WO2016025473A2 - Synthesis of tetracyclic flavonoids - Google Patents

Abstract

The disclosure provides tetracyclic flavonoids of Formula I: and methods of making and using the same.

Description

SYNTHESIS OF TETRACYCLIC FLAVONOIDS RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application serial number 62/035,929, filed August 11, 2014, and of U.S. Provisional

Application serial number 62/037,865, filed August 15, 2014, the contents of each of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present disclosure relates to tetracyclic flavonoids. BACKGROUND

[0003] Prostate cancer (PCa) is the most common age-related malignancy in men in the United States and the second leading cause of male cancer deaths. Androgen ablation therapy results in 60-80% initial response rates. However, PCa becomes unresponsive to androgen ablation therapy in majority of patients resulting in short survival rate. Majority of conventional cancer therapies are not effective against this androgen-independent prostate cancer (AIPC). Hyperactivation of the androgen receptor (AR) is the primary factor responsible for PCa progression. A subset of key molecules regulates AR activity in PCa cells in culture and includes Akt1, c-Jun, cyclin D and sirtuin 1 (Sirt1). The silent mating type information regulator 2 (Sirt1) is one of the seven members (Sirt1-Sirt7) of the sirtuin class of NAD-dependent protein deacetylases. Sirt1 deacetylates AR and suppresses its transcriptional activity and cellular proliferation.

[0004] The androgen receptor (AR) is a ligand-activated transcription factor that belongs to the superfamily of steroid receptors. These receptors have similar protein structures that are composed of an N-terminal domain (NTD) that contains AF-1 (activation function-1), a DNA- binding domain (DBD), a hinge region, and a ligand-binding domain (LBD) that contains a second activation domain, AF-2. Upon binding androgen, the AR undergoes a conformational change that results in formation of a homodimer, and recruitment of multiple transcription factors that activate the transcription of androgen-dependent genes. Different cellular mechanisms appear to be involved in the progression of PCa to androgen-ablation therapy resistance, including cytokine growth factor and kinase-mediated activation of the AR.

[0005] Prostate adenocarcinoma consists of epithelial cells arranged in acinar structures surrounded by stromal or mesenchymal cells. Metastatic or anaplastic prostate carcinoma consists of nests of anaplastic epithelial cells separated by fibrovascular stroma. Critical to the understanding of the treatment of PCa was the early observation that androgen ablation reduced PCa growth. Androgens induce proliferation and inhibit apoptosis of PCa cells. Factors thought to contribute to the autonomous growth of androgen ablation therapy-resistant tumors include the induction of activating AR mutations within the tumors, loss of tumor suppressor proteins with histone deacetylase activity, induction of kinase activity (Akt) by paracrine growth factors and increased oncogene expression (c-jun).

[0006] As with many nuclear receptors, an understanding of the aberrant function of the receptor is dependent upon an understanding of the receptor’s interacting repressors and activators. Several co-activators induce activity of the AR, including p300/CBP-associated factor (P/CAF), Ubc9, GRIP1, FHL2, BRCA1 and CARM-1. In contrast with the wild type AR, several mutant ARs found in patients with PCa are promiscuously activated by a variety of hormones (estrogen, progesterone) that would normally have no effect on the ARwt receptor. The AR mutations also caused anti-androgens such as flutamide to behave as androgen agonists.

[0007] The histone acetylase (HAT) activity of p300/CBP and P/CAF correlates with their transactivation function. Acetyl transferase activity is directed toward both histone and nonhistone proteins to regulate transcriptional activity. In addition to acetylation of core histone, p300/CBP and P/CAF acetylate select target transcription factors including nuclear receptors (ERα, AR, GR, TRβ) and transcription factors (TFIIEB, EKLF, p53, HMG1(Y), GATA1 and E2F-1).

[0008] The nuclear receptors are directly acetylated, and the ligand induces NR acetylation. The ERα is directly acetylated at lysines and the substitution mutations of these lysines to charged or polar residues enhance ligand sensitivity. An estrogen receptor mutant (K303R) was identified in a substantial proportion of premalignant human breast cancers. The ERα mutant was associated with an enhanced proliferative phenotype. This point mutation significantly enhances estrogen sensitivity. These studies indicate that direct acetylation of lysine may contribute to normal transcriptional attenuation as mutation of these residues enhances ligand sensitivity to estrogen. Through performing multiple point mutations of residues within this site it has been shown that the acetylation site of the AR determines ligand sensitivity and specificity.

[0009] The AR protein is subject to post-translational modifications, including

phosphorylation, acetylation, ubiquitination and sumoylation. The AR is subjected to acetylation in vitro and in vivo, by the histone acetyltransferases p300 and p/CAF. Acetylation of the AR is essential for its ligand-dependent activation. The KLKK acetylation motif is highly conserved between different species and is found in a subset of evolutionarily conserved nuclear receptor superfamily members. This finding suggests that acetylation is a general mechanism regulating nuclear receptor function.

[0010] Expression of p300, SRC-1, and SRC-2 are increased in clinical samples from androgen-independent PCa. These findings suggest that increased coactivator expression could be a mechanism contributing to AR activity in androgen-independent PCa. Consistent with these findings, it has been demonstrated that acetylation of the AR regulates its association with corepressor and coactivator proteins. The HDAC/Smad/NCoR complex binding to the AR is attenuated by acetylation of the AR, conversely, the AR and its co-activator binding with p300 and SRC1 is enhanced by acetylation mimic (“active”) mutants of the AR. More importantly, the AR acetylation site was shown to regulate prostate cellular growth and apoptosis and the AR acetylation site mutations conferred a growth advantage to human PCa cells.

[0011] Acetylation of histones is a reversible process involved in the regulation of transcriptional activation and silencing. The relative levels of acetylation are controlled by the actions of histone acetyl transferases (HAT) and histone deacetylases (HDAC). Histone deacetylation (HDAC) contributes to gene regulation through transcriptional repression, either directly through its deacetylase function or through the recruitment of co-repressor proteins. Based on their homology to yeast transcriptional repressors, HDACs have been divided into three distinct classes with class I and II deacetylases being homologous to Rpd3P and Hda1P proteins. Class III HDACs are homologous to the yeast transcriptional repressor Sir2p. The proteins in class I and class II are characterized by their sensitivity to the inhibitor trichostatin A (TSA). Class III HDAC activity is nicotine adenosine dinucleotide (NAD)-dependent but cannot be inhibited by TSA. TSA enhances liganded AR activity, consistent with a role for HDAC in regulating AR activity. [0012] The silent information regulator (Sir2) proteins convey transcriptional silencing at distinct loci including telomeres, the rDNA locus, and the mating-type locus. The SIR2 gene family is conserved from archaebacteria to eukaryotes. Seven human Sirt proteins (Sirt1-7) are known with homology to Sir2p, which have been named“sirtuins”. The histone deacetylation by Sir2 is coupled to cleavage of a high-energy bond in nicotine adenine dinucleotide and to the synthesis of a novel product 2’-O-acetyl- ADP-ribose. In yeast, the deacetylation of histones by Sir2p results in silencing in the ribosomal DNA, and increased dosage of the yeast Sir2 gene extends the lifespan of mother cells. Such findings led to speculation that Sir2 may inhibit functions related to aging. The enzymatic activity of Sir2 is regulated by the availability of the oxidized form of NAD+ allowing SIR2 to function in part as a redox or metabolic sensor.

Recently, several other non-histone substrates of Sirt1 have been identified, including TAF168, RelA/p65, P/CAF, MyoD and the FOXO (forkhead box class O) subfamily of transcription factors (FOXO4, FOXO3). p300 functions as a limiting coactivator of most of these Sirt1 substrates. Sirt1 inhibits and deacetylates p300 and this may contribute to the general mechanism by which Sirt1 inhibits activity of many transcription factors.

[0013] The AR is repressed by Sirt1. Addition of Sirt1 inhibitors (nicotinamide, sirtinol, splitomycin) induced AR signaling and AR abundance. Inhibition of endogenous Sirt1 with Sirtinol increased the relative amount of acetylated AR. Sirt1 deacetylated the AR with similar kinetics and Km to that described for p53. Conversely, Sirt1 activation could reverse these molecular events which underpin the progression of human PCa. Resveratrol, the most potent Sirt1 activator, has been shown to allosterically modulate Sirt1 Km values for its substrates, leading to a robust deacetylation. It was also shown that a single point-mutation of Sirt1 E223K resists stimulation by traditional Sirt1 activators. These genetic approaches showed the Sirt1- dependence of resveratrol and other activators. The repression of AR by Sirt1 should represent an important tumor suppressor mechanism in human PCa.

[0014] Sirt1 has been reported to have both oncogenic and tumor suppressor roles in PCa. Overexpression of Sirt1 is reported in androgen-refractory PC3, DU145 and LNCaP cells.

Treatment with a Sirt1 inhibitor, sirtinol as well as siRNA silencing of Sirt1 inhibited cell growth of PCa cells and increased their sensitivity to chemotherapeutics such as cisplatin. However, there is accumulating evidence that Sirt1 primarily acts as a tumor suppressor protein. [0015] Sirt1 controls hormonal function of androgen receptor by deacetylating the AR at a conserved lysine motif and inhibits androgen dependent prostate cellular growth. Mutations on the AR acetylation (ARK630T) site result in faster growth of prostate cancer cell lines when compared with wildtype AR when implanted in nude mice. Homozygous deletion of Sirt1 gene in mice results in inhibition of androgen responsive gene expression and Sirt1 -/- mice develop prostatic intraepithelial neoplasia (PIN) associated with reduced autophagy. In addition, chemical inhibition of Sirt1 by sirtinol abolishes autophagy in prostate cancer cells.

[0016] On the other hand, resveratrol, a natural Sirt1 activator, prevents development of high grade PIN by modulating Sirt1/S6K signaling (Li, et al., Cancer Prev Res (Phila) 2013, 6(1): 27- 39.). Sirt1 promotes the activity of TSC2, a repressor of mTOR. Activation of Sirt1 by resveratrol also reduces levels of H2A.Z and causes down-regulation of c-myc and other oncogenes (Baptista, et al., Oncotarget 2013, 4(10): 1673-85.). Separately, Sirt1 has also been suggested as a tumor suppressor in colon cancer due to its ability to deacetylate and inactivate oncogenic β- catenin. YK-3-237, a Sirt1 activator, functionally reduces the level of mutant p53 by deacetylation and exhibits antiproliferative effects toward triple-negative breast cancer (Yi, et al., Oncotarget 2013, 4(7): 984-94). Thus Sirt1 functions as a tissue specific regulator of cellular growth. Nevertheless, it is clear that androgen receptor plays a key role in in aberrant prostate cell growth.

[0017] The development of novel and effective therapies to prevent PCa recurrence and/or progression to AIPC is needed. SUMMARY

[0018] In one aspect, the disclosure provides a compound of Formula I:

wherein:

each R¹¹ is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl,

heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; each R¹² is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl, heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; R¹³ is hydrogen, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl, carbonyl, amide, or acyl, each of which can be optionally substituted;

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3,

provided that the compound is not Cycloartocarpin; Cyclocommunol; Cyclocommunin; a compound wherein m is 2, n is 1, each R¹¹ and R¹² is selected from Cl, F, CF₃, methyl, t-butyl, NO₂, OH, OMe or amino, and R¹³ is alkyl or aryl; or a compound wherein m and n are 0 and R¹³ is 2-methylpropenyl or phenyl; or a compound selected from the group consisting of

[0019 n ano er aspec , e sc osure prov es a me o or prepar ng a compoun o Formula I. Method generally comprising palladium-catalyzed intramolecular cyclization of a compound of Formula II:

wherein R¹¹, R¹², R¹³, m and n are as defined for Formula I.

[0020] In still another aspect, the disclosure provides a method of increasing activity or expression level of Sirt1. Generally the method comprises contacting Sirt1 with an effective amount of a compound of Formula I.

[0021] In yet another aspect, the disclosure provides a method of treating prostate cancer in a subject. Generally, the method comprises administering a therapeutically effective amount a compound of Formula I to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] FIG. 1 is a general scheme for the synthesis of demethylated 3-allyl flavonoid 6b. Reagents and conditions: a) 2-MeOC₆H₄COCl, NaH, THF, 0 °C to reflux, 3 hours (i.e., 3 h), 75%; b) NaH, THF, 0 °C to reflux, 2 h, 71%; c) allyl bromide, K₂CO₃, acetone, 48 °C, 12 h, 57%; d) AcOH, H₂SO₄, room temperature (r.t.), 1 h, 76%; e) BBr₃, CH₂Cl₂,–5 °C to r.t., 2 h, 89%. Yields provided for R¹ = 4-Me, R² = H.

[0024] FIG. 2 is a graph showing the single-crystal X-ray structure of 7f.

[0025] FIG. 3 is a proposed mechanism for palladium-catalyzed intramolecular oxidative cyclization.

[0026] FIG. 4 shows binding pockets of Resveratrol (cornflower blue colored carbon atoms) and NSC241011 (cyan) in hSirt1 (grey colored carbon atoms). Nicotine part of NAD+ is shown by green colored atom. All other atoms are atom-based colored (oxygen: red, nitrogen, blue). The AR peptide (aa641-644) are shown in hot-pink and magenta colored carbon (K(Ac)642). [0027] FIGs. 5A-5B are experimental data showing the biological effects on androgen function. (FIG. 5A) Effects of Sirt1 compounds on AR activation and Sirt1-medaited AAR inhibition. (FIG. 5B) Effects on Sirt1 agonist on ligand induced androgen receptor expression.

[0028] FIG. 6 is a plot showing the effects of SIRT1 compound on cellular proliferation of LNCaP cells: vehicle vs. DHT, Sirt1 compounds at 50 mM.

[0029] FIG. 7 is a plot showing Sirt1 expression in human prostate cancer tissue. Patients who developed prostate cancer with low Sirt1 expression (blue line) have less survival probability (*: P=0.012) than patients with high Sirt1 expression (red line). Mean ± SEM, n=154.

