WO2013028866A1 - Therapeutic compounds and methods - Google Patents

Abstract

The invention provides a method for treating or preventing cancer in an animal comprising administering (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof to the animal.

Description

3 -CHLOROACETYLINDOLE FOR MEDICAL THERAPY

Priority of Invention

This application claims priority to United States Provisional Application

Number 61/527,019, filed 24 August 2011. The entire content of this provisional application is hereby incorporated herein by reference.

Government Funding

The invention described herein was made with government support under Grant Numbers R37 CA081064, CA120388, ES016548, CA0227501, and

HHSN261200533001C-NO1-CN-53301, all awarded by The National Institutes of Health. The United States Government has certain rights in the invention.

Background of the Invention

Indole-3-carbinol (13 C) is produced in Brassica vegetables such as broccoli and cabbage and has been shown to inhibit proliferation and induce apoptosis in various cancer cells, including breast, prostate, colon, and leukemia. However, only high doses of 13 C were shown to inhibit cell proliferation (IC50 = 200-300 μΜ).

Currently there is a need for agents that are useful for treating or preventing cancer.

Summary of the Invention

A more potent antitumor agent was prepared by modifying the structure of 13 C. The new compound (3-chloroacetyl)indole (3CAI) more strongly inhibited colon cancer cell growth compared to I3C. Additionally, in screening against 85 kinases in a competitive kinase assay, 3CAI was identified as a specific AKT inhibitor.

AKT is a serine/threonine kinase that plays a pivotal role in promoting transformation and chemoresistance by inducing proliferation and inhibiting apoptosis. Therefore, AKT is regarded as a critical target for cancer therapy. 3ICA, has been found to be a potent and specific AKT inhibitor. This compound showed significant inhibition of AKT in an in vitro kinase assay and suppressed expression of AKT direct downstream targets such as mTOR and GSK3P as well as induced growth inhibition and apoptosis in colon cancer cells. Additionally, oral

administration of this potent AKT inhibitor suppressed cancer cell growth in an in vivo xenograft mouse model.

Accordingly the invention provides a method for treating or preventing cancer in an animal comprising administering (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof to the animal. The invention also provides a method for suppressing cancer cell growth comprising contacting (in vitro or in vivo) a cancer cell with (3-chloroacetyl)indole or a salt thereof.

The invention also provides a method for treating or preventing a pathological AKT mediated condition in an animal comprising administering (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof to the animal.

The invention also provides a pharmaceutical composition comprising

(3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

The invention also provides a method for inhibiting AKT in a cell, comprising contacting the cell in vitro or in vivo with an effective amount of (3-chloroacetyl)indole or a salt thereof.

The invention also provides (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of cancer.

The invention also provides (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of a pathological AKT mediated condition.

The invention also provides (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in medical therapy.

The invention also provides the use of (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for the treatment of or prevention of cancer in an animal, such as a human.

The invention also provides the use of (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for the treatment of or prevention of a pathological AKT mediated condition in an animal, such as a human.

The invention also provides processes and intermediates disclosed herein that are useful for preparing (3-chloroacetyl)indole or salts thereof.

Brief Description of the Figures

Figure 1: Anti-cancer activity of 13 C derivatives. (A) Structure of indole-3-carbinol (13 C) and (B) 4 derivatives. (C) 3CAI derivatives inhibit proliferation of colon cancer cells. Cells were treated with I3C or derivatives (#1, #2, #3 or #4) for 48 hours and harvested. Proliferation was analyzed using the MTS assay. The asterisk indicates a significant decrease in proliferation compared to untreated control. (D) 13 C and derivatives inhibit anchorage independent cell growth. Cells were incubated in 0.3% agar for 3 weeks with I3C or derivatives (#1, #2, #3 or #4). Colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. Data are represented as means ± S.D. of values from triplicates and similar results were obtained from two independent experiments. The asterisk (*) indicates a significant (p < 0.05) decrease in colony formation induced by DC or its derivatives (#1, #2 or #3) compared to untreated control.

