.33, 32.27, 29.29, 29.13, 26.65, 26.34, 25.68, 17.68, 16.12, 16.02.

Compound 7; diethyl (3-(((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l- yl)thio)propyl)phosphonate: Bromide 6 (1.0 eq.), diethylphosphite (1.0 eq.) and cesium cabonate (2.0 eq.) were all transferred to a round bottom flask and the atmosphere in the flask was replaced with argon. The contents were allowed to react with each other for 8 days. Product started appearing on TLC on day 3. The atmosphere in the flask was replaced with argon every 6-8 hours in order to maintain oxygen free conditions. Upon formation of sufficient product (as determined by TLC analysis), the reaction was quenched with 10% citric acid (5 mL) and the contents were transferred to a separatory funnel. The mixture was extracted with ethyl acetate and the aqueous phase was further extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were concentrated under reduced pressure to yield an oily mixture, which upon isocratic column chromatic elution with 50% ethyl acetate in hexanes yielded phosphonate 7 in 50% yield. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.16 (td, J= 7.7, 1.0 Hz, 1H), 5.10 - 4.89 (m, 2H), 4.06 (dd, J= 11.0, 3.9 Hz, 4H), 4.01 (t, J= 4.8 Hz, 2H), 3.08 (d, J= 7.7 Hz, 2H), 2.61 - 2.40 (m, 2H), 2.11 - 1.79 (m, 10H), 1.61 (s, 3H), 1.57 (d, J= 10.5 Hz, 3H), 1.53 (s, 6H), 1.36 - 1.12 (m, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 138.71, 135.07, 131.04, 124.11 , 123.57, 120.14, 65.90, 65.83, 63.56, 63.49, 39.51, 39.42, 30.10, 30.01, 29.10, 26.72, 26.51, 26.22, 25.51, 17.49, 16.01, 15.92, 15.89, 15.83. ³¹ P NMR (122 MHz, CDC1 ₃ ) 6 50.17

Compound 8: N-hydroxy-3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l- yI)thio)propanamide was prepared by treating compound 4 (1.0 eq.) with hydroxylamine hydrochloride (1.1 eq.) in presence of HOBT, HBTU and DIE A. DMF (5 mL/mmole of acid 4) was used as the solvent. The reagents were allowed to react at 0 °C for 8 hours.

Transferring the reaction mixture to a separatory funnel followed this and 10% citric acid (20 mL) was added to the separatory funnel. The product was extracted using ethyl acetate (3 x 20 mL). The combined organic extracts were washed with 20 mL of brine and dried over sodium sulfate (5 g). Evaporation of the organic residues yielded viscous oil that was purified using 5% methanol in methylenechloride as the chromatography solvent. 1H NMR (300 MHz, ) δ 5.21 (t, J= 7.5 Hz, 1H), 5.04 (q, J= 6.3 Hz, 2H), 4.87 (s, 2H), 3.33 - 3.18 (m, 2H), 3.04 (s, 2H), 2.23 - 1.78 (m, 8H), 1.65 (s, 3H), 1.64 (s, 3H), 1.56 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 169.32, 139.16, 135.47, 131.26, 124.26, 118.19, 40.64, 39.52, 37.68, 31.49, 26.55, 26.07, 25.84, 17.64, 16.09, 15.98.

Compound 10a: methyl 2-(((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l- yl)thio)acetate was prepared using a similar method as described in the preparation of compound 3. Methylthioglycoalte was used instead of the methylthiopropionate to afford compound 10a in 70-75% yield routinely. 1H NMR (300 MHz, CDC1 ₃ ) 6 5.24 - 5.12 (m, 1H), 5.06 (t, J= 6.0 Hz, 2H), 3.70 (s, 3H), 3.23 (t, J= 8.0 Hz, 2H), 3.14 (d, J= 2.8 Hz, 2H), 2.17 - 1.88 (m, 8H), 1.65 (s, 3H), 1.63 (s, 3H), 1.57 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 171.09, 140.35, 135.25, 131.14, 124.19, 123.58, 119.19, 52.16, 39.59, 39.52, 31.80, 29.78, 26.59, 26.24, 25.59, 17.57, 15.92, 15.85.

Compound 10b: 2-(((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl)thio)ac etamide was prepared by treating compound 10a with 5 mL of 7N ammonia in methanol at room temperature for 14 hours under an atmosphere of argon. Evaporation of the 7N

ammonia/methanol solvent under reduced pressure afforded a sticky solid that was purified using normal phase gravity column chromatography utilizing 50% ethyl acetate in hexanes as the elution solvent. Compound 10b was isolated in a 85% overall yield. ^! H NMR (300 MHz, Chloroform-d) δ 6.81 (s, 1H), 6.66 (s, 1H), 5.17 (t, J= 8.0 Hz, 1H), 5.06 (t, J= 6.7 Hz, 2H), 3.18 (d, J= 7.9 Hz, 2H), 3.11 (s, 2H), 2.02 (pq, J = 7.7, 3.6 Hz, 8H), 1.64 (s, 3H), 1.61 (s, 3H), 1.57 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 173.32, 141.73, 136.30, 132.15, 125.17, 124.51, 119.62, 40.56, 40.45, 35.35, 31.03, 27.56, 27.19, 26.58, 18.57, 16.91.

Compound 11: 2-(((2E,6E)-3,7,H-trimethyldodeca-2,6,10-trien-l-yl)thio)ace tic acid was prepared by saponification of compound 10a using the method described for preparation of compound 4. Column chromatographic purification yielded 79% of isolated product. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.29 - 5.15 (m, 1H), 5.13 - 4.98 (m, 2H), 3.28 (d, J= 7.8 Hz, 2H), 3.18 (d, J= 4.1 Hz, 2H), 2.19 - 1.87 (m, 8H), 1.66 (d, J= 0.8 Hz, 3H), 1.65 (d, J= 0.7 Hz, 3H), 1.58 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 177.22, 140.65, 135.31, 131.19, 124.21 , 123.59, 119.01, 39.61, 39.53, 32.16, 29.85, 26.61 , 26.27, 25.62, 17.60, 15.94, 15.92.