[0030] FIG. 8 is an example of established synthetic route (R₁=R₂=H). Conditions: (a) 2- methoxy benzoylchloride, NaH, 0 °C-reflux, 90%; (b) NaH, 0 °C-reflux, 75%; (c) Allyl bromide, K₂CO₃, 48 °C, 76%; (d) AcOH, H₂SO₄, 74%; (e) BBr₃, CH₂Cl₂,–5 °C to 10 °C , 95%; (f) White catalyst, Benzoquinone, AcOH, DCM, 86%; (g) Grubbs second generation catalyst (10 mol%), 2-methyl-2-butene, reflux, 65%.

[0031] FIG. 9 is a schematic representation of targeting for Sirt1 E223K Sirt1 activation resistant mouse. Hatched regions represent Sirt1 coding exonic sequences, grey rectangles Sirt1 non-coding regions. Position of E22K point mutation is indicated. DETAILED DESCRIPTION

[0032] In one aspect, the disclosure provides a compound of Formula I:

.

[0033] In compounds of Formula I, variables m and n are independently 0, 1, 2 or 3.

Without limitations, the compounds of Formula I can comprise m and n in any combination. For example, m can be 0 and n can be 0, m can be 0 and n can be 1, m can be 0 and n can be 2, m can be 0 and n can be 3, m can be 1 and n can be 0, m can be 1 and n can be 1, m can be 1 and n can be 2, m can be 1 and n can be 3, m can be 2 and n can be 0, m can be 2 and n can be 1, m can be 2 and n can be 2, m can be 2 and n can be 3, m can be 3 and n can be 0, m can be 3 and n can be 1, m can be 3 and n can be 2, or m can be 3 and n can be 3. In some embodiments, m is 0, 1 or 2. In some embodiments, n is 0 or 1.

[0034] Each R¹¹ in the compounds of Formula I can be independently selected from the group consisting of alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl, heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, O-acyl, carbonyl, amide and ester, each of which can be optionally substituted. In some embodiments, each R¹¹ is selected independently form the group consisting of from alkyl, alkenyl, alkoxy, hydroxyl, nitro, CF₃, amine, and halogen. In some exemplary compounds of Formula I, each R¹¹ is independently selected from the group consisting of methyl, t-butyl, 3-methylbutenyl, hydroxyl, methoxy, nitro, NH₂, F, Cl and Br.

[0035] Similar to R¹¹, in the compounds of Formula I, each R¹² can be independently selected from the group consisting of alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl, heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, O-acyl, carbonyl, amide and ester, each of which can be optionally substituted. In some embodiments, each R¹² is selected independently form the group consisting of from alkyl, alkenyl, alkoxy, hydroxyl, nitro, CF₃, amine, and halogen. In some exemplary compounds of Formula I, each R¹² is independently selected from the group consisting of methyl, t-butyl, 3- methylbutenyl, hydroxyl, methoxy, nitro, NH₂, F, Cl and Br. In some embodiments, R¹² is hydroxyl, methoxy, Cl or NH₂.

[0036] In compounds of Formula I, R¹³ can be selected from the group consisting of hydrogen, alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl, heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, O-acyl , carbonyl, amide and ester, each of which can be optionally substituted. In some embodiments, R¹³ can be hydrogen, alkyl, alkenyl, aryl, heteroaryl, aldehyde or amide. In some exemplary compounds of Formula I, R¹³ is hydrogen, ethenyl, 2-methylpropenyl, phenyl, formyl, tetrazol-5-yl, 1- morpholinomethanoyl, thiazolidine-2,4-dion-5-yl, or 2,5-dihydrooxazolyl.

[0037] It is noted that the carbon to which R¹³ is attached can have the R or S

stereochemistry in compounds of Formula I. Thus, in some embodiments, the carbon to which R¹³ is attached is in the R configuration. In some other embodiments, the carbon to which R¹³ is attached is in the S configuration. [0038] In some compounds of Formula I, m is 2 and one R¹¹ is alkyl and the other R¹¹ is halogen. In some other compounds of Formula I, m is 2 and one R¹¹ is alkoxy and the other R¹¹ is alkenyl or hydroxyl. In still some other compounds of Formula I, one R¹¹ is hydroxyl and the other R¹¹ is alkenyl.

[0039] In some embodiments, m is 1 and R¹¹ is alkyl, alkenyl, hydroxyl, alkoxy or halogen. In some further embodiments of this R¹¹ is methyl, hydroxyl, F, Cl, Br, or methoxy.

[0040] In some compounds of Formula I, n is 1 and R¹² is hydroxyl, alkoxy, amine, or halogen. In some further embodiments of this, R¹² is hydroxyl, methoxy or Cl.

[0041] In some embodiments, a compound of Formula I is of Formula IA, IB, IC, ID, IE, IF, IG, IH, IJ, IK, IL, IM, IN, or IO:

[0042] Without limitations, compounds of Formula I can include isomers, derivatives, analogs, prodrugs and pharmaceutically acceptable salts thereof.

[0043] In some embodiments, the compound of Formula I is selected from the group consisting of:

[0044] In some embodiments, the compound of Formula I is selected from the group

[0045] In the novel compounds of Formula I, Cycloartocarpin having the structure

is expressly excluded. Cycloartocarpin is also referred to as NSC241011 in the art. In some embodiments, the compound of Formula I is not

Cyclocommunol or Cyclocommunin.

[0046] In some embodiments, compounds where m is 2; n is 1; each R¹¹ and R¹² is selected independently from Cl, F, CF₃, methyl, t-butyl, NO₂, OMe or amino; and R¹³ is alkyl or aryl are excluded from the novel compounds of Formula I. In some embodiments, compounds wherein m and n are 0 and R¹³ is 2-methylpropenyl or phenyl are excluded from the novel compounds of Formula I. In some embodiments, the following compounds are excluded from the novel compounds of Formula I:

.

Synthesis of compounds of Formula I

[0047] The disclosure also provides a novel method of preparing compounds of Formula I. Generally the method comprises palladium-catalyzed intramolecular cyclization of a compound of Formula II:

wherein R¹¹, R¹², R¹³, m and n are as defined for Formula I.

[0048] It is to be noted that the synthesis method disclosed herein can be used to prepare any compound of Formula I including those compounds which are excluded from the novel compounds of Formula I.

[0049] Generally, the palladium catalyst is a palladium (II) catalyst. Exemplary palladium catalysts for use in the method disclosed herein include, but are not limited to, palladium catalyst is selected from the group consisting of palladium (II) acetate, palladium (II) chloride, palladium dibenzylideneacetone, dichlorobis(acetonitrile)palladium (II), dichlorobis(benzonitrile)palladium (II), dichlorodiamine palladium (II), palladium (II) acetylacetonate, palladium (II) bromide, palladium (II) cyanide, palladium (II) iodide, palladium oxide, palladium (II) nitrate hydrate, palladium (II) sulfate dihydrate, palladium (II) trifluoroacetate, tetraamine palladium (II) tetrachloropalladate, tetrakis(acetonitrile)palladium (II) tetrafluoroborate and combinations thereof. In some embodiments, the palladium catalyst is the White Catalyst ([1,2-bis- (phenylsulfinyl)ethane]palladium(II)acetate).

[0050] In some embodiments, the cyclization reaction is carried out in the presence of an oxidizing agent. Exemplary oxidizing agents include, but are not limited to, benzoquinone, benzoyl peroxide; bleach; n-bromosaccharin; n-bromosuccinimide; (e)-but-2-enenitrile; n-fluoro- 2,4,6-trimethylpyridinium triflate; n-tert-butylbenzenesulfinimidoyl chloride; tert-butyl hydroperoxide; tert-butyl hypochlorite; tert-butyl nitrite; can; cerium ammonium nitrate;

chloramine-t; chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate); 3- chloroperoxybenzoic acid; chromium compounds; chromium trioxide; Collins reagent; Corey- Suggs reagent; CMHP; copper compounds; crotononitrile; cumene hydroperoxide; DBDMH; DDQ; DEAD; Dess-Martin periodinane; DIAD; 1,3-dibromo-5,5-dimethylhydantoin; 2,3- dichloro-5,6-dicyanobenzoquinone; diethyl azodicarboxylate; DIH; 1,3-diiodo-5,5- dimethylhydantoin; diisopropyl azodicarboxylate; dimethyl sulfoxide; di-tert-butyl peroxide; DPQ; DTBP; (E)-but-2-enenitrile; ferric chloride; ferric nitrate; N-fluoro-2,4,6- trimethylpyridinium triflate; formic acid; hydrogen peroxide; hydrogen peroxide urea adduct; hydroxy(tosyloxy)iodobenzene; hypervalent bromine compounds; hypervalent iodine

compounds; HTIB; IBX; iodine; iodobenzene dichloride; iodosobenzene bis(trifluoroacetate); iodosobenzene diacetate; N-iodosuccinimide; iodosylbenzene; 2-iodoxybenzoicacid; iron(iii), (v) and (iv); Jones reagent; Koser’s reagent; magnesium monoperoxyphthalate hexahydrate;

manganese compounds; manganese(IV) oxide; MCPBA; meta-chloroperbenzoic acid; N- methylmorpholine-N-oxide; methyltrioxorhenium; MMPP• 6H₂O; molybdenum compounds; MTO; N-bromosaccharin; N-bromosuccinimide; N-chloro tosylamide sodium salt; N- chlorosuccinimide; N-iodosuccinimide; N,N,N’,N’-tetrachlorobenzene-1,3-disulfonamide; nitric acid; nitrosobenzene; N-methylmorpholine-N-oxide; NMO; AZADO; nor-AZADO; N-tert- butylbenzenesulfinimidoyl chloride; osmium tetroxide; oxalyl chloride; oxone; oxygen; ozone; PCC; PDC; peracetic acid; periodic acid; peroxy acids; phenyliodonium diacetate; PIFA; pivaldehyde; potassium ferricyanide; potassium permanganate; potassium peroxydisulfate; potassium peroxomonosulfate; 2-propanone; pyridine N-oxide; pyridinium hydrobromide perbromide; pyridinium chlorochromate; pyridinium dichromate; pyridinium tribromide;

ruthenium (III-VII) compounds; sarett reagent; selectfluor; selenium dioxide; sodium bromate; sodium chlorite; sodium dichloroiodate; sodium hypochlorite; sodium nitrite; sodium perborate; sodium percarbonate; sodium periodate; sulfur; styrene; TBCA; TBHP; TBN; TCBDA;; TCCA; TEMPO; N-tert-butylbenzenesulfinimidoyl chloride; tert-butyl hydroperoxide; tert-butyl hypochlorite; tert-butyl nitrite; tetrabutylammonium peroxydisulfate; N,N,N’N’- tetrachlorobenzene-1,3-disulfonamide; 2,2,6,6-tetramethylpiperidinyloxy; tetrapropylammonium perruthenate; 3,3’,5,5’-tetra-tert-butyldiphenoquinone; TPAP; triacetoxyperiodinane;

tribromoisocyanuric acid; trichloroisocyanuric acid; 1,1,1 -trifluoroacetone; trifluoroacetic peracid; and any combinations thereof. Generally, the oxidizing agent is chosen that is compatible with the palladium catalyst. In some embodiments, the oxidizing agent is benzoquinone.

[0051] The cyclization reaction can be carried out in a solvent. Without limitations, the solvent can a non-polar or polar solvent. Further, the solvent can be aprotic or protic solvent. In some embodiments, the solvent is a polar aprotic solvent. Solvent for the cyclization can be selected from the group consisting of dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene, toluene, 1,4-dioxane, chloroform, diethyl ether, formic acid, n-butanoel, isopropanol, n-propanol, ethanol, methanole, acetic acid, nitromethane, and any combinations thereof. In some embodiments, the solvent is dichloromethane.

[0052] Without limitations, the cyclization can be carried out at any desired temperature. For example, the cyclization can be carried out at a temperature between 0^oC to 100^oC. In some embodiment, the cyclization is conducted at room temperature or at an elevated temperature. In some embodiments, cyclization is conducted at a temperature between room temperature and 50^oC. In some embodiments, cyclization is conducted at a temperature between 30^oC and 50^oC. In some embodiments, cyclization is conducted at a temperature between 35^oC and 45^oC.

[0053] In some embodiments, R¹³ in Formula II comprises a terminal alkenyl group. Thus, in some embodiments, the method comprises further reacting the product of the cyclization reaction in order to derivatize, modify or replace the terminal alkenyl group. For example, in some embodiments, the method comprises subjecting the cyclization reaction product to olefin metathesis. Methods and reagents for olefin metathesis are well known in the art and available to one of skill in the art. In some embodiment olefin metathesis is in the presence of Grubbs Catalyst.

[0054] In some embodiments, the method further comprises purifying or isolating the desired product (e.g., compound of Formula I) from the cyclization reaction. Methods for purifying reaction products are well known in the art and available to one of ordinary skill in the art.

Exemplary purification/isolation methods include, but are not limited to, chromatography (e.g., column, HPLC, Gas, etc…), distillation, filtration, extraction, crystallization and the like. Method of use

[0055] The compounds of Formula I can act as modulators of Sirt1 activity. Thus, in one aspect, the disclosure provides a method of increasing or enhancing Sirt1 activity. Generally, the method comprises contacting Sirt1 with a compound of Formula I. In embodiments of the method, Sirt1 can be in a cell, wherein the cell expresses an endogenous Sirt1. In some other embodiments, Sirt1 can be in a cell, wherein the cell expresses an exogenous Sirt1. Without limitations, the method can be used to increase Sirt1 activity in vitro, ex-vivo, or in vivo. Thus, in some embodiments, contacting of the compound of Formula I with Sirt1 is ex-vivo. In still some other embodiments, contacting of the compound of Formula I with Sirt1 is in vivo.

