WO2013028866A1 - Composés et méthodes thérapeutiques - Google Patents

Composés et méthodes thérapeutiques Download PDF

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WO2013028866A1
WO2013028866A1 PCT/US2012/052067 US2012052067W WO2013028866A1 WO 2013028866 A1 WO2013028866 A1 WO 2013028866A1 US 2012052067 W US2012052067 W US 2012052067W WO 2013028866 A1 WO2013028866 A1 WO 2013028866A1
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3cai
indole
chloroacetyl
akt
pharmaceutically acceptable
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PCT/US2012/052067
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English (en)
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Zigand DONG
Dong Joon Kim
Ann M. Bode
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Regents Of The University Of Minnesota
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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
  • this potent AKT inhibitor suppressed cancer cell growth in an in vivo xenograft mouse model.
  • 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
  • a pharmaceutical composition comprising
  • 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.
  • FIG. 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.
  • AKT1 kinase activity in vitro 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 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
  • 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 32 P-labeled substrate was visualized by autoradiography.
  • Figure 3 Illustrates computer modeling of 3CAI and AKT 1/2; (A) Binding modes of
  • FIG. 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.
  • HCT116 colon cancer cell lysate 500 ⁇ g 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.
  • AKT-target proteins in HCTl 16 colon cancer cells were treated with 3CAI, 13 C, or an AKT inhibitor, and then harvested at various times (0.5, 1 and 3 h).
  • 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 ⁇ -actin.
  • ⁇ -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.
  • FIG. 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).
  • CAI 3-chloroacetyl)indole
  • 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.
  • a salt of a compound can be useful as an intermediate for isolating or purifying the compound.
  • administration of a compound as a pharmaceutically acceptable acid or base salt may be appropriate.
  • 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.
  • 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.
  • a sufficiently basic compound such as an amine
  • 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.
  • 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.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • 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.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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
  • 3CAI (purity: 9%) was synthesized as described (Sudipta Roy SH and Gordon W.
  • HCT116 and HT29 human colon cancer cells were cultured in McCoy's 5 A medium
  • FBS fetal bovine serum
  • GA fetal bovine serum
  • 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)P 4 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.
  • 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.
  • 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 (Pier
  • 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).
  • Recombinant human AKTs 200 ng were incubated with 3CAI-Sepharose 4B (or Sepharose 4B only as a control) beads (50 ⁇ , 50% slurry) in reaction buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 ⁇ g/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.
  • reaction buffer 50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40
  • 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 ⁇ of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% C0 2 incubator. Absorbance was measured at 492 nm.
  • Colon cancer cells were plated into 60-mm culture dishes (1 10 5 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 ⁇ 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 ⁇ of [ ⁇ - 32 ⁇ ] ⁇ with each compound in 40 ⁇ of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 10 mM MnCl 2 , and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 ⁇ 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,
  • 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
  • An DC derivative, 3CAI suppresses colon 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.
  • 3CAI is a potent inhibitor of AKT kinase activity.
  • PI3K activity was potently inhibited by LY294002, a well-known inhibitor of PI3K, and 3CAI inhibited PI3K by 60% at the highest concentration (10 ⁇ ; Fig. 2B).
  • 3CAI is a much more potent AKTl inhibitor than PI3K (60% inhibition at 1 vs 10 ⁇ , respectively).
  • I3C, 3CAI and the AKT inhibitor VIII were studied.
  • 3CAI but not DC, substantially suppressed AKT1 activity (Fig. 2C) as well as AKT2 activity (Fig. 2D) in a dose dependent manner.
  • 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)P 4 in AKT1, whereas 3CAI adopts a pose perpendicular with Ins(l,3,4,5)P 4 in AKT2 (Fig. 3B).
  • 3CAI adopts a pose perpendicular with Ins(l,3,4,5)P 4 in AKT2 (Fig. 3B).
  • 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).
  • 3CAI inhibits growth of colon cancer cells in a Xenograft mouse model
  • 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.
  • 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).
  • 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.
  • 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.
  • MDM2 directly binds to p53 and induces
  • 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).
  • 3CAI is a potent and specific AKT inhibitor and suppressed cell growth and induced apoptosis both in vitro and in vivo.

Abstract

La présente invention concerne une méthode de traitement ou de prévention du cancer chez un animal, comprenant l'administration d'un (3-chloroacétyl)indole ou de son sel pharmaceutiquement acceptable à l'animal.
PCT/US2012/052067 2011-08-24 2012-08-23 Composés et méthodes thérapeutiques WO2013028866A1 (fr)

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