Compound 12: 2-(((2E,6 E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl)thio)ethanol was prepared by DIBAL-H reduction of compound 10a using the method described for preparation of compound 5. Column chromatographic purification (30% ethyl acetate in hexanes) yielded 69% of isolated product. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.18 (tt, J= 1 1.1, 5.6 Hz, 1H), 5.13 - 4.94 (m, 2H), 3.65 (t, J= 6.1 Hz, 2H), 3.13 (t, J= 7.5 Hz, 2H), 2.64 (s, 1H), 2.61 (d, J= 6.0 Hz, 2H), 2.15 - 1.84 (m, 8H), 1.63 (d, J= 0.7 Hz, 3H), 1.61 (s, 3H), 1.54 (d, J= 10.4 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 139.04, 135.12, 131.05, 124.13, 123.54, 120.08, 60.27, 39.52, 39.41, 33.81, 28.66, 26.52, 26.18, 25.53, 17.51, 15.89, 15.85.

Compound 13: V-hydroxy-2-(((2E,6£)-3,7,l l-trimethyldodeca-2,6,10-trien-l- yl)thio)acetamide To a solution of hydroxylamine hydrochloride in methanol (10 mL) was added potassium hydroxide (10 eq.). The contents were allowed to react at 40 °C for 10 minutes and then cooled to 0 °C, followed by filtration. Ester 10a was added to the filtrate followed by 1 eq. of potassium hydroxide and the contents were allowed to react at room temperature for 30 minutes. Upon completion of reaction, the solvent was removed under reduced pressure and the residue was diluted with 10% aqueous ammonium chloride. The mixture extracted with ethyl acetate (3 x 20 mL) and the combined organic extracts were washed with brine and concentrated under reduced pressure. Chromatographic purification using 10% methanol in methylenechloride yielded compound 13 in 65% overall yield. Ή NMR (300 MHz, ) δ 5.19 (t, J= 7.6 Hz, 1H), 5.06 (q, J= 6.4 Hz, 2H), 4.90 (s, 2H), 3.35 - 3.15 (m, 2H), 3.01 (s, 2H), 2.23 - 1.76 (m, 8H), 1.64 (s, 3H), 1.63 (s, 3H), 1.56 (s, 6H). ^l3 C NMR (75 MHz, CDC1 ₃ ) δ 167.43, 141.22, 135.47, 131.26, 123.56, 118.51, 39.64, 39.52, 30.16, 26.64, 26.27, 25.65, 17.64, 16.01, 15.98.

Compound 14: 2-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l-yl)thio)ac etaldehyde was prepared by treating compound 12 (1 eq.) with Dess-Martin periodinane (1.5 eq).

Briefly, a dry round bottom flask was charged with freshly distilled methylenechloride (5 mL/mmol) and the atmosphere in the flask was replaced with argon. Compound 12 was added (as a solution in methylenechloride) and the contents were allowed to cool to 0 °C for 20 minutes. This was followed by addition of Dess-Martin periodinane and the contents were allowed to react for 2 hours. The temperature was slowly allowed to reach ambient temperature. The reaction was monitored through TLC analysis utilizing 2,4- dinitrophenylhydrazine as the visualizing reagent. Upon completion of the reaction, saturated sodium bicarbonate solution (20 mL) was added to the reaction mixture and the contents were extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine and dried with sodium sulfate. The combined organic extracts were evaporated under reduced pressure, followed by chromatographic purification (30% ethyl acetate in hexanes). Compound 13 was isolated as a viscous oil in a 64% overall yield. 1H NMR (300 MHz, Chloroform-d) δ 9.49 (dt, J= 6.9, 3.2 Hz, 1H), 5.20 (dt, J= 15.5, 7.6 Hz, 1H), 5.09 (q, J= 6.7 Hz, 2H), 3.19 - 3.05 (m, 4H), 2.04 (ddt, J= 22.6, 16.2, 6.9 Hz, 8H), 1.68 (d, J= 6.4 Hz, 3H), 1.62 (d, J= 5.0 Hz, 3H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 194.72, 140.68, 135.42, 131.31, 124.26, 123.62, 118.95, 40.48, 39.68, 39.53, 29.15, 26.67, 26.23, 25.68, 17.67, 16.12, 16.01.

Compound 15: 2-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l-yl)thio))e thyl acetate:

To a dry round bottom flask was added compound 12 (1 eq. as a solution in 5 mL methylene chloride) and the atmosphere in the flask was replaced with argon. Diisopropylethyl amine (DIEA, 1.05 eq) was added to the flask. The contents were allowed to cool to 0 °C. This was followed by slow addition of acetyl chloride (1.05 eq.) to the flask. The contents were allowed to react for 5 hours. Upon completion of the reaction, brine (20 mL) was added to the reaction mixture and the product was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated under reduced pressure to yield a viscous oil. Chromatographic purification (30% ethyl acetate in hexanes) yielded compound 15 as a viscous oil in 88% yield. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.22 (t, J= 7.7 Hz, 1H), 5.17 - 4.92 (m, 2H), 4.18 (t, J= 6.9 Hz, 2H), 3.18 (d, J= 7.8 Hz, 2H), 2.77 - 2.56 (m, 2H), 2.18 - 1.86 (m, 11H), 1.66 (s, 3H), 1.64 (d, J= 9.0 Hz, 3H), 1.58 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 170.74, 139.37, 135.27, 131.24, 124.24, 123.67, 120.06, 63.45, 39.64, 39.54, 29.39, 29.16, 26.65, 26.29, 25.64, 20.86, 17.63, 16.00, 15.96.

Compound 16: (2-bromoethyl)((2E,6E)-3,7,ll-trimethyIdodeca-2,6,10-trien-l -yl)sulfane was prepared by bromination of compound 12 using the method described for preparation of compound 6. Column chromatographic purification yielded 77% of isolated product as a viscous liquid. 1H NMR (300 MHz, CDC1 ₃ ) δ 5.64 (t, J= 7.3 Hz, 1H), 5.58 - 5.35 (m, 2H), 3.97 - 3.75 (m, 2H), 3.60 (d, J= 7.8 Hz, 2H), 3.36 - 3.19 (m, 2H), 2.66 - 2.27 (m, 8H), 2.14 - 2.04 (m, 6H), 2.05 - 1.93 (m, 6H). ^I3 C NMR (75 MHz, CDC1 ₃ ) δ 140.51, 136.24, 132.04, 125.20, 124.49, 120.82, 40.55, 40.46, 33.65, 31.45, 30.30, 27.55, 27.19, 26.66, 18.66, 17.01, 16.94.