[0056] Sirt1, also known as NAD-dependent deacetylase sirtuin-1, is a protein that in humans is encoded by the SIRT1 gene. As used herein,“Sirt1” refers to a polypeptide exhibiting nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase activity, and having an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, identical to the amino acid sequence of a known Sirt1 polypeptide. A Sirt1 protein includes yeast Sir2 (GenBank Accession No. P53685); C. elegans Sir-2.1 (GenBank Accession No. NP—501912); human Sirt1 (see, e.g., GenBank Accession No. NM—012238 or NP— 036370 (or AF083106); Frye ((1999) Biochem. Biophys. Res. Comm. 260:273); and GenBank Accession Nos. Q96EB6, AAH12499, NP—036370, and AAD40849); mouse Sirt1 (see, e.g., GenBank Accession Nos. Q923E4 and NP—062786); and equivalents and fragments thereof. [0057] Sirt1 deacetylates a variety of proteins, including, but not limited to, KU70, Nbs1, p53, NF-κB, PPARγ, PGC-1α, FOXO, and SUV39H1, and regulates genomic integrity, the inflammatory response, adipogenesis, mitochondrial biogenesis, and stress resistance. For example, Sirt1 catalyzes the deacetylation of tumor suppressor protein p53, thus promoting survival by inhibiting p53-mediated apoptosis. Sirt1 also directly interacts with PPAR-γand PGC-1α, thus regulating metabolic response. Sirt1 also inhibits androgen receptor acetylation.

[0058] In some embodiments, the compound of Formula I modulates the deacetylase activity of Sirt 1. Methods of measuring deacetylase activity of Sirt1 are well known in the art. For example, activity of Sirt1 can be determined in deacetylating proteins selected from AR, KU70, Nbs1, p53, NF-κB, PPARγ, PGC-1 α, FOXO, and SUV39H1. The deacetylation activity can be determined by methods including, but not limited to, co-immunoprecipitation, Western blotting, ELISA, immunofluorescence, radioimmunoassay, immunocytochemistry, and a combination thereof.

[0059] The effect of the compound of Formula I on Sirt1 AR deacetylase activity can be determined by measuring the amount of deacetylated AR polypeptide produced by action of the Sirt1 polypeptide in the presence of the compound of Formula I, compared to the amount of deacetylated AR polypeptide produced by action of the Sirt1 polypeptide in a control reaction sample that does not include the compound of Formula I. As an alternative to, or in addition to, measuring the amount of deacetylated AR polypeptide, the amount of remaining acetylated AR can be measured. Methods of determining the level of acetylated AR in a sample include immunological assays using antibody that is specific for acetylated form of AR, and that therefore distinguishes between acetylated AR and deacetylated AR. Any of a variety of immunological assays can be used, including, e.g., enzyme linked immunosorbent assay

(ELISA), radioimmunoassay (RIA), protein blot (“Western” blot) assays, and the like. In some embodiments, mass spectroscopy is used. Sirt1 activity can also be determined by measuring the level of NAD in the test sample. The action of Sirt1 on acetylated AR can be coupled to a second enzymatic reaction that reduces NAD to NADH, and measuring fluorescence of NADH at, e.g., 340 nm.

[0060] A compound of Formula I can increase the enzymatic activity of Sirt1 by at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, or more than 20-fold. Increase in activity can be relative to a reference or control.

[0061] A compound of Formula I can increase Sirt1 enzymatic activity at an EC₅₀ (half maximal effective concentration) of from about 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM, from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 450 nM, from about 450 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

[0062] A compound of Formula I can increase at an EC_1.5 (concentration of compound required to increase enzyme activity by 50%) of from about 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 nM to about 15 nM, from about 15 nM to about 25 nM, from about 25 nM to about 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 450 nM, from about 450 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about 100 μM, from about 100 μM to about 250 μM, from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM. For example, a compound of Formula I can have an EC_1.5 of from about 0.01 μM to about 100 μM, e.g., from about 0.01 μM to about 0.1 μM, from about 0.1 μM to about 0.5 μM, from about 0.5 μM to about 1.0 μM, from about 1.0 μM to about 10 μM, from about 10 μM to about 25 μM, from about 25 μM to about 50 μM, or from about 50 μM to about 100 μM.

[0063] In some embodiments, the compound of Formula I is a selective Sirt1 activator. For example, in some embodiments, the compound of Formula I increases the enzymatic activity of a Sirt1 polypeptide, but does not substantially increase the enzymatic activity of any other sirtuin. For example, in some embodiments, the compound of Formula I increases the enzymatic activity of a Sirt1 polypeptide, but does not substantially increase the enzymatic activity of Sirt2, Sirt4 or Sirt5.

[0064] Increased Sirt1 activity has been documented as beneficial in many disease models and human subjects. Therefore, it has been widely accepted that Sirt1-activating compounds could provide therapeutic benefits for various disease where increased Sirt1 is of benefit, e.g., cancer. Thus, in another aspect, the compounds of Formula I can be used to treat prostate cancer. Accordingly, the disclosure provides a method of treating prostate cancer in a subject, the method comprising administering to the subject a therapeutically-effective amount of a compound disclosed herein.

[0065] Prostate cancer is cancer found in the prostate, which is an exocrine gland of the male reproductive system, and exists directly under the bladder, in front of the rectum. Most prostate cancers are adenocarcinomas. Prostate cancer, as used herein, includes pre-cancer forms such as prostatic intraepithelial neoplasia. The severity of prostate cancer is generally evaluated using a Gleason score, with range from 2 to 10, obtained by adding the score for a predominant pattern to a secondary pattern, with pattern scores ranging from 1 to 5 and increasing score numbers indicating a more advanced and/or aggressive form of prostate cancer. For example, a Gleason score of 6 or more can indicate the presence of a worse than average, or severe, form of prostate cancer.

[0066] The degree of severity of prostate cancer is based on a variety of systems, one of which is disease staging, an example of which follows: Stage 1: the cancer is very small and completely inside the prostate gland which feels normal when a rectal examination is done. Stage 2: the cancer is still inside the prostate gland, but is larger and a lump or hard area can be felt when a rectal examination is done. Stage 3: the cancer has broken through the covering of the prostate and may have grown into the neck of the bladder or the seminal vesicle. Stage 4: the cancer has spread to another part of the body, where the most common site of prostate cancer spread is the bones. It does not often spread to other body organs.

[0067] In some embodiments of the various aspects disclosed herein, the subject has one or more symptoms of prostate cancer. Symptoms of prostate cancer include trouble urinating, decreased force in the stream of urine, blood in the urine, blood in the semen, general pain in the lower back, hips or thighs, discomfort in the pelvic area, bone pain, and erectile dysfunction. Additional screening and diagnostic tests can be performed to help determine if a subject has prostate cancer.

[0068] Prostate cancer can be diagnosed by methods including, but not limited to, biopsy, digital rectal examination, cystoscopy, transrectal ultrasonography, prostate imaging (e.g., ultrasound or magnetic resonance imaging), determining a Gleason score, or a combination thereof. Prostate cancer can also be diagnosed by assaying a tumor marker associated with the development of the cancer, such as BCL-2, Ki-67, and ERK5.

[0069] In some embodiments, the compounds disclosed herein can be used with another anti- prostate cancer therapy, for example, in a combinatorial therapy. Treatment for prostate cancer vary and include a range of treatment options including, but not limited to, one or more of surgery (i.e., radical prostatectomy); radiation therapy (i.e., external beam or brachytherapy); hormonal therapy, such as "androgen ablation", e.g., administration of anti-androgens; and chemotherapy. Anti-androgens most often used in the treatment of prostate cancer include, but are not limited to: leuprolide an injectable, synthetic hormone that is used to treat prostate cancer. Leuprolide (Lupron) is a gonadotropin-releasing hormone analog, which may be indicated for treatment of advanced prostate cancer. Leuprolide may be used in combination with one or both of Goserelin (Zoladex®) and Casodex (bicalutamide). Goserelin (Zoladex®) contains a synthetic decapeptide analogue of luteinizing hormone-releasing hormone (LHRH), also known as a gonadotropin releasing hormone (GnRH) agonist analogue. Casodex

(bicalutamide) is an oral non-steroidal anti-androgen which contains the active ingredient bicalutamide. It works by blocking the effects of male hormones such as testosterone.

[0070] Flutamide is also used in the treatment of advanced prostate cancer. It works by preventing testosterone from binding to androgen receptors in the prostate gland. It also acts on an area of the brain called the hypothalamus, which ultimately results in a reduction in the amount of testosterone produced by the body. In the treatment of prostate cancer, flutamide is often used in combination with an LHRH analogue. LHRH analogues are one of the standard treatments for prostate cancer and include medicines such as buserelin, goserelin, leuprorelin and triptorelin.

[0071] Another drug used in the treatment of prostate cancer is Nilutamide (Anandron®), a nonsteroidal anti-androgen with affinity for androgen receptors (but not for progestogen, estrogen, or glucocorticoid receptors). [0072] Typical means of monitoring prostate cancer in a subject, as generally known in the art, can be carried out in conjunction with evaluation of the treatment methods disclosed herein. The subject may be monitored in any of a number of ways such as an evaluation of tumor mass, tumor volume, the number of tumor cells or growth rate of the tumor. Parameters that may be evaluated include but are not limited to, direct measurement of accessible tumors, counting of tumor cells (e.g. in the blood), measurement of tumor antigens (e.g., Prostate Specific Antigen (PSA), Alpha-fetoprotein (AFP), and the like), various visualization techniques (e.g., MRI, CAT- scan and X-rays), determination of bone density or evaluation of bone metastases. The information obtained from these analyses is useful in adjusting the dose or schedule of administration in order to optimize the response of the individual and achieve an improved therapeutic outcome relative to prostate cancer. Additional doses may be given as appropriate until the desired effect is achieved. Pharmaceutical compositions

[0073] For administration to a subject, the compounds of Formula I can be provided in pharmaceutically acceptable compositions. These pharmaceutically acceptable compositions comprise a compound of Formula I, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions described herein can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), gavages, lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. Additionally, compounds can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed.“Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which is herein incorporated by reference. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquids such as suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms.

[0074] As used here, the term“pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0075] As used here, the term“pharmaceutically-acceptable carrier” means a

pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum

hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C₂-C₁₂ alchols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as“excipient”,“carrier”,“pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0076] Oral formulations. Pharmaceutical compositions comprising the compounds of Formula I can also be formulated into oral dosage forms such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's

Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).

[0077] Typical oral dosage forms are prepared by combining the pharmaceutically acceptable salt of the disclosed compounds in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of the composition desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, kaolin, diluents, granulating agents, lubricants, binders, and disintegrating agents. Due to their ease of administration, tablets and capsules represent the most advantageous solid oral dosage unit forms, in which case solid pharmaceutical excipients are used. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. These dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

[0078] For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient(s) in a free- flowing form, such as a powder or granules, optionally mixed with one or more excipients.

Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Examples of excipients that can be used in oral dosage forms of the disclosure include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[0079] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101 , AVICEL-PH-103 AVICEL RC-581 , and AVICEL- PH- 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa., U.S.A.), and mixtures thereof. An exemplary suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM.

[0080] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in

pharmaceutical compositions of the disclosure is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

[0081] Disintegrants are used in the compositions of the disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much

disintegrant may swell, crack, or disintegrate in storage, while those that contain too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) should be used to form solid oral dosage forms of the disclosure. The amount of disintegrant used varies based upon the type of formulation and mode of administration, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, preferably from about 1 to about 5 weight percent of disintegrant.

[0082] Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, agar, alginic acid, calcium carbonate,

microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, clays, other algins, other celluloses, gums, and mixtures thereof.

[0083] Lubricants that can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL^® 200, manufactured by W. R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB-O-SIL^® (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the

pharmaceutical compositions or dosage forms into which they are incorporated.

[0084] Parenteral formulations. Parenteral dosage forms can be administered to patients by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, administration DUROS^®-type dosage forms, and dose-dumping.

[0085] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0086] Compounds that alter or modify the solubility of the compounds disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0087] Controlled and delayed release formulations. The pharmaceutical compositions can be administered by controlled- or delayed-release means. Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non- controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0088] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under- dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.

[0089] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0090] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899;

3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543;

5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS^® (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, Duolite^® A568 and Duolite^® AP143 (Rohm&Haas, Spring House, Pa. USA).

[0091] The term“effective amount” as used herein refers to the amount of a therapy needed to alleviate at least one or more symptoms of the disease or disorder, e.g., prostate cancer, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of a therapy that is sufficient to cause a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact“effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the art using only routine experimentation. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

[0092] Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices, are preferred.

[0093] As used herein, the term ED denotes effective dose and is used in connection with animal models. The term EC denotes effective concentration and is used in connection with in vitro models.

[0094] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0095] The therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay.

[0096] The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. Generally, the compositions are administered so that RARγ agonist is given at a dose from 1 µg/kg to 150 mg/kg, 1 µg/kg to 100 mg/kg, 1 µg/kg to 50 mg/kg, 1 µg/kg to 20 mg/kg, 1 µg/kg to 10 mg/kg, 1µg/kg to 1mg/kg, 100 µg/kg to 100 mg/kg, 100 µg/kg to 50 mg/kg, 100 µg/kg to 20 mg/kg, 100 µg/kg to 10 mg/kg, 100µg/kg to 1mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges given here include all intermediate ranges, for example, the range 1 tmg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1mg/kg to 6 mg/kg, 1mg/kg to 7 mg/kg, 1mg/kg to 8 mg/kg, 1mg/kg to 9 mg/kg, 2mg/kg to 10mg/kg, 3mg/kg to 10mg/kg, 4mg/kg to 10mg/kg, 5mg/kg to 10mg/kg, 6mg/kg to 10mg/kg, 7mg/kg to 10mg/kg,8mg/kg to 10mg/kg, 9mg/kg to 10mg/kg , and the like. It is to be further understood that the ranges intermediate to the given above are also within the scope of this invention, for example, in the range 1 mg/kg to 10 mg/kg, dose ranges such as 2mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4mg/kg to 6mg/kg , and the like.

[0097] In some embodiments, an effective amount of a compound disclosed herein is an amount which causes the tumor size to shrink by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%.

[0098] As used herein, the term“administer” refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced. Routes of administration suitable for the methods described herein include both local and systemic administration. Generally, local administration results in more of the composition being delivered to a specific location as compared to the entire body of the subject, whereas, systemic administration results in delivery to essentially the entire body of the subject.

[0099] A compound of Formula I or composition described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. [00100] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.“Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In preferred embodiments of the aspects described herein, the compositions are administered by intravenous infusion or injection. The administration can be systemic or local.