Figure 2: Effect of (3-chloroacetyl)indol (3CAI) on AKT activity. (A) 3CAI suppresses

AKT1 kinase activity in vitro. The effect of 3CAI on AKT1, MEK1, JNK1, ERK1 or TOPK activity was assessed by an in vitro kinase assay using AKT1 (active, 100 ng), histone H2B (AKT substrate, 500 ng), MEK1 (active, 400 ng), inactive ERK2 (MEK1 substrate, 500 ng), JNK1 (active, 50 ng), c-Jun (JNK1 substrate, 500 ng), ERK1 (active, 400 ng), inactive RSK2 (ERK1 substrate, 500 ng), TOPK (active, 500 ng) or histone H2 AX (TOPK substrate, 500 ng) with

[γ-³²Ρ]ΑΤΡ. (B) 3CAI inhibits PI3K kinase activity at the highest concentration in vitro. The inhibitory effect of 3CAI or LY294002 as a PI3 inhibitor on PI3K activity was assessed by an in vitro kinase assay. The conversion of PIP4 to PIP3 was determined by autoradiography. (C) 3CAI substantially inhibits AKT1 or (D) AKT2 kinase activity in a dose-dependent manner. The inhibitory effect of 3CAI, 13 C or an AKT inhibitor VIII on AKT1 or AKT2 activity was assessed by an in vitro kinase assay. The ³²P-labeled substrate was visualized by autoradiography. Band density was measured using the image J program. All data are represented as means ± S.D. of values from three independent experiments. The asterisk (*) indicates a significant (p < 0.05) decrease caused by 3CAI, LY294002 or AKT inhibitor VIII compared to untreated control.

Figure 3: Illustrates computer modeling of 3CAI and AKT 1/2; (A) Binding modes of

3CAI with PH domains of AKT1 and 2; (A-a) binding mode of 3CAI with the AKT1 PH domain; (A-b) binding mode of 3CAI with the AKT2 PH domain; In the full images of AKT1/2 and 3CAI (A-c, A-d), 3CAI is represented as spheres and the carbon atoms are colored white. In the images of the binding site, 3CAI is represented as sticks; (B) Docked conformation of 3CAI compared with Ins(l,3,4,5)P₄ from 1UNQ. Left: docked conformation of 3CAI with the AKT1 PH domain; Right: docked conformation of 3CAI with the AKT2 PH domain. The binding sites are represented as surfaces and the ligands are represented as sticks.

Figure 4: 3CAI directly binds to AKT1 or 2 in an ATP non-competitive manner. (A) 3CAI directly binds to AKT1 or (B) AKT2 in an ATP non-competitive manner. Recombinant AKT1 (200 ng) was incubated with 3CAI- or DC-conjugated Sepharose 4B beads, or with Sepharose 4B beads alone. The pulled down proteins were analyzed by Western blotting. (C) 3CAI directly binds to endogenous AKT1 and (D) AKT2. An HCT116 colon cancer cell lysate (500 μg) was incubated with 3CAI- or BC-conjugated Sepharose 4B beads, or with Sepharose 4B beads alone, and then the pulled down proteins were analyzed by Western blotting. Similar results were obtained from two independent experiments.

Figure 5: Effect of 3CAI on the AKT signaling pathway. (A) 3CAI inhibits

AKT-target proteins in HCTl 16 colon cancer cells. Cells were treated with 3CAI, 13 C, or an AKT inhibitor, and then harvested at various times (0.5, 1 and 3 h). (B) 3CAI regulates pro- or anti-apoptotic proteins in HCTl 16 colon cancer cells. Cells were treated with 3CAI or I3C, and then harvested at various times (6, 12 and 24 h). The cells were immunoblotted with antibodies to detect GSKp, p-GSK3p (Ser9), mTOR, p-mTOR (Ser2448), AKT, p-AKT (Thr308), p53, p21 , Bcl2, Bad, ASKl(Ser83) and β-actin. β-Actin was used to verify equivalent loading of protein. Band density and ratio (phosphorylation/total protein) was measured using the Image J software program. Similar results were obtained from two independent experiments. (C) 3CAI induces apoptosis in colon cancer cells. HCTl 16 or HCT29 colon cancer cells were seeded with 3CAI, I3C or AKT inhibitor in 1% FBS and medium and then incubated for 4 days. Cells were stained with annexin V and propidium iodide (PI) and apoptosis was determined by Fluorescence Activated Cell Sorting (FACS). The asterisk (*) indicates a significant difference (p < 0.05) between untreated controls and treated cells.

Figure 6: Athymic nude mice were inoculated in the right flank with HCTl 16 cells. Tumors were allowed to grow to an average of 40 mm3 and then mice were divided into 3 equal groups with the same average tumor volume. Treatment was initiated on Day 8 and continued to Day 20. The asterisk (*) indicates that the tumors from the vehicle-treated group were significantly larger in volume than the group treated with 10 mg/kg (3-chloroacetyl)indole (CAI). The two asterisks (**) indicate that tumors from the vehicle-treated group were larger than those in either of the other two groups. This result suggests that CAI could be an effective therapeutic against colon cancer cell growth.