Compound 17: diethyl (2-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l- yl)thio)ethyl)phosphonate was prepared using the method described for preparation of compound 7. Column chromatographic purification yielded 43% of isolated product as a viscous liquid. 1H NMR (300 MHz, Chloroform-d) δ 5.18 (tdd, J= 7.7, 2.8, 1.4 Hz, 1H), 5.04 (dddt, J= 5.4, 4.3, 3.0, 1.6 Hz, 2H), 4.18 - 3.96 (m, 4H), 3.14 (d, J= 7.7 Hz, 2H), 2.69 (t, J= 7.1 Hz, 2H), 2.13 - 1.86 (m, 8H), 1.62 (dd, J = 3.1, 1.5 Hz, 6H), 1.55 (d, J = 1.3 Hz, 6H), 1.30 (td, J= 7.1, 1.0 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 139.44, 135.24, 131.17, 124.21, 123.61, 119.94, 66.34, 63.79, 63.71, 39.51, 30.55, 30.45, 29.42, 26.60, 26.28, 25.61, 17.59, 15.99, 15.92. ³¹ P NMR (122 MHz, CDC1 ₃ ) δ 49.88.

Compound 18: (2-azidoethyl((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l- yl)sulfane

To a dry round bottom flask was added compound 16 as a solution in DMF (3 mL/mmol). The atmosphere in the flask was replaced with argon and sodium azide (1.2 eq) was added to the reaction vessel. The contents were heated to 50 °C and were allowed to react for 10 hours. The reaction was monitored by TLC. Upon completion of the reaction, 10% citric acid (20 mL) was added to the reaction flask and the contents were extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated under reduced pressure to yield a viscous oil. Column chromatographic purification (2% ethyl acetate in hexanes) yielded compound 18 in 61% overall yield. -1H NMR (300 MHz, Chloroform-d) δ 5.39 - 5.17 (m, 1H), 5.09 (q, J= 7.0, 6.6 Hz, 2H), 3.55 - 3.34 (m, 2H), 3.19 (d, J= 7.8 Hz, 2H), 2.66 (dt, J= 14.3, 6.5 Hz, 2H), 2.06 (m, , 8H), 1.77 - 1.69 (m, 3H), 1.66 (d, J= 4.5 Hz, 6H), 1.60 (s, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.44, 136.34, 132.23, 125.22, 124.60, 121.00, 52.14, 40.64, 40.51, 30.93, 30.43, 27.64, 27.28, 26.65, 18.63, 17.06.

Synthesis of compounds 20a-h.

Briefly, to a dry round bottom flask was added 7N ammonia methanol, or DIEA in methylene chloride and the atmosphere in the flask was replaced with argon. This was followed by addition of the commercially available thiol (1.0 eq, except 10 eq. for compound 20c) and the reactants were allowed to stir at 0 °C for 2 minutes. A solution of farnesyl chloride (1.2 eq.) in methylene chloride (or methanol) was added to the reaction mixture and the contents were allowed to stir for 5 hours. Upon completion of the reaction (as monitored by TLC, visualized by vanillin staining), the contents of the reaction flask were evaporated under reduced pressure. Brine (20 mL) was added to the reaction vessel and the contents were extracted with ethyl acetate (3 x 20 mL) using a separatory funnel. The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated under reduced pressure to yield viscous oils. All compounds were purified using column chromatography (1% ethyl acetate in hexanes (20a-c), 30% ethyl acetate in hexanes (20d-f and 20h or 5% methanol in methylene chloride (20g)) to yield between 65-81% of isolated product.

Compound 20a: isopropyl((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl)s uIfane: 1H

NMR (300 MHz, Chloroform-d) δ 5.26 (t, J= 7.7 Hz, 1H), 5.14 - 5.04 (m, 2H), 3.12 (d, J= 7.7 Hz, 2H), 2.87 (m, 1H), 2.01 - 1.88 (m, 8H), 1.64 (s, 3H), 1.62 (s, 3H), 1.59 - 1.49 (m, 6H), 1.19 (d, J= 7.1 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 137.17, 136.15, 131.98, 125.24, 124.75, 121.70, 40.56, 40.17, 38.71, 26.78, 26.43, 24.47, 24.28, 23.95, 18.59, 16.38, 15.71.

Compound 20b: propyl((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl)suIf ane: 1H

NMR (300 MHz, Chloroform-d) 5 5.23 (t, J- 7.8 Hz, 1H), 5.15 - 5.02 (m, 2H), 3.12 (d, J=

7.7 Hz, 2H), 2.49 - 2.33 (m, 2H), 1.68 - 1.65 (s, 3H), 1.64 - 1.63 (s, 3H), 1.62 - 1.49 (m, 8H), 0.96 (t, J= 7.3 Hz, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 139.29, 136.10, 132.13, 125.24, 124.75, 121.70, 40.63, 40.54, 33.96, 30.14, 27.64, 27.34, 26.61, 23.94, 18.59, 16.92, 14.53.

Compound 20c: 2-(((2E,6E)-3,7,ll-trimethvIdodeca-2,6,10-trien-l-yl)thio)et hanethiol:

1H NMR (300 MHz, Chloroform-d) δ 5.29 - 5.16 (m, 1H), 5.15 - 4.99 (m, 2H), 3.16 (d, J =

7.8 Hz, 2H), 2.69 (s, 1H), 2.68 (s, 2H), 2.17 - 1.90 (m, 8H), 1.67 (s, 3H), 1.65 (s, 3H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.14, 136.31, 132.24, 125.24, 124.64, 121.18, 40.65, 40.54, 35.90, 30.13, 27.65, 27.31, 26.66, 25.75, 18.65, 17.01

Compound 20d: (2-methoxvethyl)((2E,6E)-3,7,11-trimethyldodeca-2.6.10-trien -l- yl)sulfane: 1H NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 7.8 Hz, 1H), 5.15 - 5.02 (m, 2H), 3.53 (t, J= 6.8 Hz, 2H), 3.35 (s, 3H), 3.18 (d, J= 7.8 Hz, 2H), 2.64 (td, J= 6.8, 3.4 Hz, 2H), 2.15 - 1.89 (m, 8H), 1.67 (s, 3H), 1.65 (s, 3H), 1.59 (s, 6H) ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 139.95, 136.25, 132.24, 125.27, 124.73, 121.39, 73.10, 59.64, 40.66, 31.19, 30.61, 27.66, 27.37, 26.66, 18.65, 17.03, 16.98.