[00101] The amount of a compound of Formula I that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally out of one hundred percent, this amount will range from about 0.01% to 99% of the compound, preferably from about 5% to about 70%, most preferably from 10% to about 30%.

[00102] In some embodiments, the compositions are administered at a dosage so that the compound of Formula I or a metabolite thereof has an in vivo concentration of less than 500nM, less than 400nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20, nM, less than 10 nM, less than 5nM, less than 1 nM, less than 0.5 nM, less than 0.1nM, less than 0.05, less than 0.01, nM, less than 0.005 nM, less than 0.001 nM after 15 mins, 30 mins, 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs or more of time of administration.

[00103] With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment or make other alteration to treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the compound. The desired dose can be administered every day or every third, fourth, fifth, or sixth day. The desired dose can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. Such sub-doses can be administered as unit dosage forms. In some embodiments of the aspects described herein, administration is chronic, e.g., one or more doses daily over a period of weeks or months. Examples of dosing schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[00104] Specific elements of any of the embodiments described in the disclosure can be combined or substituted for elements in other embodiments described in the disclosure.

Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments can also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. Some selected definitions

[00105] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.

[00107] As used herein the term“comprising” or“comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[00108] The singular terms“a,”“an,” and“the” include plural referents unless context clearly indicates otherwise. Similarly, the word“or” is intended to include“and” unless the context clearly indicates otherwise.

[00109] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.” The term“about” when used in connection with percentages may mean ±5% of the value being referred to. For example, about 100 means from 95 to 105.

[00110] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term“comprises” means“includes.” The abbreviation,“e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the

abbreviation“e.g.” is synonymous with the term“for example.”

[00111] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00112] The terms“decrease”,“reduce”,“reduction”, or“inhibit” are all used herein generally to mean a decrease by a statistically significant amount. For example,“decrease”,“reduce”, “reduction”, or“inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[00113] The terms“increased” ,“increase”,“enhance”, or“activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of doubt, the terms“increased”,“increase”,“enhance”, or“activate” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2- fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker, protein expression or symptom is meant a statistically significant increase in such level.

[00114] The term“statistically significant” or“significantly” refers to statistical significance and generally means at least two standard deviation (2SD) away from a reference level. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.

[00115] As used herein, the term“aliphatic” means a moiety characterized by a straight or branched chain arrangement of constituent carbon atoms and can be saturated or partially unsaturated with one or more (e.g., one, two, three, four, five or more) double or triple bonds.

[00116] As used herein, the term“alicyclic” means a moiety comprising a nonaromatic ring structure. Alicyclic moieties can be saturated or partially unsaturated with one or more double or triple bonds. Alicyclic moieties can also optionally comprise heteroatoms such as nitrogen, oxygen and sulfur. The nitrogen atoms can be optionally quaternerized or oxidized and the sulfur atoms can be optionally oxidized. Examples of alicyclic moieties include, but are not limited to moieties with C₃-C₈ rings such as cyclopropyl, cyclohexane, cyclopentane,

cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptene, cycloheptadiene, cyclooctane, cyclooctene, and cyclooctadiene.

[00117] As used herein, the term“alkyl” means a straight or branched, saturated aliphatic radical having a chain of carbon atoms. C_x alkyl and C_x-C_yalkyl are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C₁-C₆alkyl includes alkyls that have a chain of between 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and the like). Alkyl represented along with another radical (e.g., as in arylalkyl) means a straight or branched, saturated alkyl divalent radical having the number of atoms indicated or when no atoms are indicated means a bond, e.g., (C₆- C₁₀)aryl(C₀-C₃)alkyl includes phenyl, benzyl, phenethyl, 1-phenylethyl 3-phenylpropyl, and the like. Backbone of the alkyl can be optionally inserted with one or more heteroatoms, such as N, O, or S.

[00118] In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The term “alkyl” (or“lower alkyl”) as used throughout the specification, examples, and claims is intended to include both“unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.

[00119] Unless the number of carbons is otherwise specified,“lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise,“lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

[00120] Substituents of a substituted alkyl can include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters),-CF3, -CN and the like.

[00121] As used herein, the term“alkenyl” refers to unsaturated straight-chain, branched- chain or cyclic hydrocarbon radicals having at least one carbon-carbon double bond. C_x alkenyl and C_x-C_yalkenyl are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C₂-C₆alkenyl includes alkenyls that have a chain of between 1 and 6 carbons and at least one double bond, e.g., vinyl, allyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3- butenyl, 2-methylallyl, 1-hexenyl, 2-hexenyl, 3- hexenyl, and the like). Alkenyl represented along with another radical (e.g., as in arylalkenyl) means a straight or branched, alkenyl divalent radical having the number of atoms indicated. Backbone of the alkenyl can be optionally inserted with one or more heteroatoms, such as N, O, or S.

[00122] As used herein, the term“alkynyl” refers to unsaturated hydrocarbon radicals having at least one carbon-carbon triple bond. C_x alkynyl and C_x-C_yalkynyl are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C₂-C₆alkynyl includes alkynls that have a chain of between 1 and 6 carbons and at least one triple bond, e.g., ethynyl, 1- propynyl, 2-propynyl, 1-butynyl, isopentynyl, 1,3-hexa-diyn-yl, n-hexynyl, 3-pentynyl, 1-hexen- 3-ynyl and the like. Alkynyl represented along with another radical (e.g., as in arylalkynyl) means a straight or branched, alkynyl divalent radical having the number of atoms indicated. Backbone of the alkynyl can be optionally inserted with one or more heteroatoms, such as N, O, or S. [00123] The terms“alkylene,”“alkenylene,” and“alkynylene” refer to divalent alkyl, alkelyne, and alkynylene” radicals. Prefixes C_x and C_x-C_y are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C₁-C₆alkylene includes methylene, (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),

tetramethylene (—CH₂CH₂CH₂CH₂—), 2-methyltetramethylene (—CH₂CH(CH₃)CH₂CH₂—), pentamethylene (—CH₂CH₂CH₂CH₂CH₂—) and the like).

[00124] As used herein, the term“alkylidene” means a straight or branched unsaturated, aliphatic, divalent radical having a general formula =CR_aR_b. C_x alkylidene and C_x-C_yalkylidene are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C₂-C₆alkylidene includes methylidene (=CH₂), ethylidene (=CHCH₃), isopropylidene

(=C(CH₃)₂), propylidene (=CHCH₂CH₃), allylidene (=CH—CH=CH₂), and the like).

[00125] The term“heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[00126] As used herein, the term“halogen” or“halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term“halogen radioisotope” or“halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

[00127] A“halogen-substituted moiety” or“halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more“halo” atoms, as such terms are defined in this application. For example, halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C₁-C₃)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (-CF₃), 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).

[00128] The term“aryl” refers to monocyclic, bicyclic, or tricyclic fused aromatic ring system. C_x aryl and C_x-C_yaryl are typically used where X and Y indicate the number of carbon atoms in the ring system. Exemplary aryl groups include, but are not limited to, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,

dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1 , 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent.

[00129] The term“heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1 -3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively. C_x heteroaryl and C_x-C_yheteroaryl are typically used where X and Y indicate the number of carbon atoms in the ring system. Heteroaryls include, but are not limited to, those derived from benzo[b]furan, benzo[b] thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno[2,3-c]pyridine, thieno[3,2- b]pyridine, thieno[2, 3-b]pyridine, indolizine, imidazo[l,2a]pyridine, quinoline, isoquinoline, phthalazine, quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole, indoline, benzoxazole, benzopyrazole, benzothiazole, imidazo[l,5-a]pyridine, pyrazolo[l,5-a]pyridine, imidazo[l,2-a]pyrimidine, imidazo[l,2-c]pyrimidine, imidazo[l,5-a]pyrimidine, imidazo[l,5- c]pyrimidine, pyrrolo[2,3-b]pyridine, pyrrolo[2,3cjpyridine, pyrrolo[3,2-c]pyridine, pyrrolo[3,2- b]pyridine, pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrrolo [2,3-b]pyrazine, pyrazolo[l,5-a]pyridine, pyrrolo[l,2-b]pyridazine, pyrrolo[l,2-c]pyrimidine, pyrrolo[l,2- a]pyrimidine, pyrrolo[l,2-a]pyrazine, triazo[l,5-a]pyridine, pteridine, purine, carbazole, acridine, phenazine, phenothiazene, phenoxazine, l,2-dihydropyrrolo[3,2,l-hi]indole, indolizine, pyrido[l,2-a]indole, 2(lH)-pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,

dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H- quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,

tetrahydropyranyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Some exemplary heteroaryl groups include, but are not limited to, pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, 2-amino-4-oxo-3,4-dihydropteridin-6-yl, tetrahydroisoquinolinyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring may be substituted by a substituent.

[00130] The term“cyclyl” or“cycloalkyl” refers to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons. C_xcyclyl and C_x-C_ycylcyl are typically used where X and Y indicate the number of carbon atoms in the ring system. The cycloalkyl group additionally can be optionally substituted, e.g., with 1, 2, 3, or 4 substituents. C₃-C₁₀cyclyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl, cyclooctyl,

bicyclo[2.2.2]octyl, adamantan-l-yl, decahydronaphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo [2.2.1]hept-l-yl, and the like.

[00131] Aryl and heteroaryls can be optionally substituted with one or more substituents at one or more positions, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

[00132] The term“heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). C_xheterocyclyl and C_x-C_yheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent.

Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl,

perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like.

[00133] The terms“bicyclic” and“tricyclic” refers to fused, bridged, or joined by a single bond polycyclic ring assemblies.

[00134] The term“cyclylalkylene” means a divalent aryl, heteroaryl, cyclyl, or heterocyclyl.

[00135] As used herein, the term“fused ring” refers to a ring that is bonded to another ring to form a compound having a bicyclic structure when the ring atoms that are common to both rings are directly bound to each other. Non-exclusive examples of common fused rings include decalin, naphthalene, anthracene, phenanthrene, indole, furan, benzofuran, quinoline, and the like. Compounds having fused ring systems can be saturated, partially saturated, cyclyl, heterocyclyl, aromatics, heteroaromatics, and the like.

[00136] As used herein, the term“carbonyl” means the radical—C(O)—. It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including aldehyde (e.g., formyl), acids, acid halides, amides, esters, ketones, and the like. In some embodiments, the carbonyl group is substituted with a heterocyclyl. For example, the carbonyl group can be in the form of an ester or amide when connected to an oxygen or nitrogen atom of heterocyclyl.

[00137] The term“carboxy” means the radical—C(O)O—. It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. The term“carboxyl” means–COOH

[00138] The term“cyano” means the radical—CN.

[00139] The term,“heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include—N=,—NR^N—,—N⁺(O-)=,—O—,—S— or —S(O)₂—,—OS(O)₂—, and—SS—, wherein R^N is H or a further substituent.

[00140] The term“hydroxy” means the radical—OH.

[00141] The term“imine derivative” means a derivative comprising the moiety—C(NR)—, wherein R comprises a hydrogen or carbon atom alpha to the nitrogen.

[00142] The term“nitro” means the radical—NO₂.

[00143] An“oxaaliphatic,”“oxaalicyclic”, or“oxaaromatic” mean an aliphatic, alicyclic, or aromatic, as defined herein, except where one or more oxygen atoms (—O—) are positioned between carbon atoms of the aliphatic, alicyclic, or aromatic respectively.

[00144] An“oxoaliphatic,”“oxoalicyclic”, or“oxoaromatic” means an aliphatic, alicyclic, or aromatic, as defined herein, substituted with a carbonyl group. The carbonyl group can be an aldehyde, ketone, ester, amide, acid, or acid halide.

[00145] As used herein, the term,“aromatic” means a moiety wherein the constituent atoms make up an unsaturated ring system, all atoms in the ring system are sp² hybridized and the total number of pi electrons is equal to 4n+2. An aromatic ring canbe such that the ring atoms are only carbon atoms (e.g., aryl) or can include carbon and non-carbon atoms (e.g., heteroaryl).

[00146] As used herein, the term“substituted” refers to independent replacement of one or more (typically 1 , 2, 3, 4, or 5) of the hydrogen atoms on the substituted moiety with substituents independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. In general, a non-hydrogen substituent can be any substituent that can be bound to an atom of the given moiety that is specified to be substituted. Examples of substituents include, but are not limited to, acyl, acylamino, acyloxy, aldehyde, alicyclic, aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene, alkylthios, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl, aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl, arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls

(including ketones, carboxy, carboxylates, CF₃, cyano (CN), cycloalkyl, cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl, heterocyclyl, hydroxy, hydroxy, hydroxyalkyl, imino, iminoketone, ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (including phosphonate and phosphinate), silyl groups, sulfonamido, sulfonyl (including sulfate, sulfamoyl and sulfonate), thiols, and ureido moieties, each of which may optionally also be substituted or unsubstituted. In some cases, two substituents, together with the carbon(s) to which they are attached to, can form a ring.

[00147] The terms“alkoxyl” or“alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, n-propyloxy, iso-propyloxy, n-butyloxy, iso-butyloxy, and the like. An“ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by–O- aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above for alkyl.

[00148] The term“aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[00149] The term“alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term“alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups. [00150] The term“sulfinyl” means the radical—SO—. It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.

[00151] The term“sulfonyl” means the radical—SO₂—. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO₃H), sulfonamides, sulfonate esters, sulfones, and the like.

[00152] The term“thiocarbonyl” means the radical—C(S)—. It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

[00153] As used herein, the term“amino” means -NH₂. The term“alkylamino” means a nitrogen moiety having at least one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen. For example, representative amino groups include —NH₂,—NHCH₃,—N(CH₃)₂,—NH(C₁-C₁₀alkyl),—N(C₁-C₁₀alkyl)₂, and the like. The term “alkylamino” includes“alkenylamino,”“alkynylamino,”“cyclylamino,” and

“heterocyclylamino.” The term“arylamino” means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example—NHaryl, and—N(aryl)₂. The term

“heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example—NHheteroaryl, and—N(heteroaryl)₂. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.