Detailed Description

In cases where compounds are sufficiently basic or acidic, a salt of a compound can be useful as an intermediate for isolating or purifying the compound. Additionally, administration of a compound as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

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

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

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

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

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

microorganisms.

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

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

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

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

Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

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

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of

administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

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

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

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

The biological activity (e.g. anti-cancer properties) of a compound can be evaluated using assays that are known or using the assays described in the Examples below.

The invention will now be illustrated by the following non-limiting Example.

Example

Example 1

Materials and Methods

Reagents

I3C (purity: 95%) was purchased from Sigma-Aldrich (St Louis, MO). 3CAI (purity: 95%), 5-methoxy-3CAI (purity: 95%), 5-fluoro-3CAI (purity: 95%) and

2-(4-(2-hydroxyethyl)piperazin-l-yl)-l-(5-methoxy-lH-indol-3-yl)ethanone) (purity: 95%) were purchased from InterBioScreen (Moscow, Russia). CNBr-Sepharose 4B beads were purchased from GE Healthcare (Piscataway, NJ). The active AKTs, active MEKl , active JNKl , active ERKl human recombinant protein, histone H2B and H2 AX for kinase assays were purchased from Millipore (Temecula, CA). The active TOPK human recombinant protein for the kinase assay was purchased from SignalChem (Richmond, BC). PI3K was obtained from Upstate Biotechnology (Lake placid, NY). AKT, p-AKT (Thr308), mTOR, p-mTOR (Ser2448), GSKp, p-GSK3p D(Ser9), Bad, Bcl2 and p-ASKl (Ser83) and CDKNIA antibodies were purchased from Cell

Signaling Technology (Beverly, MA). Antibodies to detect p53 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 was purchased from Gibco BRL (Grand Island, NY). AKT inhibitor VIII was purchased from Merck KGaA (Darmstadt, Germany). Synthesis of3CAI

3CAI (purity: 95%) was synthesized as described (Sudipta Roy SH and Gordon W.

Gribble, Synthesis, 2006, 23, 3948-54) and purity and structure were analyzed and verified using HPLC and NMR.

Cell culture

Cells were purchased from American Type Culture Collection (ATCC; Manassas, VA). HCT116 and HT29 human colon cancer cells were cultured in McCoy's 5 A medium

supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.

Molecular modeling

The crystal structure of the pleckstrin homology (PH) domain of AKTl was obtained from the RCSB Protein Data Bank, PDB entry 1UNQ (Milburn CC, et al., Biochem J, 2003, 75, 531 -8), which is a complex structure of the AKTl PH domain and Ins(l,3,4,5)P₄ and has an atomic resolution of 0.98 A. The crystal structure was prepared using the Protein Preparation Wizard in Maestro v.9.2. Hydrogens were added to the protein structure consistent with a pH of 7. All water molecules in the crystal structure were removed. Then the crystal structure was minimized with an RMSD cutoff value of 0.3 A. The structure of the AKT2 PH domain used in this study was modeled with the template structure of 1UNQ using Prime v.3.0. Energy grids for docking were computed for each protein structure using default settings in Glide v.5.7. 3CAI was prepared using LigPrep v.2.5 and then was docked into the PH domains of AKTl and 2 with Glide extra precision (XP) mode.

Anchorage independent cell growth

Cells (8 10 per well) suspended in complete growth medium (McCoy's 5 A

supplemented with 10% FBS and 1% antibiotics) were added to 0.6% agar with different doses of each compound in a base layer and a top layer of 0.3% agar. The cultures were maintained at 37°C in a 5% C0₂ incubator for 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v. 4) program (Media Cybernetics).

Western blot analysis

Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl pH 7.4, 1 % NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 protease inhibitor tablet). Equal amounts of protein were determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. After washing with PBS containing 0.1% Tween 20, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody at 1 : 5,000 dilution and the signal detected with a chemiluminescence reagent (Amersham Biosciences Corp).

In vitro pull-down assay

Recombinant human AKTs (200 ng) were incubated with 3CAI-Sepharose 4B (or Sepharose 4B only as a control) beads (50 μΐ, 50% slurry) in reaction buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 μg/mL bovine serum albumin). After incubation with gentle rocking overnight at 4°C, the beads were washed 5 times with buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40) and binding was visualized by Western blotting.