Compound 20e: 1-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l--yl)thio)p ropan-2-ol

1H NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 7.9 Hz, 1H), 5.10 - 5.01 (m, 2H), 4.05 - 3.66 (m, 1H), 3.16 (qd, J= 13.3, 7.6 Hz, 2H), 2.80 - 2.54 (m, 2H), 2.51 - 2.30 (m, 1H), 2.23 - 1.87 (m, 8H), 1.67 (s, 3H), 1.63 (d, J= 7.4 Hz, 3H), 1.59 (s, 6H), 1.39 - 1.01 (m, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 132.32, 125.27, 124.67, 121.11, 78.43, 78.00, 77.62, 77.58, 66.39, 41.47, 40.68, 40.57, 30.26, 27.70, 27.35, 26.69, 22.99, 18.68, 17.08, 17.03.

Compound 20f: 3-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-l-yl)thio)pr opane-l,2- diol 1H NMR (300 MHz, Chloroform-d) δ 5.22 (q, J= 8.2 Hz, 1H), 5.07 (t, J= 6.8 Hz, 2H), 3.87 - 3.64 (m, 2H), 3.48 (dt, J= 26.9, 3.8 Hz, 2H), 3.14 (ttd, J= 11.7, 8.0, 4.3 Hz, 3H), 2.69 - 2.44 (m, 2H), 2.23 - 1.84 (m, 8H), 1.66 (t, J- 7.2 Hz, 6H), 1.59 (d, J= 8.6 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.50, 136.26, 132.19, 125.19, 124.59, 120.80, 71.21, 66.35, 40.59, 40.50, 35.32, 30.43, 27.60, 27.30, 26.61, 18.59, 17.04, 16.93.

Compound 20g: 2-(((2E,6E)-3,7,ll-trimethyIdodeca-2,6,10-trien-l-yl)thio)et hanamine

1H NMR (300 MHz, Chloroform-d) δ 5.21 (q, J= 7.6 Hz, 1H), 5.07 (q, J= 7.1, 6.6 Hz, 2H), 4.54 (bs, 2H), 3.28 - 3.07 (m, 2H), 3.09 - 2.82 (m, 2H), 2.67 (s, 2H), 2.00 (dq, J- 23.7, 8.1, 7.4 Hz, 8H), 1.64 (s, 3H), 1.63 (d, J= 5.0 Hz, 3H), 1.57 (d, J= 6.7 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.30, 136.20, 132.16, 125.18, 124.62, 120.94, 40.59, 40.49, 29.82, 27.59, 27.31, 26.59, 18.58, 17.05, 16.92.

Compound 20h: methyl 2-methyl-2-(((2E,6E)-3,7,ll-trimethyIdodeca-2,6,10-trien-l- yI)thio)propanoate 1H NMR (300 MHz, Chloroform-d) δ 5.19 (d, J= 8.2 Hz, 1H), 5.06 (t, J = 6.8 Hz, 2H), 3.70 (s, 3H), 3.24 (t, J= 7.4 Hz, 2H), 2.02 (tdd, J= 18.5, 8.6, 5.1 Hz, 8H), 1.66 (dd, J= 9.2, 4.0 Hz, 6H), 1.56 (d, J= 3.8 Hz, 6H), 1.49 (s, 6H). ¹³ C NMR (75 MHz, CDCI3) δ 175.54, 140.92, 136.11, 132.09, 125.25, 124.65, 119.40, 53.20, 48.00, 40.58, 40.46, 28.80, 27.60, 27.18, 26.59, 26.53, 18.59, 17.04, 16.90. Compound 21: ethyl (2-(((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trieii-l- yl)thio)ethyl)carbamate To a dry round bottom flask was added compound 20g as a solution in acetone (3 mL/mmol). The atmosphere in the flask was replaced with argon and potassium carbonate (1.2 eq.) was added to the flask. This was followed by slow (1 drop/3 seconds) addition of ethyl chloroformate. The contents were allowed to react at ambient temperature for 10 hours under an atmosphere of argon. Upon completion of the reaction (as monitored by TLC, visualized by vanillin staining), the contents of the reaction flask were evaporated under reduced pressure. Brine (20 mL) was added to the reaction vessel and the contents were extracted with ethyl acetate (3 x 20 mL) using a separatory funnel. The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated under reduced pressure to yield viscous oils. Compound 21 was purified using column chromatography (2% methanol in methylene chloride) to yield between 76% of isolated product as a viscous oil. 1H NMR (300 MHz, Chloroform-d) δ 5.22 (t, J= 7.8 Hz, 1H), 5.15 3 4.98 (m, 3H), 4.11 (q, J= 7.1 Hz, 2H), 3.35 (q, J= 6.3 Hz, 2H), 3.15 (d, J= 7.8 Hz, 2H), 2.60 (t, J= 6.4 Hz, 2H), 2.03 (dtt, J= 25.3, 10.6, 6.0 Hz, 8H), 1.67 (d, J= 2.5 Hz, 3H), 1.65 (s, 3H), 1.59 (s, 6H), 1.23 (t, J = 7.1 Hz, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.32, 136.34, 125.27, 78.42, 78.00, 77.58, 61.82, 40.68, 29.97, 27.34, 26.69, 17.09.

Compound 22: 2-methvl-2-(((2£'.6E)-3.7.11-trimethvldodeca-2.6.10-trien-l - yl)thio)propanoic acid was prepared by saponification of compound 20h using the method described for preparation of compound 4. The only change was that the reaction contents were heated to 40 °C through the duration of the reaction to afford complete saponification. Column chromatographic purification yielded 61% of isolated product. 1H NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 8.0 Hz, 1H), 5.09 (d, J= 6.7 Hz, 2H), 3.32 (d, J= 7.8 Hz, 2H), 2.01 (tt, J= 15.4, 7.5 Hz, 8H), 1.67 (s, 6H), 1.59 (d, J= 3.9 Hz, 6H), 1.53 (s, 6H). ¹³ C NMR (75 MHz, CDCls) δ 174.69, 141.39, 136.29, 132.28, 125.32, 124.72, 119.20, 48.00, 40.67, 40.52, 29.08, 27.70, 27.27, 26.69, 26.29, 18.69, 17.05, 17.01.