[00154] The term“aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl . For example, an (C₂-C₆) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

[00155] The term“alkoxyalkoxy” means–O-(alkyl)-O-(alkyl), such as–OCH₂CH₂OCH₃, and the like.

[00156] The term“alkoxycarbonyl” means–C(O)O-(alkyl), such as–C(=O)OCH₃,–

C(=O)OCH₂CH₃, and the like. [00157] The term“alkoxyalkyl” means -(alkyl)-O-(alkyl), such as -- CH₂OCH₃,– CH₂OCH₂CH₃, and the like.

[00158] The term“aryloxy” means–O-(aryl), such as–O-phenyl,–O-pyridinyl, and the like.

[00159] The term“arylalkyl” means -(alkyl)-(aryl), such as benzyl (i.e.,–CH₂phenyl),–CH₂- pyrindinyl, and the like.

[00160] The term“arylalkyloxy” means–O-(alkyl)-(aryl), such as–O-benzyl,–O–CH₂- pyridinyl, and the like.

[00161] The term“cycloalkyloxy” means–O-(cycloalkyl), such as–O-cyclohexyl, and the like.

[00162] The term“cycloalkylalkyloxy” means–O-(alkyl)-(cycloalkyl, such as–

OCH₂cyclohexyl, and the like.

[00163] The term“aminoalkoxy” means–O-(alkyl)-NH₂, such as–OCH₂NH₂,–

OCH₂CH₂NH₂, and the like.

[00164] The term“mono- or di-alkylamino” means–NH(alkyl) or–N(alkyl)(alkyl), respectively, such as–NHCH₃,–N(CH₃)₂, and the like.

[00165] The term“mono- or di-alkylaminoalkoxy” means–O-(alkyl)-NH(alkyl) or–O- (alkyl)-N(alkyl)(alkyl), respectively, such as–OCH₂NHCH₃,–OCH₂CH₂N(CH₃)₂, and the like.

[00166] The term“arylamino” means–NH(aryl), such as–NH-phenyl,–NH-pyridinyl, and the like.

[00167] The term“arylalkylamino” means–NH-(alkyl)-(aryl), such as–NH-benzyl,–NHCH₂- pyridinyl, and the like.

[00168] The term“alkylamino” means–NH(alkyl), such as–NHCH₃,–NHCH₂CH₃, and the like.

[00169] The term“cycloalkylamino” means–NH-(cycloalkyl), such as–NH-cyclohexyl, and the like.

[00170] The term“cycloalkylalkylamino”–NH-(alkyl)-(cycloalkyl), such as–NHCH₂- cyclohexyl, and the like.

[00171] It is noted in regard to all of the definitions provided herein that the definitions should be interpreted as being open ended in the sense that further substituents beyond those specified may be included. Hence, a C₁ alkyl indicates that there is one carbon atom but does not indicate what are the substituents on the carbon atom. Hence, a C₁ alkyl comprises methyl (i.e.,—CH3) as well as—CR_aR_bR_c where R_a, R_b, and R_c caneach independently be hydrogen or any other substituent where the atom alpha to the carbon is a heteroatom or cyano. Hence, CF₃, CH₂OH and CH₂CN are all C₁ alkyls.

[00172] Unless otherwise stated, structures depicted herein are meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure except for the replacement of a hydrogen atom by a deuterium or tritium, or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon are within the scope of the invention.

[00173] A“pharmaceutically acceptable salt”, as used herein, is intended to encompass any compound described herein that is utilized in the form of a salt thereof, especially where the salt confers on the compound improved pharmacokinetic properties as compared to the free form of compound or a different salt form of the compound. The pharmaceutically acceptable salt form can also initially confer desirable pharmacokinetic properties on the compound that it did not previously possess, and may even positively affect the pharmacodynamics of the compound with respect to its therapeutic activity in the body. An example of a pharmacokinetic property that can be favorably affected is the manner in which the compound is transported across cell

membranes, which in turn may directly and positively affect the absorption, distribution, biotransformation and excretion of the compound. While the route of administration of the pharmaceutical composition is important, and various anatomical, physiological and pathological factors can critically affect bioavailability, the solubility of the compound is usually dependent upon the character of the particular salt form thereof, which it utilized. One of skill in the art will appreciate that an aqueous solution of the compound will provide the most rapid absorption of the compound into the body of a subject being treated, while lipid solutions and suspensions, as well as solid dosage forms, will result in less rapid absorption of the compound.

[00174] Pharmaceutically acceptable salts include those derived from inorganic acids such as sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. See, for example, Berge et al.,“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19 (1977), the content of which is herein incorporated by reference in its entirety. Exemplary salts also include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, succinate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and

laurylsulphonate salts and the like. Suitable acids which are capable of forming salts with the compounds of the disclosure include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and the like; and organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2- naphthalenesulfonic acid, 3-phenylpropionic acid, 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, 4,4’-mefhylenebis(3-hydroxy-2-ene-l-carboxylic acid), acetic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid,

cyclopentanepropionic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid , naphthalene sulfonic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p- chlorobenzenesulfonic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, tertiary butylacetic acid, trifluoroacetic acid, trimethylacetic acid, and the like. Suitable bases capable of forming salts with the compounds of the disclosure include inorganic bases such as sodium hydroxide, ammonium hydroxide, sodium carbonate, calcium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, N-methylglucamine, pyridine, picoline, dicyclohexylamine, N,N’-dibezylethylenediamine, and the like), and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine, trierhanolamine and the like).

[00175] In some embodiments, the compounds described herein can be in the form of a prodrug. The term“prodrug” as used herein refers to compounds that can be converted via some chemical or physiological process (e.g., enzymatic processes and metabolic hydrolysis) to compound described herein. Thus, the term“prodrug” also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug can be inactive when administered to a subject, i.e. an ester, but is converted in vivo to an active compound, for example, by hydrolysis to the free carboxylic acid or free hydroxyl. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound, as described herein, may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. For example, a compound comprising a hydroxy group can be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Suitable esters that can be converted in vivo into hydroxy compounds include acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, formates, benzoates, maleates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates,

cyclohexylsulfamates, quinates, esters of amino acids, and the like. Similarly, a compound comprising an amine group can be administered as an amide, e.g., acetamide, formamide and benzamide that is converted by hydrolysis in vivo to the amine compound. See Harper,“Drug Latentiation” in Jucker, ed. Progress in Drug Research 4:221-294 (1962); Morozowich et al, “Application of Physical Organic Principles to Prodrug Design” in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977); Bioreversible Carriers in Drug in Drug Design, Theory and Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al.“Prodrug approaches to the improved delivery of peptide drug” in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti et al. (1997) Improvement in peptide bioavailability:

Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998)“The Use of Esters as Prodrugs for Oral Delivery of (3-Lactam antibiotics,” Pharm. Biotech. ll,:345-365; Gaignault et al. (1996)“Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,” Pract. Med. Chem. 671-696; Asgharnejad,“Improving Oral Drug Transport”, in Transport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al.,“Prodrugs for the improvement of drug absorption via different routes of administration”, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko,“Involvement of multiple transporters in the oral absorption of nucleoside analogues”, Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne, “Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1-12 (1997); Bundgaard,

“Bioreversible derivatization of drugs— principle and applicability to improve the therapeutic effects of drugs”, Arch. Pharm. Chemi 86(1): 1-39 (1979); Bundgaard H.“Improved drug delivery by the prodrug approach”, Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. “Prodrugs as a means to improve the delivery of peptide drugs”,Arfv. Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al.“Improved oral drug delivery: solubility limitations overcome by the use of prodrugs”, Arfv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al.“Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81, (1985); Farquhar D, et al.,“Biologically Reversible Phosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983); Freeman S, et al., “Bioreversible Protection for the Phospho Group: Chemical Stability and Bioactivation of Di(4- acetoxy-benzyl) Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun., 875- 877 (1991); Friis and Bundgaard,“Prodrugs of phosphates and phosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives of phosphate- or phosphonate containing drugs masking the negative charges of these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro- drug, molecular structure and percutaneous delivery”, Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977); Nathwani and Wood,“Penicillins: a current review of their clinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993); Sinhababu and Thakker,“Prodrugs of anticancer agents”, Adv. Drug Delivery Rev. 19(2): 241-273 (1996); Stella et al.,“Prodrugs. Do they have advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tan et al.“Development and optimization of anti-HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure-activity relationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3): 117-151 (1999); Taylor,“Improved passive oral drug delivery via prodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino and Borchardt, “Prodrug strategies to enhance the intestinal absorption of peptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treating cephalic HIV infection”, Adv. Drug Delivery Rev.: 39(l-3):63-80 (1999); Waller et al.,“Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989), content of all of which are herein incorporated by reference in its entirety. [00176] The term“protected derivatives” means derivatives of compounds described herein in which a reactive site or sites are blocked with protecting groups. Protected derivatives are useful in the preparation of compounds or in themselves can be active. A comprehensive list of suitable protecting groups can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

[00177] “Isomers” mean any compound having identical molecular formulae but differing in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and stereoisomers that are nonsuperimposable mirror images are termed“enantiomers” or sometimes “optical isomers”. A carbon atom bonded to four nonidentical substituents is termed a“chiral center”. A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of the two enantiomeric forms is termed a“racemic mixture”. A compound that has more than one chiral center has 2^n-1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as ether an individual diastereomers or as a mixture of diastereomers, termed a“diastereomeric mixture”. When one chiral center is present a stereoisomer may be characterized by the absolute configuration of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (e.g., see“Advanced Organic Chemistry”, 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).

[00178] The term“enantiomer” is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable. Other terms used to designate or refer to enantiomers include“stereoisomers” (because of the different arrangement or stereochemistry around the chiral center; although all enantiomers are stereoisomers, not all stereoisomers are enantiomers) or“optical isomers” (because of the optical activity of pure enantiomers, which is the ability of different pure enantiomers to rotate planepolarized light in different directions). Enantiomers generally have identical physical properties, such as melting points and boiling points, and also have identical spectroscopic properties. Enantiomers can differ from each other with respect to their interaction with plane-polarized light and with respect to biological activity.

[00179] The designations“R” and“S” are used to denote the absolute configuration of the molecule about its chiral center(s). The designations may appear as a prefix or as a suffix; they may or may not be separated from the isomer by a hyphen; they may or may not be hyphenated; and they may or may not be surrounded by parentheses.

[00180] The designations or prefixes“(+)” and“(-)” are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) meaning that the compound is levorotatory (rotates to the left). A compound prefixed with (+) is dextrorotatory (rotates to the right).

[00181] The term“racemic mixture,”“racemic compound” or“racemate” refers to a mixture of the two enantiomers of one compound. An ideal racemic mixture is one wherein there is a 50:50 mixture of both enantiomers of a compound such that the optical rotation of the (+) enantiomer cancels out the optical rotation of the (-) enantiomer.

[00182] The term“resolving” or“resolution” when used in reference to a racemic mixture refers to the separation of a racemate into its two enantiomorphic forms (i.e., (+) and (-); 65 (R) and (S) forms). The terms can also refer to enantioselective conversion of one isomer of a racemate to a product.

[00183] The term“enantiomeric excess” or“ee” refers to a reaction product wherein one enantiomer is produced in excess of the other, and is defined for a mixture of (+)- and (-)- enantiomers, with composition given as the mole or weight or volume fraction F(+) and F(-) (where the sum of F(+) and F(-) = 1). The enantiomeric excess is defined as * F(+) -F(-)* and the percent enantiomeric excess by 100x* F(+) -F(-)*. The“purity” of an enantiomer is described by its ee or percent ee value (% ee).

[00184] Whether expressed as a“purified enantiomer” or a“pure enantiomer” or a“resolved enantiomer” or“a compound in enantiomeric excess”, the terms are meant to indicate that the amount of one enantiomer exceeds the amount of the other. Thus, when referring to an enantiomer preparation, both (or either) of the percent of the major enantiomer (e.g. by mole or by weight or by volume) and (or) the percent enantiomeric excess of the major enantiomer may be used to determine whether the preparation represents a purified enantiomer preparation.

[00185] The term“enantiomeric purity” or“enantiomer purity” of an isomer refers to a qualitative or quantitative measure of the purified enantiomer; typically, the measurement is expressed on the basis of ee or enantiomeric excess.

[00186] The terms“substantially purified enantiomer,”“substantially resolved enantiomer” “substantially purified enantiomer preparation” are meant to indicate a preparation (e.g. derived from non-optically active starting material, substrate, or intermediate) wherein one enantiomer has been enriched over the other, and more preferably, wherein the other enantiomer represents less than 20%, more preferably less than 10%, and more preferably less than 5%, and still more preferably, less than 2% of the enantiomer or enantiomer preparation.

[00187] The terms“purified enantiomer,”“resolved enantiomer” and“purified enantiomer preparation” are meant to indicate a preparation (e.g. derived from non-optically active starting material, substrates or intermediates) wherein one enantiomer (for example, the R-enantiomer) is enriched over the other, and more preferably, wherein the other enantiomer (for example the S- enantiomer) represents less than 30%, preferably less than 20%, more preferably less than 10% (e.g. in this particular instance, the R-enantiomer is substantially free of the S-enantiomer), and more preferably less than 5% and still more preferably, less than 2% of the preparation. A purified enantiomer may be synthesized substantially free of the other enantiomer, or a purified enantiomer may be synthesized in a stereo-preferred procedure, followed by separation steps, or a purified enantiomer may be derived from a racemic mixture.

[00188] The term“enantioselectivity,” also called the enantiomeric ratio indicated by the symbol“E,” refers to the selective capacity of an enzyme to generate from a racemic substrate one enantiomer relative to the other in a product racemic mixture; in other words, it is a measure of the ability of the enzyme to distinguish between enantiomers. A nonselective reaction has an E of 1, while resolutions with E's above 20 are generally considered useful for synthesis or resolution. The enantioselectivity resides in a difference in conversion rates between the enantiomers in question. Reaction products are obtained that are enriched in one of the enantiomers; conversely, remaining substrates are enriched in the other enantiomer. For practical purposes it is generally desirable for one of the enantiomers to be obtained in large excess. This is achieved by terminating the conversion process at a certain degree of conversion.

