N-desmethyl ruboxistaurin as a RSK inhibitor for therapeutic use

Abstract

Aspects of this invention are related to the use of N-desmethyl ruboxistaurin and pharmaceutically acceptable formulations thereof to modulate RSK signaling. Some aspects of the invention relate to the use of N-desmethyl ruboxistaurin to inhibit RSK. Some aspects of the invention provide methods of using N-desmethyl ruboxistaurin in the treatment of subjects with cancer, including breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, leukemia, and bladder cancer. The disclosed methods extend beyond disease treatment, including supportive care during radiation chemotherapy and prevention of cancer relapse. N-desmethyl ruboxistaurin administration, alone or in combination with other cancer therapies, inhibits RSK and shows a safety profile that supports its long-term use.

Description

N-DESMETHYL RUBOXISTAURIN AS A RSK INHIBITOR FOR THERAPEUTIC USECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 672,446 filed July 17, 2024, the disclosure of which is incorporated by reference.FIELD OF THE INVENTION

[0002] Aspects of this invention are related to methods for treating cancers, including breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, leukemia, and bladder cancer, where N-desmethyl ruboxistaurin is clinically useful.BACKGROUND OF THE INVENTION

[0003] The various aspects discussed herein relate to the use of N-desmethyl ruboxistaurin for the treatment of cancer.

[0004] Ruboxistaurin, which has been shown to modulate GSK3 signaling and inhibit protein kinase C, has been investigated in several clinical trials for the treatment of diabetes mellitus and its complications. See A. Girach, US Patent Publication No. 2008 / 0096923, incorporated herein by reference in its entirety. Inhibition of protein kinase C has also been proposed as beneficial in the treatment of heart failure and atherosclerosis. While ruboxistaurin has not been studied in clinical trials for the treatment of cancer, inhibition of GSK3 signaling and inhibition of protein kinase C signaling have each been proposed as beneficial for certain cancer types that have aberrant signaling of GSK3 and protein kinase C pathways.

[0005] RSK1 is known to disrupt the cell cycle in cancer cells and presents an opportune target in breast cancer therapy with potential applications across diverse cancer pathologies, including ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, leukemia, and bladder cancer. RSK1 modulation has been a subject of interest in cancer research due to its impact on tumor progression and development. Recent research showed benefits in metastatic breast cancer, triple-negative breast cancer, myeloproliferative neoplasms and secondary acute myeloid leukemia. RSK2 has been shown to be dysregulated in breast cancer, and a dual RSK1 / RSK2 inhibitor has shown evidence of therapeutic benefit, with progression free survival enhanced among those with high RSK2 activity identified by a companion diagnostic with histological analysis of tumor samples. Furthermore, blocking RSK2 overcomes resistance to Gl / S inhibitors such as CDK4 / CDK6 inhibitors or antibody drug conjugates.

[0006] RSK1 is a downstream component of the Ras-MAPK cascade, and RSK1 inhibition provides a selective means of targeting this important cascade. Tumor types characterized by activation of the Ras- MAPK cascade have aberrant RSK1 signaling. Two RSK1 inhibitors are currently in clinical trials for metastatic and triple-negative breast cancer, highlighting the importance of this target in cancer therapeutics.

[0007] The present invention addresses these imperatives by introducing N-desmethyl ruboxistaurin as a novel RSK inhibitor for the treatment of cancer, where N-desmethyl ruboxistaurin is clinically useful.BRIEF SUMMARY OF THE INVENTION

[0008] Aspects of this invention are related to the use of N-desmethyl ruboxistaurin as a therapeutic agent for the treatment of cancer.

[0009] One aspect of the invention is directed to a method of treating a disease / disorder characterized by aberrant signaling of RSK, comprising administering to a subject in need thereof a therapeutically effective dose of N-desmethyl ruboxistaurin or a pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof. Stated another way, the invention provides N-desmethyl ruboxistaurin or a pharmaceutically acceptable salt thereof, for use in treating a disease / disorder characterized by aberrant signaling of RSK, by administering to a subject in need thereof a therapeutically effective dose of the N-desmethyl ruboxistaurin or a pharmaceutically acceptable salt thereof.