Compound 23: 2-methvl-2-(f(2£'.6E)-3.7.11-trimcthvldodeca-2.6.10-trien-l - yl)thio)propan-l-ol was prepared by DIBAL-H reduction of compound 20h using the method described for preparation of compound 5. Column chromatographic purification (30% ethyl acetate in hexanes) yielded 55% of isolated product. 1H NMR (300 MHz, Chloroform-d) δ 5.34 - 5.19 (m, 1H), 5.17 - 4.98 (m, 2H), 3.38 (dd, J= 12.9, 6.5 Hz, 2H), 3.19 - 3.02 (m, 2H), 2.40 (dt, J= 12.5, 6.7 Hz, 1H), 2.03 (dddd, J- 21.6, 17.1, 8.9, 4.9 Hz, 8H), 1.77 - 1.63 (m, 6H), 1.57 (s, 6H), 1.34 - 1.14 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.16, 136.18, 132.12, 125.24, 124.62, 120.58, 69.65, 49.22, 40.59, 40.44, 27.62, 26.61, 26.35, 18.60, 16.97, 16.92.

Compound 25: S-((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien-l-yl) ethanethioate NMR data matches with reported data. ²³

Compound 26: (2E,6E)-3,7,ll-trimethyldodeca-2,6,10-triene-l-thioI was prepared according to the method described earlier by Schmidt et. al. ²³ 1H NMR (300 MHz,

Chloroform-d) δ 5.34 (t, J= 7.9 Hz, 1H), 5.09 (t, J= 6.9 Hz, 2H), 3.15 (t, J= 7.4 Hz, 2H), 2.16 - 1.90 (m, 8H), 1.68 (s, 3H), 1.65 (s, 3H), 1.60 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 138.34, 136.17, 132.16, 125.26, 124.66, 124.26, 40.62, 40.32, 27.64, 27.20, 26.63, 23.04, 18.62, 16.95, 16.69.

Compound 26a: 1-(((2E,6E)-3,7,1 l-trimethyldodeca-2,6,10-trien-l-yl)thio)propan-2-one Compound 26a was prepared in a similar fashion as compound 28. Isocratic column chromatographic purification was performed using ethyl acetate (10%) and hexanes (90%) to yield 65% of isolated 26a as a colorless oil. 1H NMR (300 MHz, Chloroform-d) δ 5.19 (t, J= 8.1 Hz, 1H), 5.08 (t, J= 6.6 Hz, 2H), 3.20 - 3.07 (m, 4H), 2.28 (s, 3H), 2.12 - 1.97 (m, 8H), 1.67 (s, 3H), 1.64 (s, 3H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 205.12, 141.36, 136.33, 132.28, 125.25, 124.65, 120.14, 42.04, 40.67, 40.55, 30.60, 28.84, 27.66, 27.30, 26.67, 18.66, 16.99.

Compound 26b: 2-((((2E,6E)-3 ,7, 11 -trimethyldodeca-2,6, 10-trien- 1 -yl)thio)methyl)- 1,3- dioxolane Ή NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 7.8 Hz, 1H), 5.14 - 5.07 (m, 2H), 5.04 (t, J= 4.6 Hz, 1H), 4.05 - 3.81 (m, 4H), 3.24 (d, J= 7.8 Hz, 2H), 2.64 (d, J= 4.6 Hz, 2H), 2.17 - 1.90 (m, 8H), 1.66 (d, J= 4.7 Hz, 6H), 1.59 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 139.22, 135.17, 131.15, 124.25, 123.70, 120.21, 65.13, 39.61, 39.54, 33.83, 29.95, 26.62, 26.33, 25.62, 17.61, 15.97, 15.94. Compound 28: methyl 2,2-difluoro-2-(((2E,6E)-3,7,ll-trimethyldodeca-2,6,10-trien -l- yl)thio)acetate To a dry round bottom flask was added methyl 2-bromo-2,2-difluoroacetate (compound 27 1.0 eq.) as a solution in anhydrous DMF. The atmosphere in the flask was replaced with argon, followed by addition of cesium carbonate (1.01 eq.). The contents of the flask were cooled to 0 °C. Farnesyl mercaptan (26, 1.01 eq.) was added slowly to the reaction mixture as a solution in DMF. The contents of the flask were allowed to react for 6 hours and the reaction was monitored by TLC. Upon completion of the reaction, 10% citric acid (20 mL) was added to the reaction flask and the contents were extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure to yield a viscous oil. Column

chromatographic purification (20% ethyl acetate in hexanes) yielded compound 28 in 81% overall yield. 1H NMR (300 MHz, Chloroform-d) δ 5.33 - 5.22 (m, 1H), 5.19 - 4.99 (m, 2H), 3.80 (s, 3H), 3.57 (t, J= 8.3 Hz, 2H), 2.06 (dddd, J= 25.5, 21.2, 9.7, 5.0 Hz, 8H), 1.68 (d, J= 3.6 Hz, 6H), 1.60 (t, J= 5.5 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 174.07, 143.01, 136.45, 132.25, 125.28, 124.44, 118.00, 54.80, 40.67, 40.63, 27.80, 27.66, 27.11, 26.65, 18.63, 17.00, 16.98.

Compound 29: 2,2-difluoro-2-( 2E.6E)-3.7.11-trimethvIdodeca-2.6.10-trien-l- yl)thio)ethanol was prepared by DIBAL-H reduction of compound 28 using the method described for preparation of compound 5. Column chromatographic purification (30% ethyl acetate in hexanes) yielded 48% of isolated product. 1H NMR (300 MHz, Chloroform-d) δ 5.24 (t, J= 7.8 Hz, 1H), 5.09 (t, J= 6.9 Hz, 2H), 3.70 (q, J= 5.8 Hz, 2H), 3.18 (t, J= 9.9 Hz, 1H), 2.69 (q, J= 5.7, 4.5 Hz, 1H), 1.67 (s, 6H), 1.60 (d, J= 3.9 Hz, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.38, 136.38, 132.76, 125.27, 124.68, 121.15,

61.22, 40.69, 40.58, 35.18, 29.73, 27.70, 27.34, 26.76, 18.69, 17.09.

Synthesis of Compounds 31a-c

General preparation: To a dry round bottom flask was added dry methylene chloride (5 mL/mmol of electrophile) and the atmosphere in the flask was replaced with argon.