[00189] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms,“patient”,“individual” and“subject” are used interchangeably herein.

[00190] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.

[00191] The terms“disease”,“disorder”, or“condition” are used interchangeably herein, refer to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, affectation.

[00192] As used herein, a“subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms,“patient” and“subject” are used interchangeably herein. The terms,“patient” and“subject” are used interchangeably herein. A subject can be male or female.

[00193] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders associated with autoimmune disease or inflammation. In addition, the methods and compositions described herein can be used to treat domesticated animals and/or pets.

[00194] In some embodiments, the method of treating prostate cancer further comprises diagnosing the subject for prostate cancer before onset of treatment regime or administration of compound of Formula I. [00195] As used herein, the terms "treat,” "treatment," "treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition, disease or disorder, e.g., prostate cancer. The term“treating” is not intended to cure prostate cancer. The term“treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder, e.g., prostate cancer. Treatment is generally“effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective" if the progression of a disease is reduced or halted. That is,“treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. For example, treatment is considered effective if the size of prostate tumor is reduced, or the progression of prostate cancer is halted. The term "treatment" of a disease also includes providing relief from the symptoms or side- effects of the disease (including palliative treatment).

[00196] Some exemplary embodiments of various aspects of the invention disclosed herein can be described by one or more of the following numbered paragraphs:

1. A compound of Formula I:

wherein:

each R¹¹ is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl,

heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; each R¹² is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl,

heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; R¹³ is hydrogen, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, or heteroaryl, each of which can be optionally substituted;

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3,

provided that the compound is not Cycloartocarpin; Cyclocommunol; Cyclocommunin; a compound wherein m is 2, n is 1, each R¹¹ and R¹² is selected from Cl, F, CF₃, methyl, t-butyl, NO₂, OH, OMe or amino, and R¹³ is alkyl or aryl; or a compound wherein m and n are 0 and R¹³ is 2-methylpropenyl or phenyl; or a compound selected from the group consisting of

2. The compound of paragraph 1 , wherein m is 0 and n is 0, m is 0 and n is 1, m is 0 and n is 2, m is 0 and n is 3, m is 1 and n is 0, m is 1 and n is 1, m is 1 and n is 2, m is 1 and n is 3, m is 2 and n is 0, m is 2 and n is 1, m is 2 and n is 2, m is 2 and n is 3, m is 3 and n is 0, m is 3 and n is 1, m is 3 and n is 2, or m is 3 and n is 3

3. The compound of paragraph 1 or 2, wherein m is 0, 1 or 2.

4. The compound of any of paragraphs 1-3, wherein n is 0 or 1.

5. The compound of any of paragraphs 1-4, wherein each R¹¹ is selected independently from alkyl, alkenyl, alkoxy, hydroxyl or halogen.

6. The compound of any of paragraphs 1-5, wherein each R¹¹ is selected independently from methyl, 3-methylbutenyl, hydroxyl, methoxy, Cl or Br. 7. The compound of any of paragraphs 1-6, wherein m is 2 and

(i) one R¹¹ is alkyl and the other R¹¹ is halogen

(ii) one R¹¹ is alkoxy and the other R¹¹ is alkenyl or hydroxyl; or

(iii) one R¹¹ is hydroxyl and the other R¹¹ is alkenyl.

8. The compound of any of paragraphs 1-6, wherein m is 1 and R¹¹ is alkyl, alkenyl,

hydroxyl, alkoxy or halogen.

9. The compound of paragraph 8, wherein R¹¹ is alkyl, hydroxyl, or halogen.

10. The compound of any of paragraphs 1-9, wherein R¹² is hydroxyl.

11. The compound of any of paragraphs 1-10, wherein R¹³ is hydrogen, alkenyl, cyclyl, heterocyclyl, aryl, or heteroaryl.

12. The compound of any of paragraphs 1-11, wherein R¹³ is hydrogen, ethenyl, 2- methylpropenyl, phenyl, formyl, tetrazol-5-yl, 1-morpholinomethanoyl, thiazolidine-2,4- dion-5-yl, or 2,5-dihydrooxazolyl.

13. The compound of any of paragraphs 1-12, wherein the compound is of Formula IA, IB, IC, ID, IE, IF, IG, IH, IJ, IK, IL, IM, IN, or IO:

14. The compound of paragraph 1, wherein the compound is selected from the group consisting of

15. A pharmaceutical composition comprising a compound of any of paragraphs 1-14 and a pharmaceutically acceptable excipient.

16. A method of preparing a compound of Formula I, the method comprising intramolecular cyclization of a compound of Formula II in the presence of a palladium catalyst,

1²

n

wherein:

R¹¹, R¹², R¹³, m and n are as defined for Formula I.

17. The method of paragraph 16, wherein said palladium catalyst is selected from the group consisting of palladium (II) acetate, palladium (II) chloride, palladium

dibenzylideneacetone, dichlorobis(acetonitrile)palladium (II),

dichlorobis(benzonitrile)palladium (II), dichlorodiamine palladium (II), palladium (II) acetylacetonate, palladium (II) bromide, palladium (II) cyanide, palladium (II) iodide, palladium oxide, palladium (II) nitrate hydrate, palladium (II) sulfate dihydrate, palladium (II) trifluoroacetate, tetraamine palladium (II) tetrachloropalladate, tetrakis(acetonitrile)palladium (II) tetrafluoroborate and combinations thereof.

18. The method of paragraph 16 or 17, wherein said palladium catalyst is White Catalyst. 19. The method of any of paragraphs 16-18 wherein said cyclization is in the presence of an oxidizing agent.

20. The method of paragraph 19, wherein the oxidizing agent is benzoquinone.

21. The method of any of paragraphs 16-20, wherein the cyclization is in the presence of an acid.

22. The method of any of paragraphs 16-22, wherein the cyclization is in the presence of acetic acid.

23. A method of increasing activity or expression level of Sirt1, the method comprising contacting a cell with a compound of Formula I.

24. The method of paragraph 23, wherein the contacting is in vivo.

25. The method of paragraph 23, wherein the contacting is in vitro. 26. A method of treating prostate cancer, the method comprising a therapeutically effective amount of a compound of Formula I to a subject in need thereof.

27. The method of paragraph 26, wherein the subject is a mammal.

28. The method of paragraph 27, wherein the mammal is a human.

29. The method of any of paragraphs 26-28, wherein the administering is systemic.

30. The method of any of paragraphs 26-28, wherein the administering is local.

31. Use of a compound of Formula I for the preparation of a medicament for the treatment of prostate cancer.

32. Use of a compound of Formula I for the treatment of prostate cancer.

33. A pharmaceutical composition comprising a compound of Formula I for the treatment of prostate cancer.

[00197] The disclosure is further illustrated by the following examples which should not be construed as limiting. The examples are illustrative only, and are not intended to limit, in any manner, any of the aspects described herein.

EXAMPLES

[00198] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention. Example 1: Synthesis of Tetracyclic Flavonoids via Palladium-Catalyzed Intramolecular Oxidative Cyclization

[00199] Palladium-catalyzed oxidative cyclization of terminal-olefin-tethered phenols was achieved in good yields (61–90% yields). The reaction was optimized and White catalyst {[1,2- bis-(phenylsulfinyl)ethane]palladium(II)acetate} provided the best yield in the presence of benzoquinone as an oxidant. The reaction tolerated both electron-donating and electron- withdrawing substituents on the substrates, and the structure of the tetracyclic flavonoids was confirmed using single-crystal X-ray analysis. The study represents the first example of successful allylic C–H activation– C–O bond formation using terminal-olefin-tethered phenols. [00200] Direct functionalization of ubiquitous C–H bonds is an attractive strategy to efficiently and selectively synthesize biologically relevant compounds. Recent developments in transition-metal-catalyzed activation of inert C–H bonds and subsequent functionalization have dramatically improved the ability to form C–O, C–N, and C–C bonds.¹ Allylic C–H activation is a special subcategory and can be achieved efficiently using palladium(II) catalysis.² Since its introduction, the electrophilic palladium(II) catalysis has been utilized for allylic C–H

oxygenations,³ amina-tions,⁴ alkylations,⁵ and introduction of various other functional groups to monosubstituted olefins.⁶ The methodology is now being efficiently applied to the synthesis of biologically interesting molecules.^3p,7 Allylic C–H activation–C–O bond formation has been achieved mainly using carboxylates as nucleophiles. Wacker-type oxidative intramolecular cyclization employing phenol as nucleophile has been reported using internal di- and

trisubstituted olefins.⁸ This work summarizes the development of intra-molecular allylic C–H activation–C–O bond formation using terminal-alkene-tethered phenols to provide the tetracyclic flavonoid core.

[00201] Prenylated flavonoids isolated from Artocar-pus species particularly those from leaves, bark, and stem possess several useful biological properties including antibacterial, anticancer, antiplatelet, and cytotoxic proper-ties.⁹ Recently, construction of the D ring of flavones and coumarins was reported employing palladium-catalyzed C–H alkenylation followed by C–O bond formation.¹⁰ Retrosynthetically, we proposed the D ring in tetracyclic flavonoids can be formed by palladium-catalyzed intra-molecular allylic alkylation of the corresponding phenol by a C–H activation and C–O bond-formation strategy.

[00202] We synthesized the desired 3-allyl flavones 6a–g in a series of steps from commercially available 2-hydroxy-ace-tophenones (FIG.1). Acetophenones 1a–g were acylated using o-anisoyl or substituted o-anisoyl acid chlorides.¹¹ Baker–Venkataraman rearrangement of 2a–g provided diketones 3a–g in moderate to good yields. C-Allylation of 3a–g with allyl bromide in the presence of K₂CO₃ followed by intramolecular condensation of 4a–g using catalytic H₂SO₄ and AcOH as solvent provided the desired 3-allylflavones 5a–g in excellent yields.¹² Lewis acid catalyzed demethylation of 5a–g using BBr₃ in di-chloromethane afforded the desired precursors 6a–g in excellent yields.

[00203] Next, in order to demonstrate the key cyclization by palladium-catalyzed C–H activation–C–O bond formation, we performed optimization studies on substrate 6b as shown in Table 1. Pd(OAc)₂ (10 mol%) in the presence of benzoquinone (BQ) and AcOH resulted in a clean reaction with 73% yield of the desired product 7b (Table 1, entry 1). Changing the palladium source to Pd(TFA)₂ resulted in complete conversion of the starting material but provided lower yield of 7b and the reaction gave a complex mixture of byproducts (Table 1, entry 2). White catalyst provided excellent yield (86%) in shorter reaction time (Table 1, entry 3). To confirm the role of

benzoquinone, the reaction was performed in the absence of BQ using deoxygenated

dichloromethane. The reaction provided 13% yield of 7b, and starting material was recovered (Table 1, entry 4). The reaction in the absence of AcOH provided 7b in moderate yield but required longer time to complete (Table 1, entry 5). Finally, no reaction was observed without the palladium source (Table 1, entry 6).

[00204] With the above optimized conditions, we next studied the substrate scope of various demethylated flavonoids 6a–g for intramolecular cyclization as shown in Table 2. Unsubstituted flavonoid 6a underwent cyclization smoothly in four hours to provide the desired product 7a in excellent yield (Table 2, entry 1). Substrate with an electron-donating substituent such as methyl on the A ring (6b) also provided the product 7b in excellent yield (Table 2, entry 2). Substrates with a hydroxy substituent on either the A or D ring (6c and 6g) took relatively longer time to undergo cy- clization but provided the desired products 7c and 7g in excellent yields (Table 2, entries 3 and 7). Electron-withdrawing substituents such as Cl and Br on the A ring (6d– f) also underwent cyclization in short reaction time and provided the products 7d–f in good to excellent yields (Table 2, entries 4–6). The regioselectivity of the cyclization was confirmed by single-crystal X-ray analysis of compound 7f (FIG.2).¹⁴

[00205] Without wishing to be bound by a theory, the reaction can proceed via a palladium(II)/palladium(0) catalytic cycle (FIG.3).^3q Palladium(II) promotes allylic C–H cleavage to furnish π-allyl–palladium species II. Stoichiometric BQ serves both as a ligand and an oxidant. BQ first coordinates to complex II and generates the activated complex III. Next, displacement of anionic acetate ligand by phenolic OH results in complex IV which

subsequently undergoes C–O bond formation and the palladium(0) species V is formed. BQ in the presence of AcOH then oxidizes palladium(0) to provide active palladium(II) species to reinitiate the catalytic cycle.

[00206] In summary, we have developed a novel and efficient palladium-catalyzed

intramolecular C–H activation followed by C–O bond formation of 3-allyl flavonoids to afford cy- clized products in good to excellent yields. To our knowledge, this is the first report on

intramolecular allylic oxygenation of terminal olefins using phenols as nucleophiles by employing C–H activation. We are currently developing the asymmetric version of this reaction and its application to complete the total synthesis of naturally occurring tetracyclic flavonoids.

[00207] General Procedure for Compound 7f: A dry two-neck round-bottom flask was charged with White catalyst (0.027 g, 0.053 mmol, 0.2 equiv) and benzoquinone (0.059 g, 0.55 mmol, 2 equiv). Compound 6f (0.09 g, 0.27 mmol, 1 equiv) and AcOH (0.018 g, 0.29 mmol, 1.1 equiv) in CH₂Cl₂ (5 mL) was added dropwise to the above mixture and refluxed for 5 h. To the reaction mixture, sat. NH₄Cl was added, and the mixture was extracted with CH₂Cl₂. The organic phase was washed with brine, dried over Na₂SO₄, and evaporated. The crude product was purified by silica gel column chromatography by eluting with EtOAc–hexanes (5%) to afford 7f (79 mg, 87%).