[0010] In one embodiment, the subject has cancer or a history of cancer. In another embodiment, the subject has a cancer with evidence of dysregulated RSK activity identified by a companion diagnostic. The companion diagnostic can be either a histological assessment of the tumor sample for evidence of excess RSK activity, or it can be an evaluation of the tumor sample for KRAS, NRAS, or BRAF mutations, or other markers that identify hyperactivation of the MAPK pathway.

[0011] In another embodiment, N-desmethyl ruboxistaurin, or a pharmaceutically acceptable salt thereof is administered in an amount of about 32 to about 320 mg once daily, or about 16 to about 160 mg twice daily. In another embodiment, N-desmethyl ruboxistaurin, or a pharmaceutically acceptable salt thereof is administered in combination with other cancer therapies.

[0012] The diseases subject to treatment include but are not limited to breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, leukemia, or bladder cancer. Furthermore, the invention extends to providing supportive care during radiation or chemotherapy and preventing cancer relapse.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 displays the chemical structure of ruboxistaurin.

[0014] FIG. 2 displays the chemical structure of N-desmethyl ruboxistaurin.

[0015] FIG. 3 shows a graph of the mean plasma concentration versus time profile following single-dose administration of 32 mg ruboxistaurin in healthy subjects. Results in the insert are in the fed state.

[0016] FIG. 4 shows a synthetic scheme for N-desmethyl ruboxistaurin.

[0017] FIG. 5 displays N-desmethyl ruboxistaurin inhibition of RSK1.DETAILED DESCRIPTION

[0018] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The broader term "RSK" refers to RSK1, RSK2, RSK3, and RSK4, the 90 kDa ribosomal S6 kinase (RSK) family of proteins that consists of four human isoforms. Depending on context, it may refer to one or more of the isoforms.

[0019] As disclosed herein, several ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention. The term "about" generally includes up to plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 20" can mean from 18 to 22. Preferably "about" includes up to plus or minus 6% of the indicated value. Alternatively, "about" includes up to plus or minus 5% of the indicated value. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" can also mean from 0.5 to 1.4.

[0020] The term "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Such salts include acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such as formic acid, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2- ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'- methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Additional information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference.

[0021] As used herein, the term "therapeutically effective amount" means an amount of compound of the present invention which is capable of alleviating the symptoms of the various pathological conditions herein described. The specific dose of a compound administered according to this invention will, of course, be determined by the particular circumstances surrounding the case including, for example, the compound(s) administered, the route of administration, the state of being of the patient, and the pathological condition being treated. Dosing can be once per day, or administered in multiple sub-doses per day, e.g., two, three, or more doses per day.

[0022] Reference to tumors characterized by "aberrant signaling" of RSK means that the tumor type is one where a high proportion of cancers is known to have activation of RSK or the ras-MAPK cascade, or where the tumor is associated with a biomarker (such as a blood test, tumor biopsy result, or PET imaging study) indicating increased activity of RSK or the ras-MAPK cascade.

[0023] The effective dose of N-desmethyl ruboxistaurin, or pharmaceutically acceptable salt thereof, is about 32 to about 320 mg once daily, or about 16 to about 160 mg twice daily for monotherapy. A pharmaceutical composition of N-desmethyl ruboxistaurin, or pharmaceutically acceptable salt thereof, further comprises at least one pharmaceutically acceptable adjuvant or excipient.