Cell proliferation assay

Cells were seeded (1 10 cells per well) in 96- well plates and incubated for 24 h and then treated with different doses of each compound. After incubation for 48 h, 20 μΐ of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% C0₂ incubator. Absorbance was measured at 492 nm.

Apoptosis assay

Colon cancer cells were plated into 60-mm culture dishes (1 10⁵ cells/dish) and incubated for 1 day in medium containing 10% FBS. The culture medium was then replaced with a 1% serum medium and cultured for 4 days with 3CAI, I3C or a commercial AKT inhibitor. The cells were collected by trypsinization and washed with phosphate buffered saline (PBS). The cells were resuspended in 200 μΐ of binding buffer. Annexin V staining was accomplished following the product instructions (Clontech, Palo Alto, CA). The cells were observed under a fluorescence microscope using a dual filter set for FITC and propidium iodide and then analyzed by flow cytometry. In vitro kinase assay

The kinase assay was performed in accordance with instructions provided by Upstate Biotechnology (Billerica, MA). Briefly, the reaction was carried out in the presence of 10 μθί of [γ-³²Ρ]ΑΤΡ with each compound in 40 μΐ of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl₂, 10 mM MnCl₂, and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 μΐ protein loading buffer and the mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relative amounts of incorporated radioactivity were assessed by autoradiography. Hematoxylin-eosin staining and immunohistochemistry

Tumor tissues from mice were embedded in a paraffin block and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. Tumor tissues were de-paraffinized and hydrated then permeabilized with 0.5% Triton X- 100/1 PBS for 10 min, hybridized with Ki-67 (1 :500) as the primary antibody and horse-radish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG antibody was used as secondary antibody. After developing with 3,

3'-diaminobenzidine, the sections were counterstained with H&E. All sections were observed by microscope and the Image-Pro Plus software (v. 4) program (Media Cybernetics).

Xenograft mouse model

Athymic nude mice (6 week old nu/nu female mice, Harlan Laboratory, Minneapolis, MN) were inoculated in the right flank with HCT116 cells (1.5*10⁶ cells/mouse). Mice were maintained under "specific pathogen-free" conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Tumors were allowed to grow to an average of -40 mm and then mice were divided into 3 equal groups with the same average tumor volume (n=T0 of each group; group 1 = vehicle only; group 2 = 3CAI at 1 mg/kg; group 3 = 3CAI at 10 mg/kg). Treatment with 3CAI was initiated on Day 8 and continued to Day 20 (~2weeks) and was administered by intraperitoneal injection (i.p.) 3 times a week. Tumor volume was measured 3 times a week and body weight was measured 2 times a week. The 3CAI compound was prepared in 10% tween-20 in 90% D.D.W. The tumors from the vehicle-treated group were significantly larger in volume than either treated group. No toxicity was observed in any mice. Statistical analysis

All quantitative results are expressed as mean values ± S.D. Statistically significant differences were obtained using the Student's t test or by one-way ANOVA. A value of p < 0.05 was considered to be statistically significant.

Results

An DC derivative, 3CAI, suppresses colon cancer cell growth.

To compare the effects of 13 C (Fig. 1 A) and its derivatives (Fig. IB) on cancer cell growth, HCT116 colon cancer cells were treated with various concentrations of 13 C or each of its derivatives for 48 h. Proliferation was assessed by MTS assay and results indicated that growth was significantly decreased by 13 C derivative #1 , #2 or #3, but 13 C or derivative #4 had little effect (Fig. 1C). Additionally, we compared the effect of the 5 compounds on anchorage-independent cell growth. HCT116 colon cancer cells were seeded with I3C or its derivatives in 0.3% agar and incubated for 3 weeks. Data showed that only the high dose of I3C (200 μΜ) or I3C derivatives (# 1 , #2 or #3), but not derivative #4, strongly suppressed anchorage-independent cell growth (Fig. ID). Interestingly, I3C derivatives #2 or #3 had inhibitory effects on growth similar to derivative #1. Therefore, these findings suggested that methoxy and fluoro modification of 3CAI was not important for inhibiting proliferation or anchorage-independent cell growth. Based on these results, 3CAI appears to be the most effective anti-colon cancer compound of the 4 derivatives tested and was used in further studies.

3CAI is a potent inhibitor of AKT kinase activity.