[00208] Spectral Data: White solid; yield 79 mg (87%); mp 171–172 °C. IR (KBr): 3020 (w), 1635 (s), 1612 (s), 1216 (s), 832 (m), 772 (s) cm–1. 1H NMR (400 MHz, CDCl₃): δ = 2.51 (s, 3 H), 5.16 (dt, J = 10.4, 1.3 Hz, 1 H), 5.34 (dt, J = 17.1, 1.2 Hz, 1 H), 5.99 (ddd, J = 17.1, 10.5, 5.0 Hz, 1 H), 6.15 (dt, J = 5.0, 1.5 Hz, 1 H), 7.01 (dd, J = 8.3, 0.7 Hz, 1 H), 7.07 (td, J = 7.6, 1.0 Hz, 1 H), 7.41 (ddd, J = 8.3, 7.4, 1.7 Hz, 1 H), 7.44 (s, 1 H), 7.78 (dd, J = 7.8, 1.5 Hz, 1 H), 8.16 (s, 1 H). ¹³C NMR (100 MHz, CDCl₃): δ = 20.8, 72.9, 111.9, 115.6, 117.0, 117.7, 119.9, 121.7, 123.2, 123.7, 125.5, 131.9, 134.0 (2×), 142.9, 153.9, 154.9, 156.1, 173.5. ESI-MS: m/z = 325.1 [M + H]+. References for Example 1:

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[00209] Subtype specific and highly potent small molecule activators of Sirt1 enzyme function should inhibit androgen receptor (AR) expression in prostate cancer (PCa) cells and prevent progression to androgen-independent prostate cancer (AIPC). Designing and testing of safe, novel agents that can deacetylate the AR by Sirt1 dependent mechanism are based on computer-aided molecular design and chemical synthesis. Discoveries can not only help develop targeted drugs to treat early-stage PCa recurrence and progression, but should also help validate a hitherto unexplored AR mediated transcription protein complex essential for AIPC progression.

[00210] in-silico drug design is employed and coupled with chemical synthesis to activate deactelyation of AR by Sirt1.

[00211] Sirt1 has been reported to have both oncogenic and tumor suppressor roles in PCa. A true potent activator in in vitro and in vivo settings is used to confirm and validate the tumor suppressor role of Sirt1.

[00212] The role of Sirt1 in regulating AR function in vivo is unknown due to the embryonic lethality of genetic deletion. Using novel Sirt1 point-mutant mice resistant to Sirt1 activators can determine if the designed compounds activate Sirt1 to regulate endogenous AR gene expression and reverse pathophysiology in laboratory PCa models. This represents a previously unexplored transcription complex that plays an important role in progression of PCa to AIPC and it can fulfill a long unmet medical need. Further, using novel genetic models of rodent and human PCa allows for an ideal experimental design to determine Sirt1 activator analogs to treat this disease.

[00213] The lead NSC241011 itself has never been synthesized. A new method to complete the first synthesis of NSC241011 is developed. This method can also be used for efficient and quick synthesis of NSC241011 analogs. [00214] Discoveries can pave the way for the first use of Sirt1 activator for prostate cancer in clinical studies.

[00215] Design and synthesis of new analogs of cycloartocarpin (NSC241011): (1 ) NSC241011 has been identified as a potential Sirt1 activator through in silico screening. in-silico model for NSC241011 bound to Sirt1 -substrate-NAD+ complex is refined and novel analogs are designed. (2) A synthetic route to prepare NSC241011 analogs has been established. Proposed analogs of NSC241011can be synthesized using this route.

[00216] Determination of activity of the new analogs in biological and biophysical assays: (1) NSC241011 inhibits dihydrotestosterone (DHT) induced AR activation and enhances Sirt1- mediated AR inhibition (data not shown). The effects of Sirt1 agonists on AR expression can be determined; (2) The AR is a substrate of NAD-dependent deacetylase, Sirt1, in vitro and in vivo. To determine whether NSC241011 and its analogs affect Sirt1 activity, the de-acetylation of AR is investigated; (3) Sirt1 activation increases mitochondrial numbers and activity. Using highly specific staining for mitochondrial number and membrane potential in PCa cells using flow cytometry and flourometric methods, one can determine whether NSC241011 and its analogs increase Sirt1 mediated mitochondrial biogenesis and activity; (4) NSC241011 inhibits cellular proliferation of LNCaP cells (data not show). Effects of NSC241011 analogs on proliferation of both androgen-independent and androgen-responsive cancer cell lines can be determined; (5) Using nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), one can validate binding of analogs to Sirt1 and determine Kd.

[00217] Evaluation of safety, toxicity and in vivo significance of Sirt1 activators: (1) The five most potent Sirt1 activators can be evaluated for in vitro plasma and microsomes stability. Separately, the compounds can be screened in normal cell cytotoxicity assay (PBMCs) and AMES genotoxicity assay. Finally, in vivo rat pharmacokinetic studies can be conducted to assess preliminary ADME properties of these five potent Sirt1 activators; (2) One can use a novel Sirt1 point-mutant knock-in with targeted insertion of E222K mutation in Exon 3 that renders this model unresponsive to Sirt1 activators. Crossing these mice and WT Sirt1 mice to the Pb (Prostate epithelial cell expressing) Cre and the APC Min+ PCa model will allow one to test the five most potent Sirt1 activators for Sirt1-dependent efficacy on PCa pathophysiology; (3) The AR acetylation site functions as a growth switch of the AR in prostate cancer cells in culture and in nude mice. Sirt1 inhibits AR-mediated prostate cancer cell growth. The functional significance of the five most potent Sirt1 activators in prostate cancer cell growth can be determined in vivo using mice.

[00218] Identification of potential ligands through an in-silico docking. For identifying potential ligands, the reported homology model of human Sirt1 complexed with NAD+ and acetylated lysine (39) was used for the in-silico docking studies. The most potent activator of Sirt1, resveratrol, was used to probe in and around the binding pocket of acetylated lysine to find potential binding pocket (FIG. 4). Since resveratrol is an activator, not an inhibitor, of Sirt1 and a rigid molecule consisting of two aromatic rings with a linker containing a double bond, we assumed the following: 1) the pocket should be closer to the acetylated lysine and stabilize its orientation either with acyl group or/and with the nicotine ring in order to activate the enzyme reaction of Sirt1 and 2) the aromatic rings should be able to stack with other aromatic rings in Sirt1 (aromatic rings do not pack tighter compared to hydrophobic non-aromatic side chains) near the binding site of acetylated lysine. Based on these assumptions, a pocket on Sirt1 formed by a cluster of aromatic and hydrophobic residues F287, P288, P291 and F414 was identified. Resveratrol was docked into this pocket using the docking module of MOE (Molecular

Operating Environment, Chemical Computing Group, Quebec, CA) and the whole complex was energy minimized using the force field (MMFF94x) with a reaction field model for solvation calculations. From the docking position and the orientation of resveratrol, in the binding pocket, critical interacting residues were identified involved in the binding of this compound. Using these ligand-Sirt1 interactions and the size of the resveratrol binding pocket, a 3D stereo- electronic pharmacophore was developed. This pharmacophore contained two aromatic ring systems connected by a linker of 2 to 3 atoms and one of the rings can have OH substitutions in any of the three positions of the ring with respect to the linker. Queries from this pharmacophore were used to identify 100 compounds that matched more than 75% of the features of the queries from the NCI database (http://dtp.cancer.gov/index.html). Molecules tested previously (49) and molecules that had large substitution in the rings potentially interfering with the binding of acetylated lysine or NAD+ were excluded. The molecules were docked into the predicted binding site using resveratrol and 26 potential activators were identified for Sirt1. Of these, twelve compounds were obtained from Developmental Therapeutics Program (DTP) at NCI/NIH (http://dtp.cancer.gov/index.html) and tested in this study. [00219] NSC241011 represses AR function (FIG. 5). AR function is repressed by the Sirt1. Sirt1 expression and function are regulated by DHT suggesting Sirt1 may function as a key regulator of AR function through deacetylation of the AR. NSC115554, NSC118076, and NSC241011 inhibit DHT induced AR activation and enhances Sirt1 mediated AR inhibition. In addition, all three compounds repress AR expression. NSC241011 (C6) was again the most potent compound of the twelve tested.

[00220] NSC241011 inhibits PCa cells from growing in response to DHT (FIG. 6):

Proliferation was assessed using MTT assay. LNCaP, C4W2 or NeuT transformed prostate primary epithelial cell lines were treated with twelve newly identified Sirt1 activator compounds at 50 μM in absence or in presence of DHT (100nM). Compounds, C5 (NSC118076), C6 (NSC241011) and C10 (NSC115554) were identified as three structurally distinct compounds affecting the growth of PCa cells. NSC241011 (C6) was the most potent compound of the twelve tested.

[00221] Reduced Sirt1 expression results in poor prognosis in prostate cancer patients (FIG. 7): In order to determine the predictive value and the biological significance of altered Sirt1 expression in human PCa samples, 154 patient samples were examined. The relative expression of Sirt1 was reduced in patients with poor prognosis (82% vs. 67%, P= 0.012).

[00222] Refinement of the docking of NSC241011 and AR acetylated peptide into the recently available crystal structure of the catalytic domain of Sirt1 complexed with NAD+ : The lead compound NSC241011 was identified based on our homology model of the catalytic domain of Sirt1 complex (39). Recently the 3D coordinates of a crystal structure of the Sirt1 catalytic domain bound to NAD and an inhibitor EX527 analog was made available in the Protein Data Base (PDBID: 4I5I; http://www.rcsb.org). On comparing our homology model with that of the crystal structure, except 10 amino acids in the C-terminal which is not involved in the active site, the root-mean-square deviation was less than 1 Å between the c-alpha atoms of these two structures. Thus, the structural data generated using the homology model can be easily transferred to the recent crystal structure of Sirt1. Since the crystal structure does not have a peptide with K(Ac) bound to the pocket and the binding of an inhibitor analog, the position of NAD+ shifted to interact with the inhibitor. We use the crystal structure of the catalytic domain and transfer the information about the position and orientation of AR peptides (627- GARK(Ac)LKKLG-635) containing the K(Ac), NAD+ and NSC241011 from our homology model. The coordinates of the whole complex can be energy refined using the software, MOE with R-field electrostatic potential. Similarly, the modeling using crystal structure of Sirt1 complexes is repeated for other known activators.

[00223] Identification of pockets available near and around the binding site of AR peptide and NAD+ : After superposing all the Sirt1:NAD+:ARpeptide:activator models, chemical groups that are critical for the activation of deacetylation of AR-peptide can be generated. Available sub-pockets near and around the K(Ac) amino acid in the substrate AR- peptide and the bound lead compounds can be identified. From the stereo-electronic nature of these sub-pockets, the chemical groups that can occupy these sub-pockets and have favorable interactions with Sirt1 and AR peptide can be identified as well.

[00224] In-silico screening of analogs based on the four ring core structure of NSC24011: Using the virtual screening available in MOE, the four ring core of NSC241011 and the chemical groups identified in 1.1.2, around 500 analogs of NSC241011 can be generated and docked into the binding pocket of Sirt1:ARpeptide:NAD+ pocket and energy minimize these 500 complexes. These complexes can be ranked based on a scoring that contains their binding energies and occluded surface area to account for solvation. 100 analogs of the top ranking complexes are selected and approximately 40 potential analogs are visually select for feasibility of chemical synthesis. Analogs can be carefully selected such that the analogs do not block with the side chain of the (+2) position of the acetylated AR-peptide.

[00225] NSC241011 (also called as cycloartocarpin) is a prenylated flavonoid first isolated from Artocarpus integrifolia in 1962 (50) and subsequently assigned the correct structure in 1964 (51). Flavonoids isolated from Artocarpus species particularly those from leaves, bark, stem and fruit possess several useful biological properties including antibacterial, antitubercular, antiviral, antifungal, antiplatelet, antiarthritic, tyrosinase inhibitory and cytotoxicity (52). No total synthesis or systematic structure-activity relationships for NSC241011 have been reported to date. This proposal aims to synthesize NSC241011 analogs to improve binding with AR.

[00226] We have established a synthetic route to prepare NSC241011 analogs. The synthesis begins with commercially available acetophenones and in five previously reported steps is converted to the phenolic intermediate 6 in high yields (53-55). The key reaction then involves cyclization of the intermediate 6 using a selective CH activation method (56). The synthesis is completed by alkene-metathesis using Grubb’s catalyst (57). The synthetic scheme is outlined below in FIG. 8. The sequence has a large substrate scope and tolerates a variety of substituents (R₁ and R₂ in FIG. 8).

[00227] AR activity plays a prominent role in the pathogenesis of PCa. Androgen ablation therapy effectively inhibits tumor cell growth for many patients, however most of these tumors re-grow despite castrate levels of androgens, accompanied by hyperactivated AR. AR activity is inhibited by the NAD-dependent histone deacetylase, Sirt1. Sirt1 also inhibits the activity of most AR mutations that arise in patients with PCa. The role of Sirt1 in regulating AR function in vivo is unknown due to the embryonic lethality of genetic deletion. These studies can determine if the designed compounds activate Sirt1 to regulate endogenous AR gene expression. This constitutes a novel approach to the inhibition of AR mutations that arise in patients with PCa and enables the identification of better therapies for these patients.

[00228] PCa cell lines can be used to identify effects of NSC241011 analogs on AR activation and Sirt1 mediated AR inhibition. LNCaP cells are transfected with pcDNA3, hSirt1, hSirt1 E223K activationresistant mutant and the PSA-LUC. The cells are then be incubated with vehicle or physiological concentrations of DHT and NSC241011 analogs. The DHT induced fold change of PSA-LUC is then measured.

[00229] Effects of NSC241011 analogs on ligand-induced AR expression are measured. Western blotting of endogenous AR abundance, AR acetylation and histone acetylation in LNCaP cell-treated with vehicle, DHT or NSC241011 analogs is performed. The cell lysates are blotted with AR antibody (AR-H280) and with anti-acetylated-lysine after immunoprecipitation with the AR antibody as previously described (14).

[00230] Effects of NSC241011 analogs on AR recruitment to androgen response element (ARE) are examined using chromatin immunoprecipitation assay (ChIP assay). HEK293T cells are transfected with FLAG-ARwt and ligand-induced recruitment of the AR to the ARE of the PSA promoter in response to the ligand DHT, the histone deacetylase inhibitor TSA, the protein kinase inhibitor H89 or NSC241011 analogs are studied as previously described (41).