[0024] Ruboxistaurin has been investigated in several clinical trials for the treatment of diabetes mellitus and its complications, including diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy. See A. Girach, US Patent Publication No. 2008 / 0096923, incorporated herein by reference in its entirety. Its safety has been described as excellent, with a lower incidence of serious adverse events than placebo. Further development of ruboxistaurin has been encouraged because of its clinical safety profile, andbecause of growing interest in the use of treatments that can inhibit GSK3. However, careful review of ruboxistaurin clinical and preclinical data has identified its potential to prolong the Q.T interval in human subjects, and this can increase the risk of dangerous cardiac arrhythmias, particularly in the case of an accidental or intentional overdose of ruboxistaurin, or when administered with other drugs that prolong the QT interval, or when very high ruboxistaurin levels are induced by co-administration of a drug inhibiting CYP3A4. Recently it was discovered that N-desmethyl ruboxistaurin is more stable, has superior pharmacokinetics, and its metabolism is affected much less by CYP3A4 inhibition. Therefore, administration of N-desmethyl ruboxistaurin, either alone or in combination with other agents, is desirable as an alternative to ruboxistaurin where inhibition of GSK3 is useful.

[0025] Preclinical development of N-desmethyl ruboxistaurin revealed its robust ability to inhibit RSK1. High expression (aberrant signaling) of RSK is associated with various cancers, including breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, bladder cancer, and leukemia. For example, approximately, 85% of triple-negative breast cancer samples have activated RSK. Ras-MAPK cascaTwo RSK1 inhibitors are currently in clinical trials for metastatic and triple-negative breast cancer, and multiple other RSK inhibitors are in various stages of clinical development, highlighting the importance of this target in cancer therapy.

[0026] Preparation of N-desmethyl ruboxistaurin (Compound-1) generally follows the methods illustrated in Fig. 4. As shown, starting material 1 is reacted with vinyl Grignard in the presence of copper iodide giving alcohol intermediate 2. One of ordinary skill in the art will recognize that alternatives to vinyl Grignard are useful in effecting the same transformation. Such alternatives include, but are not limited to, vinyl zinc reagents, vinyl cuprate reagents and vinyl lithium reagents. One of ordinary skill in the art will recognize that alternatives to copper iodide are useful in facilitating conversion of intermediate 1 to intermediate 2. Such alternatives include, but are not limited to, alternate Lewis acid reagents and chelating agents such as crown ethers.

[0027] Following isolation of intermediate 2, Fig. 4 illustrates conversion to intermediate 3 on reaction with allyl bromide. One of ordinary skill in the art will recognize that alternate al lylating agents are useful in the allylation of intermediate 2 to intermediate 3. Such allylating agents generally employ alternates to the bromide leaving group and include, but are not limited to, allyl chloride, allyl iodide and allyl mesylate. One of ordinary skill in the art will also recognize that alternates to the illustrated potassium tert-butoxide base are useful in effecting the reaction of intermediate 2 with an allylating agent. Suchbases include, but are not limited to, hydride reagents, carbonate reagents, bicarbonate reagents, lithium diisopropylamide, sodium hexamethyl disilazide and the like.

[0028] Fig. 4 further illustrates the 2-step conversion of intermediate 3 to intermediate 4. As shown, the first step is an ozonolysis reaction leading to the cleavage of a bis-olefin to a bis-aldehyde and the second step is a sodium borohydride reduction of a bis-aldehyde to a bis-alcohol. One of ordinary skill in the art will recognize that ozonolysis is only one of many reactions or reaction combinations suitable for cleavage of an olefin to an aldehyde. Such conversions include, but are not limited to, dihydroxylation of an olefin followed by cleavage of the resulting diol to an aldehyde. Suitable reagents for the dihydyroxylation of an olefin include, but are not limited to, osmium tetroxide and the like. Suitable reagents for the cleavage of a diol to an aldehyde include, but are not limited to, sodium periodate, lead tetraacetate and the like. One of ordinary skill in the art will further recognize that alternates to the sodium borohydride reducing agents are suitable for the reduction of aldehydes to alcohols. Such reagents include, but are not limited to, lithium aluminum hydride, diisopropyl aluminum hydride, lithium borohydride, borane and the like. One of ordinary skill in the art will additionally recognize that alternatives to boron and aluminum-based reducing agents are also useful for the reduction of aldehydes to alcohols. Such alternatives include, but are not limited to, samarium iodide and triethylsilane.