To identify the direct molecular target of 3CAI, 85 kinases were screened against 3CAI in a high-throughput substrate-competitive assay (www.kinomescan.com/). Results identified 3CAI as a potential inhibitor of AKT. Based on these screening data, the effect of 3CAI on the kinase activities of AKTl, MEKl, JNKl, ERKl and TOPK were tested using in vitro kinase assays. The results showed that 3CAI (1 μΜ) suppressed only AKTl kinase activity and the other kinases tested were not affected by 3CAI (Fig. 2A). The effect of 3CAI on kinases upstream of AKT was also studied. PI3K activity was potently inhibited by LY294002, a well-known inhibitor of PI3K, and 3CAI inhibited PI3K by 60% at the highest concentration (10 μΜ; Fig. 2B). These data suggest that 3CAI is a much more potent AKTl inhibitor than PI3K (60% inhibition at 1 vs 10 μΜ, respectively). Additionally, the effect of I3C, 3CAI and the AKT inhibitor VIII on AKTl and 2 activities were studied. 3CAI, but not DC, substantially suppressed AKT1 activity (Fig. 2C) as well as AKT2 activity (Fig. 2D) in a dose dependent manner. These data showed that 3CAI is a potent and specific AKT1 and AKT2 inhibitor. 3CAI directly binds with AKT1 or AKT2 in an ATP non-competitive manner.

A molecular docking study with 3CAI and AKT1 and AKT2 was performed in order to determine its binding orientation. The docking score of 3CAI with AKT1 was -2.03 Kcal/mol, which was a little less favorable than the docking score of 3CAI with AKT2 (-2.25 Kcal/mol). 3CAI forms a hydrogen bond with Glul7 in the AKT1 PH domain, whereas 3CAI forms three hydrogen bonds with Lysl4, Leu52 and Arg86 in the AKT2 PH domain (Fig. 3 A). The structures of AKT1 or AKT2 were aligned and superimposed to compare the docked conformation of 3CAI. 3CAI adopts apose parallel to Ins(l,3,4,5)P₄in AKT1, whereas 3CAI adopts a pose perpendicular with Ins(l,3,4,5)P₄ in AKT2 (Fig. 3B). To confirm the results of the computer docking modelJw vitro pull-down assays were carried out using 3CAI or DC-conjugated Sepharose 4B beads. These results showed that 3CAI directly bound to recombinant AKT1 (Fig. 4A) and AKT2 (Fig. 4B) in an ATP non-competitive manner. 13 C showed no binding. Similar results were obtained using an HCT116 colon cancer cell lysate (Fig. 4C and 4D). These results suggest that 3CAI binds to an AKT allosteric site and not the ATP pocket. 3CAI inhibits down-stream targets of AKT and induces apoptosis.

The effect of 3CAI on down-stream targets of AKT, including the phosphorylation of mTOR and GSK3 was investigated. Results indicated that the AKT-mediated phosphorylations of mTOR (Ser2448) and GSK3P (Ser9) were substantially decreased by 3CAI in a time-dependent manner (Fig. 5A). However, phosphorylation of AKT (Thr308) was not changed. Furthermore, pro-apoptotic marker proteins p53 and p21 were also upregulated by 3CAI after 12 or 24 hours of treatment. Additionally, the anti-apoptotic marker protein Bcl2 and AKT-mediated

phosphorylation of ASK1 (Ser83) were significantly decreased (Fig. 5B). These findings suggested that pro- or anti-apoptotic marker proteins are regulated by 3CAI in colon cancer cells. Next, to determine whether apoptosis was induced by 3CAI, the effect on cell death of this compound, I3C and an AKT inhibitor were compared. HCT116 and HT29 colon cancer cells were seeded on 6 cm dishes in 1% FBS/McCoy's 5A (HCT116) with 3CAI, I3C or the AKT inhibitor and then incubated for 4 days. Results showed that the number of apoptotic cells was significantly increased by 3CAI in HCTl 16 and HT29 colon cancer cells compared with untreated control cells (Fig. 5C).

3CAI inhibits growth of colon cancer cells in a Xenograft mouse model

Athymic nude mice (6 week old nu/nu female mice, Harlan Laboratory, Minneapolis, MN) were inoculated in the right flank with HCTl 16 cells (1.5*10⁶ cells/mouse). Mice were maintained under "specific pathogen-free" conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Tumors were allowed to grow to an average of -40 mm and then mice were divided into 3 equal groups with the same average tumor volume (n=10 of each group; group 1 = vehicle only; group 2 = 3CAJ at 1 mg/kg; group 3 = 3CAI at 10 mg/kg). Treatment with 3CAI was initiated on Day 8 and continued to Day 20 (~2 weeks) and was administered by intraperitoneal injection (i.p.) 3 times a week. Tumor volume was measured 3 times a week and body weight was measured 2 times a week. The 3CAI compound was prepared in 10% tween-20 in 90% D.D.W. The tumors from the vehicle-treated group were significantly larger in volume than either treated group (Figure 6). No toxicity was observed in any mice.