[00231] The AR is a substrate of Sirt1 NAD-dependent deacetylase activity in vitro and in vivo. The peptide studies and homology model structures of Sirt1-AR“KLKK”peptide-NAD+ complex, demonstrated that the acetylated lysine residue at K630 of the AR is the preferential substrate for Sirt1 enzyme activity. The effect of NSC241011 and its analogs on deacetylation of the AR within PCa cells can be determined. [00232] The AR triacetylated peptide corresponding to AR 630-638 (KAcLKAcKAcLGNLK- ) are incubated in presence of NAD+ with NSC241011 or its analogs and subsequently injected onto a C18 column and analyzed using LC/MS for monodeacetylated and di-deacetylated products. NSC241011 analogs that promote de-acetylation of AR 630-638 in an NAD+ - dependent manner can be identified.

[00233] Fluor de Lys kit (AK-555; Biomol) are used to assess deacetylation of p53. The assay uses a fluorogenic peptide encompassing residues 379 to 382 of p53, acetylated on lysine 382 (KI-177; Biomol). The acetylated lysine residue is coupled to an aminomethylcoumarin moiety. The peptide when deacetylated followed by the addition of a proteolytic developer releases the fluorescent aminomethylcoumarin which can then be measured using fluorescence spectroscopy. As previously shown, the Sirt1 point-mutant E223K is resistant to chemically induced activation and resulting deacetylation of p53 (40). Using this novel genetic and biochemical approach, NSC241011 analogs that promote de-acetylation of p53 can be identified.

[00234] To determine Sirt1 activation of new analogs on sirtuin-mediated mitochondrial biogenesis and membrane potential. Sirt1 activation increases mitochondrial numbers and activity (58). Using highly specific staining for mitochondrial number and membrane potential in PCa cells using flow cytometry, it can be determined whether NSC241011 and its analogs increase Sirt1 mediated mitochondrial biogenesis and activity. LNCaP cells are transfected with Sirt1-IRESGFP, chemical resistant Sirt1 E223K-IRESGFP or MSCV-IRESGFP control vectors. GFP- positive cells are sorted and stained for markers of mitochondrial number (MitoTracker, Deep Read and NAO) and mitochondrial membrane potential using TMRM and quantified using fluorometric methods.

[00235] Experiments are conducted in PCa cell lines (LNCaP). LNCaP cells are infected with MSCV-Sirt1-IRESGFP or MSCV-IRESGFP control vector. The GFP positive cells are isolated by FACS sorting and treated with DHT, TSA and NAD for 24h. The effect of NSC241011 and its analogs on AR acetylation are determined by immunoprecipitation of AR by anti-AR antibody and subsequent western blotting of anti-acetylated lysine antibody. NSC241011 analogs that attenuate AR acetylation in LNCaP cells in an NAD-dependent manner can be identified.

[00236] The AR acetylation site functions as a growth switch of the AR in PCa cells in culture and in nude mice. Sirt1 inhibits AR-mediated prostate cellular growth. The functional significance of NSC241011 analogs on prostate cancer cell growth can be determined. [00237] Effects of NSC241011 analogs on cellular proliferation of LNCaP, PC3, DU145, C4W2 and NeuT-transformed prostate primary epithelial cells are studied using thiazolyl blue (MTT) assay. Cells are treated with DHT or vehicle in combination with NSC241011 analogs. The cellular proliferation is measured using OD at 560 nm. Dose response curves for selected analogs are obtained.

[00238] Colony formation using LNCaP cells are assessed in presence or absence of DHT. LNCaP cells are seeded at low density onto 35 mm agar plates and incubated with vehicle or NSC241011 analogs. The sizes and numbers of GFP-positive colonies on soft agar plates are scored.

[00239] Surface Plasmon Resonance (SPR) Binding Studies of NSC241011 and its Analogs to AR-triacetylated Peptide Substrates: SPR experiments are performed using a Biacore 3000 instrument (GE Healthcare). Two AR triacetylated peptides corresponding to AR 630-638 (KAcLKAcKAcLGNLK-) peptides are used in this study. These peptides have identical amino acid sequence and differ only in the TAMRA group. The peptides are captured onto a neutravidin surface prepared through standard amine coupling to a CM5 sensor (GE Healthcare) and analogs of NSC241011 are injected over the surfaces from 3 to 50 µM. Equilibrium binding parameters are obtained from global fits of the data to a saturation binding model.

[00240] Isothermal Titration Calorimetry (ITC) Binding Studies of NSC241011 Analogs to Sirt1 Peptide Substrate Complex: ITC experiments are performed as previously reported (59). The TAMRA-AR 630-638 peptide or native AR 630-638 peptide are dissolved in 50 mM Tris- HCl buffer and added to Sirt1 to a final concentration of 1 mM. Titrations in presence or absence of NSC241011 analogs are performed. All titrations are performed in a Microcal VP-ITC (GE Healthcare) at 25 °C. The data are fit to a simple 1:1 interaction model in Origin ITC.

[00241] NMR Binding Studies of NSC241011 Analogs to AR-630-638 Peptide Substrates: NMR chemical shift perturbation of the AR-630-638 peptide substrates are used to monitor the molecular interaction of NSC241011 analogs in the absence of Sirt1 enzyme. NMR binding experiments are also carried out TAMRA-AR 630-638 peptide substrates. ¹H NMR spectra can be recorded on a 400-MHz Bruker Ascend spectrometer at 25 °C.

[00242] Five most potent Sirt1 activators are evaluated in the following assays: a) Plasma Stability Assay: Test compounds and controls (propranolol) are tested at 1000 nM in

mouse/human plasma (Bioreclamation LLC, Westbury, NY) at 37 ºC. Samples are removed and analyzed at various time points using LC-MS/MS. Half-life are determined by the slope of the line for the percent remaining parent compound over time course. (60) b) Microsome Stability Assay: Test compounds and controls (propranolol) are tested at 500 nM in pooled mouse and human liver microsomes (BD Biosciences, Woburn, MA). Using a 96-well plate, the reaction is initiated by adding NADPH (final concentration of 1 mg/mL) and subsequently stopped by adding acetonitrile at designated time points and analyzed by LC-MS/MS (60). c) Normal Cell Cytotoxicity Assay: Human PBMC cells (Zenbio, RTP, NC) are incubated with test compounds and subsequently treated with MTT. The cellular toxicity is measured using OD at 560 nm. d) AMES Genotoxicity Assay: Five different concentrations of each test compound are assessed using the strain, Salmonella typhimurium TA 98, in the presence or absence of S9 mix from rat liver, using EBPI Test kit (EBPI, Canada) (61). e) In vivo Rat Pharmacokinetic Studies:

Sprague-Dawley rats are administered the test compounds through oral gavage and the blood samples are withdrawn from the tail at the pre-determined time periods after the administration. The plasma samples are analyzed by HPLC and the PK parameters are calculated by the noncompartmental analysis (62).

[00243] To determine the effects of Sirt1 activators on a rodent PCa model with and without genetic manipulation designed to resist Sirt1 analogs. We have recently developed a novel Sirt1 point mutant knock-in mouse designed to be resistant to chemical Sirt1 activation (40). Targeted insertion of E222K mutation into Exon 3 renders this model unresponsive to Sirt1 activators (FIG. 9). Crossing these mice and WT Sirt1 mice to the Pb (Prostate epithelial cell expressing) Cre/APC Min+ PCa model permits the testing of the 5 most potent Sirt1 activators for Sirt1- dependent efficacy on PCa progression.

[00244] The functional significance of five most potent Sirt1 activators in prostate cancer cell growth can be determined in vivo using SCID mice (63). Five- to nine-week old male CB17 SCID mice (Charles River Laboratories, Hollister, CA) are castrated and allowed to recover for an additional 5 days before inoculation with tumor cells. LNCaP cells co-expressing exogenous AR and the AR-dependent reporter construct are used to generate a xenograft model of human prostate cancer. Tumor growth are monitored to the volume of 100 mm3 and subsequently animals are treated with the two Sirt1 activators developed in this study. Body weight and tumor volumes are measured. Each group of mice (n = 8) is treated daily for 28 consecutive days with 1, 10, or 50 mg/kg NSC241011 analogs, vehicle control, or 50 mg/kg bicalutamide. At the end of the treatment period or when tumor volume exceeds 1,000 mm³, animals are euthanized and tissue samples are collected for analysis according to the procedures approved by the

institutional Animal Care committee.

[00245] Statistical analyses for all the experiments are performed using the Mann-Whitney U test and p<0.05 is considered statistically significant. Examples of statistical analyses are presented in FIG. 5A and FIG. 7. References for Example 2:

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62. Gibaldi M. and Perrier, D., 1982. Pharmacokinetics, 2nd Ed., Marcel Dekker, New York and Basel 63. Guerrero, J., Alfaro, I. E., Gomez, F., Protter, A. A. and Bernales, S. (2013) Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer. Prostate 73(12): 1291-305. [00246] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00247] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

Claims

What is claimed is: 1. A compound of Formula I:

wherein:

each R¹¹ is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl,

heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; each R¹² is independently alkyl, alkenyl, alkoxy, hydroxyl, halogen, acyl, cyclyl,

heterocyclyl, aryl, heteroaryl, amino, alkylamino, CF₃, nitro, cyano, alkylthio, sulfonyl, sulfonyl, CO₂H, or O-acyl, each of which can be optionally substituted; R¹³ is hydrogen, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, or heteroaryl, each of which can be optionally substituted;

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3,

provided that the compound is not Cycloartocarpin; Cyclocommunol; Cyclocommunin; a compound wherein m is 2, n is 1, each R¹¹ and R¹² is selected from Cl, F, CF₃, methyl, t-butyl, NO₂, OH, OMe or amino, and R¹³ is alkyl or aryl; or a compound wherein m and n are 0 and R¹³ is 2-methylpropenyl or phenyl; or a compound selected from the group consisting of

2. The compound of claim 1, wherein m is 0 and n is 0, m is 0 and n is 1 , m is 0 and n is 2, m is 0 and n is 3, m is 1 and n is 0, m is 1 and n is 1, m is 1 and n is 2, m is 1 and n is 3, m is 2 and n is 0, m is 2 and n is 1, m is 2 and n is 2, m is 2 and n is 3, m is 3 and n is 0, m is 3 and n is 1, m is 3 and n is 2, or m is 3 and n is 3

3. The compound of claim 1 or 2, wherein m is 0, 1 or 2.

4. The compound of any of claims 1-3, wherein n is 0 or 1.

5. The compound of any of claims 1-4, wherein each R¹¹ is selected independently from alkyl, alkenyl, alkoxy, hydroxyl or halogen.

6. The compound of any of claims 1-5, wherein each R¹¹ is selected independently from methyl, 3-methylbutenyl, hydroxyl, methoxy, Cl or Br.

7. The compound of any of claims 1-6, wherein m is 2 and

(iv) one R¹¹ is alkyl and the other R¹¹ is halogen

(v) one R¹¹ is alkoxy and the other R¹¹ is alkenyl or hydroxyl; or

(vi) one R¹¹ is hydroxyl and the other R¹¹ is alkenyl.

8. The compound of any of claims 1-6, wherein m is 1 and R¹¹ is alkyl, alkenyl, hydroxyl, alkoxy or halogen.

9. The compound of claim 8, wherein R¹¹ is alkyl, hydroxyl, or halogen.

10. The compound of any of claims 1-9, wherein R¹² is hydroxyl.

11. The compound of any of claims 1-10, wherein R¹³ is hydrogen, alkenyl, cyclyl, heterocyclyl, aryl, or heteroaryl.

12. The compound of any of claims 1-11, wherein R¹³ is hydrogen, ethenyl, 2- methylpropenyl, phenyl, formyl, tetrazol-5-yl, 1-morpholinomethanoyl, thiazolidine-2,4- dion-5-yl, or 2,5-dihydrooxazolyl.

13. The compound of any of claims 1-12, wherein the compound is of Formula IA, IB, IC, ID, IE, IF, IG, IH, IJ, IK, IL, IM, IN, or IO:

14. The compound of claim 1, wherein the compound is selected from the group consisting of

15. A

pharmaceutically acceptable excipient.

16. A method of preparing a compound of Formula I, the method comprising intramolecular cyclization of a compound of Formula II in the presence of a palladium catalyst,

wherein:

R¹¹, R¹², R¹³, m and n are as defined for Formula I.

17. The method of claim 16, wherein said palladium catalyst is selected from the group

consisting of palladium (II) acetate, palladium (II) chloride, palladium

dibenzylideneacetone, dichlorobis(acetonitrile)palladium (II),

dichlorobis(benzonitrile)palladium (II), dichlorodiamine palladium (II), palladium (II) acetylacetonate, palladium (II) bromide, palladium (II) cyanide, palladium (II) iodide, palladium oxide, palladium (II) nitrate hydrate, palladium (II) sulfate dihydrate, palladium (II) trifluoroacetate, tetraamine palladium (II) tetrachloropalladate, tetrakis(acetonitrile)palladium (II) tetrafluoroborate and combinations thereof.

18. The method of claim 16 or 17, wherein said palladium catalyst is White Catalyst.

19. The method of any of claims 16-18 wherein said cyclization is in the presence of an oxidizing agent.

20. The method of claim 19, wherein the oxidizing agent is benzoquinone.

21. The method of any of claims 16-20, wherein the cyclization is in the presence of an acid.

22. The method of any of claims 16-22, wherein the cyclization is in the presence of acetic acid.

23. A method of increasing activity or expression level of Sirt1, the method comprising

contacting a cell with a compound of Formula I.

24. The method of claim 23, wherein the contacting is in vivo.

25. The method of claim 23, wherein the contacting is in vitro.

26. A method of treating prostate cancer, the method comprising a therapeutically effective amount of a compound of Formula I to a subject in need thereof.

27. The method of claim 26, wherein the subject is a mammal.

28. The method of claim 27, wherein the mammal is a human.

29. The method of any of claims 26-28, wherein the administering is systemic.

30. The method of any of claims 26-28, wherein the administering is local.

31. Use of a compound of Formula I for the preparation of a medicament for the treatment of prostate cancer.

32. Use of a compound of Formula I for the treatment of prostate cancer.

33. A pharmaceutical composition comprising a compound of Formula I for the treatment of prostate cancer.