Methylthiopropionate (9, 0.95 eq.) was added to the flask and the contents were allowed to cool to 0 °C. This was followed by addition of DIEA (2.0 eq.). The contents were allowed to stir for 5 minutes. The electrophile (30a-c) was then added slowly as a solution in methylene chloride (1 mL/mmol) to the reaction mixture. The contents were allowed to stir for 5 hours (15 hours for compound 31a). The reaction progress was monitored by TLC analysis. Upon completion of the reaction (as monitored by TLC, visualized by vanillin staining), the contents of the reaction flask were evaporated under reduced pressure. Brine (20 mL) was added to the reaction vessel and the contents were extracted with ethyl acetate (3 x 20 mL) using a separatory funnel. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure to yield viscous oils.

Compound 21 was purified using column chromatography (15% ethyl acetate in hexanes) to yield 63 - 81% of isolated product as a viscous oil.

Compound 31a: methyl 2-(((3E,7E)-4,8,12-trimethyltrideca-3,7,ll-trien-l- yl)thio)acetate: Ή NMR (300 MHz, Chloroform-d) 6 5.19 - 4.99 (m, 3H), 3.77 (s, 3H), 3.21 (d, J= 1.8 Hz, 2H), 2.61 (td, J= 7.5, 1.8 Hz, 2H), 2.28 (q, J= 7.4 Hz, 2H), 2.13 - 1.89 (m, 8H), 1.65 (s, 3H), 1.60 (s, 3H), 1.57 (s, 6H). ¹³ C NMR (75 MHz, CDC1 ₃ ) 6 171.87, 138.02, 135.93, 132.09, 125.25, 124.89, 122.78, 53.21, 40.60, 40.53, 34.30, 33.58, 28.62, 27.63, 27.36, 26.60, 18.58, 17.03, 16.90.

Compound 31b: (E)-methyl 2-((3,7-dimethylocta-2,6-dien-l-yl)thio)acetate: 1H NMR

(300 MHz, Chloroform-d) 6 5.26 - 5.12 (m, 1H), 5.04 (tt, J= 7.3, 5.7, 2.1 Hz, 1H), 3.70 (s, 2H), 3.24 (d, J= 7.8 Hz, 2H), 3.15 (d, J= 1.9 Hz, 2H), 2.15 - 1.89 (m, 4H), 1.65 (d, J= 1.2 Hz, 3H), 1.62 (d, J= 1.2 Hz, 3H), 1.57 (d, J= 1.4 Hz, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 172.11, 141.32, 132.63, 124.76, 120.21, 53.18, 40.52, 32.73, 30.76, 27.28, 26.59, 18.58, 16.80.

Compound 31c: methyl 2-(f(2E.6E.10E)-3.7.11.15-tetramethvIhexadeca-2.6.10.14- tetraen-l-yl)thio)acetate: 1H NMR (300 MHz, Chloroform-d) 6 5.58 - 5.38 (m, 1H), 5.13 (dd, J- 12.7, 7.1 Hz, 3H), 3.78 (s, 3H), 4.1 1 (t, J= 7.4 Hz, 2H), 2.33 - 1.91 (m, 12H), 1.85 - 1.54 (m, 15H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 171.24, 137.41, 136.34, 131.15, 124.10, 121.25, 119.18, 78.42, 54.32, 42.15, 40.10, 37.12, 26.66, 22.13, 19.94, 17.96.

Compound 32b (E)-2-((3,7-dimethylocta-2,6-dien-l-yl)thio)acetic acid was prepared by saponification of compound 31b using the method described for preparation of compound 4. Column chromatographic purification (80%) ethyl acetate in hexanes) yielded 60% of isolated product. 1H NMR (300 MHz, Chloroform-d) δ 11.30 (bs, 1H), 5.20 (t, J= 7.7 Hz, 1H), 5.07 (t, J= 6.5 Hz, 1H), 3.29 (d, J= 7.8 Hz, 2H), 3.20 (s, 2H), 2.08 (q, J= 5.9 Hz, 4H), 1.68 (s, 3H), 1.66 (s, 3H), 1.60 (s, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 178.33, 141.72, 132.78, 124.81, 120.09, 40.59, 27.60, 27.36, 26.69, 24.35, 18.68, 16.95.

Synthesis of compounds 33a-c was carried out using DIBAL-H reduction of the

corresponding esters (30a-c) using the procedure as described for synthesis of compound 5. Chromatographic purification using 30% ethyl acetate in hexanes yielded between 48-66% of isolated product as clear or pale yellow oils.

Compound 33a: 2-(((3£',7E)-4,8,12-trimethyltrideca-3,7,ll-trien-l-yI)thio )ethanol: 1H

NMR (300 MHz, Chloroform-d) δ 5.12 (m, 3H), 3.69 (q, J= 4.8 Hz, 2H), 2.71 (t, J= 5.9 Hz, 2H), 2.50 (dd, J= 8.7, 6.1 Hz, 2H), 2.26 (q, J= 7.5 Hz, 2H), 2.12 - 1.90 (m, 8H), 1.63 (dd, J = 24.7, 8.1 Hz, 12H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 138.10, 122.92, 78.42, 78.00, 77.58, 61.13, 40.60, 36.17, 32.91, 32.81, 29.33, 27.57, 27.49, 27.34, 16.90.

Compound 33b: (E)-2-((3,7-dimethylocta-2,6-dien-l-yl)thio)ethanol: 1H NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 7.8 Hz, 1H), 5.15 - 5.01 (m, 1H), 3.70 (t, J= 5.9 Hz, 2H), 3.16 (d, J= 7.8 Hz, 2H), 2.69 (td, J= 5.9, 3.4 Hz, 2H), 2.19 (bs, 1H), 2.14 - 1.99 (m, 4H), 1.69 (s, 3H), 1.65 (s, 3H), 1.60 (s, 3H). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 140.29, 132.74, 124.82, 121.17, 61.23, 40.55, 35.08, 29.69, 27.37, 26.68, 18.69, 17.03.

Compound 33c: 2-(((2E,6E,10E)-3,7,ll,15-tetramethylhexadeca-2,6,10,14-tetr aen-l- yl)thio)ethanol: 1H NMR (300 MHz, Chloroform-d) δ 5.23 (t, J= 7.4 Hz, 1H), 5.09 (t, J= 6.5 Hz, 3H), 3.70 (p, J= 5.6 Hz, 2H), 3.15 (d, J= 7.6 Hz, 2H), 2.68 (q, J= 5.7 Hz, 2H), 2.25 (bs, 1H), 2.04 (ddd, J= 22.8, 11.2, 6.2 Hz, 12H), 1.73 - 1.55 (m, 15H). ¹³ C NMR (75 MHz, CDC1 ₃ ) 6 140.34, 136.37, 135.92, 132.23, 125.32, 125.11, 124.67, 121.11, 61.22, 40.67, 40.57, 35.13, 29.71, 27.71, 27.57, 27.34, 26.67, 18.66, 17.07, 17.02, 16.97.