Discussion

The natural phytochemical, I3C, has been reported to exert potent anti-proliferative activities in cell-based studies and has been implicated as a potential therapy for human cancers. However, only high concentrations of 13 C can induce anti-cancer activity and appear to involve a non-specific broad range of targets. Therefore, interest in developing more potent synthetic DC-based compounds has grown. However, only a few 13 C analogues have been reported and exert only a low enhancement of potency in biological activity (Brandi G, et al., Cancer Res., 2003, 63, 4028-36; and Chao WR, et al., J Med Chem., 2007, 50, 3412-5). In contrast, l-benzyl-I3C was reported as the most potent synthetic derivative of 13 C with an approximate 1000-fold increased potency against breast cancer. The investigators suggested that l-benzyl-I3C inhibited CDK2 enzymatic activity and CDK6 activity through the downregulation of CDK6 transcription and protein expression (Nguyen HH, et al., Chem Biol Interact., 2010, 186, 255-66). However, direct targets of l-benzyl-BC or its specificity were not determined. A potent derivative of I3C, 3CAI, has been identified from a high throughput screening of 85 kinases. Of several kinases tested, 3CAI inhibited only AKT kinase activity (Fig 2 A, B), suggesting that 3CAI is a specific AKT inhibitor.

Additionally, the binding orientation between 3CAI and AKT was determined using a computer docking model. About 20 crystal structures of AKT2 and 10 of AKTl are available. The molecular alignment of the protein sequences using EMBOSS (Rice P, et al., Trends Genet. , 2000, 16, 276-7) showed that they possess about 85% and 92% identity.

Previous studies showed that HER-2 mediated AKT activation to induce translocation of MDM2 from the cytoplasm to the nucleus. MDM2 directly binds to p53 and induces

ubiquitination (Mayo LD and Dormer DB, Proc Natl Acad Sci USA, 2001, 98, 11598-603; and Zhou BP, et al., Nat Cell Biol., 2001, 3, 973-82). In other reports, an effect of AKT on MDM2 subcellular localization from the cytoplasm to the nucleus was not detected, but AKT was shown to facilitate the function of MDM2 to promote p53 ubiquitination by phosphorylation of Serl86 (Ogawara Y, et al., J Biol Chem. , 2002, 277, 21843-50). The protein level of p53 was substantially increased by 3CAI in a time-dependent manner, as was the abundance of p21 , a target of p53 (Fig. 4B). Whether inhibition of AKT kinase activity by 3CAI could induce stability of p53 by suppressing phosphorylation of MDM2 (Serl66) was also investigated. The phosphorylation of MDM2 and p53 protein level using an immunofluoresence assay and Western blot analysis of cytoplasmic and nuclear protein fractions was confirmed. However, no significant translocation of MDM2 was observed. Importantly, 3CAI suppressed colon cancer cell growth and induced apoptosis more potently than I3C or a commercially available AKT inhibitor (Fig. 4). Results of a xenograft mouse model showed that oral administration of 3CAI at 30 mg/kg B.W. for 21 days significantly inhibited colon cancer cell growth and was not toxic (Fig. 5).

In conclusion, 3CAI is a potent and specific AKT inhibitor and suppressed cell growth and induced apoptosis both in vitro and in vivo.

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

Claims

What is claimed is: 1. A method for treating or preventing cancer in an animal comprising administering (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof to the animal.

2. A method for suppressing cancer cell growth comprising contacting (in vitro or in vivo) a cancer cell with (3-chloroacetyl)indole or a salt thereof.

3. A pharmaceutical composition comprising (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

4. A method for inhibiting AKT in a cell, comprising contacting the cell in vitro or in vivo with an effective amount of (3-chloroacetyl)indole or a salt thereof.

5. (3-Chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of cancer.

6. (3-Chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of a pathological AKT mediated condition.

7. (3-Chloroacetyl)indole or a pharmaceutically acceptable salt thereof for use in medical therapy.

8. The use of (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for the treatment of or prevention of cancer in an animal, such as a human.

9. The use of (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for the treatment of or prevention of a pathological AKT mediated condition in an animal, such as a human.

10. A method for treating or preventing a pathological AKT mediated condition in an animal comprising administering (3-chloroacetyl)indole or a pharmaceutically acceptable salt thereof to the animal.