Isolation of membrane fraction from yeast cells: Membrane fractions from yeast cells were isolated as previously described. ²⁷ Crude membrane protein concentration was determined using Coomassie Plus Protein Assay Reagent (Pierce) according to the manufacturer's instructions, and compared with a bovine serum albumin standard curve. Brief Procedure for biochemical evaluation of analogs using the vapor diffusion assay:

Briefly, membrane protein (5 μg) was added to a solution of Tris-HCl buffer (100 m , pH 7.4) and AFC (200 μΜ). After 5 min incubation on ice, 20 of S-adenosyl-L- [methyl- ¹⁴ C]methionine ([ ¹⁴ C]SAM) (50-60 mCi/mmol) (60 μΜ) is added and the solution is incubated at 30 °C in a water bath for 30 min. After 30 min the reaction is stopped by the addition 50 μΐ _^ of 1 M NaOH/1% SDS. The reaction mixture is vortexed and spotted on to a pleated filter paper. The filter paper is lodged into the neck of a scintillation vial filled with 10 mL of scintillation fluid and capped. The filter papers were removed after 2.5 h and the radioactivity was measured using a Packard 1600CA Liquid Scintillation Analyzer. The IC50 was calculated using GraphPad Prism 5.0

Fluorescence Microscopy: Jurkat T cells (1.5 x 10 ⁷ cells/500 11 RPMI), transiently transfected with 20 μg of either GFP-K-Ras construct (a kind gift from M. Phillips, NYU School of Medicine) using the Cell Porator (Life Technologies, 800 μF, 250 V, low ohms), were treated with delivery group (DMSO), simvastatin alone (45 μΜ), or compound 12 (10 μΜ) for 24 h in an incubator at 37 °C. The cells were harvested, washed with PBS three times, and plated on polylysine coverslips for 10 min. The coverslips were washed with PBS for 10 min (3 times) and the cells were fixed with 3% formaldehyde for 10 min. The coverslips were washed again for 10 min with PBS (3 times). After mounting the coverslips onto the slides using FluorSave™ Reagent (Calbiochem), the slides were dried for 45 min at room temperature. Images of cells were viewed using an Olympus BH- 2 microscope and captured with a Qlmaging Microimager II. The captured images were viewed using Northern Eclipse software. Cells were classified as either full (fully mislocalized GFP-K-Ras construct with diffuse cellular staining), partial (partial proper localization), and normal (normal, primarily plasma membrane localization of GFP-K-Ras) by visual inspection. ³² ' ³³ The subcellular localization of GFP K-Ras was quantified using fluorescence microscopy (performed on an Olympus BH-2RFCA) as previously described. ³⁴ ' ³⁵

References for Example 1

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Example 2

Design, synthesis and biological evaluation of aryl-alkyl AFC analogs

To probe the prenyl-binding site of Icmt, a series of aryl-alkyl

AFC analogs were synthesized.

The synthesis of the aryl-alkyl series began with commercially available 4- iodobenzylbromide. This was alkylated with the anion of TMS-propyne affording

intermediate 2.7 (Scheme 2.1). To establish the various alkyl groups, the palladium-catalyzed Kumada coupling was utilized. This was followed by potassium hydroxide deprotection of the silyl protecting group to afford alkynes 2.8 - 2.11. The next task was the installation of the tri-substituted olefin; this was accomplished via a zirconium assisted carboalumination (ZACA) reaction pioneered by Negishi. ⁷⁷ The zirconium catalyst joins with trimethyl aluminum and aids in the delivery of the alane across the double bond, adding in a cis- fashion. The allylic alcohols 2.12 - 2.15 were generated in a stereospecific fashion. Corey- Kim chlorination, ⁷⁸ followed by S- alkylation of N- Acetyl Cysteine ⁷⁹ afforded our prenyl modified AFC analogs 2.16 - 2.19.

Scheme 2.1 (a) nBuLi, TMS-propyne, -78_C, 90% (b) R-MgBr, Cl2Pd(PPh3)2 (c) KOH, MeOH, 45-80% 2 steps (d) Me3Al, Cp2ZrCl2, (HCO)n, 0_C, 45-65% (e) NCS, Me2S, DCM (f) 7N NH3-MeOH, N-Ac-Cys 0_C, 40- 60% two steps. The analogs were screen for biological activity by utilizing a well established

14C-methanol vapor diffusion assay. ⁸⁰ For evaluation as substrates of Icmt, the

analogs (at 25 μΜ) are incubated with hlcmt (5 μg) and 20 μΜ S-adenosyl-[14Cmethyl] methionine (14C-SAM). After incubation a solution of 1M NaOH a 1% SDS solution is added thus cleaving the transferred methyl groups. The solution is then aliquoted onto filter paper and placed atop a vial containing scintillation fluid. The 14C-methanol diffuses into the scintillation fluid and can be quantified using a scintillation counter. To be evaluated as inhibitors of Icmt, an analogous process is used. The analogs, this time at 10 μΜ, are incubated with AFC (25μΜ), 5 μg Icmt, and 14C-SAM (20 μΜ). If the analog inhibits the methylation of AFC, a decrease in methylation will be quantified.

When evaluated as substrates, none of the analogs 2.16 - 2.19 were efficiently methylated by Icmt. As inhibitors, however, all are capable of reducing the methylation of AFC. The shortest analog 2.16 is closest in overall length to that of AFC while 2.19 more closely resembles that of AGGC, an alternative substrate for Icmt41 that mimics the C- terminus of geranylgeranylated proteins. The increased tail length was generally found to be beneficial to Icmt inhibition for this series of AFC analogs.

This increased lipidic character appears to be a requirement for significant inhibitory activity. The most potent of this series, 2.19, was found to have an IC50 of 35muM, nearly ten times lower than that of 2.2, the previous prenyl modified lead Icmt inhibitor. Although two of the isoprene groups were replaced, increased lipidic character from an n-octyl chain is necessary for potency. Note that 2.16 exhibited a much poorer inhibitory potency than 2.19, and has nearly 15% less inhibitory activity at 10 muM. This does reveal that non-prenyl groups, bearing sufficient hydrophobic bulk, can suffice as farnesyl proxies and occupy the prenylcysteine-binding site. This finding advances our understanding of Icmt inhibitors.

When evaluated as substrates, none of the analogs 2.16 - 2.19 were efficiently methylated by Icmt. As inhibitors, however, all are capable of reducing the methylation of AFC. The shortest analog 2.16 is closest in overall length to that of AFC

while 2.19 more closely resembles that of AGGC, an alternative substrate for Icmt41 that mimics the C-terminus of geranylgeranylated proteins. The increased tail length was generally found to be beneficial to Icmt inhibition for this series of AFC analogs. This increased lipidic character appears to be a requirement for significant inhibitory activity. The most potent of this series, 2.19, was found to have an IC50 of 35

muM, nearly ten times lower than that of 2.2, the previous prenyl modified lead

Icmt inhibitor. Although two of the isoprene groups were replaced, increased lipidic character from an n-octyl chain is necessary for potency. Note that 2.16 exhibited a much poorer inhibitory potency than 2.19, and has nearly 15% less inhibitory activity at 10 muM. This does reveal that non-prenyl groups, bearing sufficient hydrophobic bulk, can suffice as farnesyl proxies and occupy the prenylcysteine-binding site. This finding advances our understanding of Icmt inhibitors.

Example 3

FTx triazoles inhibit Icmt

A 1 ,4-disubstituted 1,2, 3 -triazole was positioned as thioether replacement and cysteine backbone modifier that would join the lipid and carboxylate region of the FTP/FTA analog together. This would also employ the replacement of the thioether

with a heteroaryl triazole. This design was advantageous because only two simple parts are needed for synthesis; one possessing and azide and one an alkyne. As outlined in Scheme 4.1 the azide starting materials were quickly assembled in one step. The azido-esters were generated by sodium azide displacement of readily available bromo-esters resulting in the azido-esters 4.1 - 4.2. Conditions of DMF as the solvent and elevated temperatures were found to be best suited for the displacement of the bromide, especially for

the unactivated bromo-esters. Proton, carbon, and IR spectrometry confirmed the

transformation from the halides to the desired intermediate azides.

n = 2

4.7

Scheme 4.1 Synthesis of FTP triazole starting materials. Reagents and

conditions, (a) NaN3, DMF 60-90% (b) nBuLi, Mes-Cl (c) ethynylmagnesium

chloride, CuBr - Me2S 2 steps 75% (d) TMS-propyne, nBuLi 85% (e) TBAF, THF 90%.

The prenyl alkynes were also quickly generated as seen in Scheme 4.1, resulting in synthons with varied spacing between the a-isoprene and alkyne moiety. The nature of alkylations at an allylic position dictated the choice of organometallic reagents.

Incorrect reaction conditions would generate side products resulting in SN20. Farnesol was activated as the mesylate and was displaced by the ethynyl cuprate, formed from the corresponding Grignard reagent. This resulted in the famesyl alkyne being inserted at the allylic position, as reported previously in the synthesis of (-)-terpestacin. ⁹⁶ This formed the desired SN2 product. It is hypothesized that using this synthon would afford FTP/FTA analogs where the triazole would occupy the space where the sulfur of the thioether would reside. To modify this spacing methylene spacers were introduced between the isoprene and alkyne. The anion of TMSprOpyne was used to alkylate the desired position of either geranyl bromide or famesyl chloride. Deprotection with TBAF where necessary afforded the requisite terminal alkynes 4.6-4.7 in good yields.

x = l, y = 0, z

4.8

x = 2, y = 0, z = 2

4.9

x = 3, y = 0, z

4.10

x = 4, y = 0, z

4.11

x = 2, y = 1, z

4.12 x = 2, y = 1, z

4.13

x = 3, y = 1 , z

4.14

Scheme 4.2 Synthesis of FTP triazoles. Reagents and conditions, (a) CuS0 ₄ , sodium ascorbate, tBuOH/H ₂ 0 (4/1) 55-95% (b) aq. NaOH 50-90%

Copper (I) mediated dipolar cyclization of the azide and alkyne in aqueous t - butanol was facilitated by the in situ reduction of copper (II) sulfate pentahydrate

with sodium ascorbate. The reactions were allowed to stir overnight at room tem37 perature and afforded the 1,4-disubstituted triazoles, demonstrating a mild and facile synthesis. Saponification afforded the desired FTx triazoles with varying attachment lengths between carboxylate and prenyl chain. These analogs were first evaluated versus Icmt as substrates in the vapor diffusion assay, they were found to not possess substrate activity. However, they were all micromolar inhibitors Icmt. Adjusting the spacing between carboxylate and triazole, an optimal linkage was found to be two carbons, demonstrated by 4.9. As this linkage is changed, activity is diminished. Interestingly, when the linkage is increased to four carbons, inhibition is higher than with three carbons but does not reach the level of 4.11. One possible explanation is extra flexibility is requisite for the carboxylate to bind with proper orientation when there is rotational restriction from the triazole; as seen in the Icmt inhibitor FTS. Modulating the spacing between the triazole and first isoprene was also important to inhibitory potency. With 4.9 as the starting point, homolog 4.12 investigated the linking region between the triazole and a-isoprene. Similar to the above compounds, it was also not a substrate but rather an inhibitor of Icmt. The lengthened isoprene linker proved to be deleterious to binding. As controls, shortened prenyl compounds were also synthesized and evaluated. Analogs 4.14 and 4.13 were also utilized to investigate the possibility that the triazole was functioning as a first-isoprene mimetic. Continuing the trend for all FTP-triazoles, 4.14 and 4.13 were not substrates and only inhibitors of Icmt, but dramatically reduced in activity. We hypothesize that with the reduced length in prenyl tail, despite a similar length to FTP, these two compounds likely do not possess enough lipid bulk to bind to the enzyme.

These data indicate FTP analogs incorporating a triazole for sulfur substitution present a potential approach for the design of Icmt inhibitors. The positioning of the triazole between the carboxylate and the isoprene is important for activity.

However,analogs of this type appear to require extensive isoprene character and this leaves room for improvement.

References for Examples 2 and 3

- -

All references disclosed herein are incorporated by reference in relevant part in the present application.