[0029] As illustrated in Fig. 4, the diol of intermediate 4 is converted to the bis-mesylate intermediate 5 on reaction with methanesulfonyl chloride and triethylamine. One of ordinary skill in the art will recognize that mesylates, as leaving groups, are generally useful as are common alternative leaving groups including, but not limited to, chlorides, bromides, iodides, tosylates and the like. One of ordinary skill in the art will also recognize that alternatives to triethylamine are useful in the conversion of alcohols to mesylates with such alternatives including, but not being limited to, diisopropyl ethylamine, pyridine, carbonate reagents, bicarbonate reagents and the like.

[0030] The reaction of the bis-mesylate intermediate 5 with the bis-indolyl maleimide intermediate 6 forming intermediate 7 is illustrated in Fig. 4 using cesium carbonate as the base. One of ordinary skill in the art will recognize that alternative bases are useful for effecting the illustrated reaction to intermediate 7. Such bases include, but are not limited to, hydrides, alkoxides, carbonates, bicarbonates and the like.

[0031] The process of converting the methyl maleimide to its demethylated version involves initial hydrolysis of intermediate 7 to the maleic anhydride intermediate 8. As illustrated in Fig. 4, this transformation is effected using potassium hydroxide in ethanol. One of ordinary skill in the art will recognize that conversion of intermediate 7 to intermediate 8 can employ alternates to potassiumhydroxide include, but are not limited to, sodium hydroxide and lithium hydroxide. Furthermore, one of ordinary skill in the art will recognize that ethanol can be exchanged for any protic solvent including, but not limited to, methanol and water.

[0032] According to Fig. 4, maleic anhydride intermediate 8 is to the corresponding maleimide intermediate 9 is accomplished on reaction of intermediate 8 with hexamethyldisilazane. One of ordinary skill in the art will recognize that maleic anhydrides can be converted to maleimides using alternate reagents including, but not limited to, ammonia, sodamide and the like.

[0033] With the maleimide established, Fig. 4 illustrates cleavage of the trityl protecting group from intermediate 9 to alcohol intermediate 10. While Fig. 4 highlights hydrochloric acid as the reagent effecting trityl cleavage, one of ordinary skill in the art will recognize that alternate acids can be used. Said alternates include, but are not limited to, hydrobromic acid, trifluoroacetic acid, acetic acid and the like.

[0034] As illustrated in Fig. 4, the alcohol of intermediate 10 is converted to the mesylate intermediate 11 on reaction with methanesulfonyl chloride and pyridine. One of ordinary skill in the art will recognize that mesylates, as leaving groups, are generally useful as are common alternative leaving groups including, but not limited to, chlorides, bromides, iodides, tosylates and the like. One of ordinary skill in the art will also recognize that alternatives to triethylamine are useful in the conversion of alcohols to mesylates with such alternatives including, but not being limited to, diisopropyl ethylamine, pyridine, carbonate reagents, bicarbonate reagents and the like.

[0035] In the final stage of the synthesis, Fig. 4 illustrates conversion of intermediate 11 to Compound-1 on reaction with methylamine. While not illustrated in Fig. 4, the methylamine is further converted to its corresponding hydrochloride salt. One of ordinary skill in the art will understand that alternate strategies for conversion of compounds such as intermediate 11 to structures such Compound-1 exist. Such strategies are generally recognizable by one skilled in the art and said strategies are generally supported by resources such as Comprehensive Organic Transformations (Larock, Wiley).

[0036] One of ordinary skill in the art will recognize that there are many additional reactions, in addition to those described above and illustrated in Fig. 4, that are useful for the preparation of N-desmethyl ruboxistaurin (Compound-1). Suitable reactions are readily identified by one skilled in the art and are available in resources such as Comprehensive Organic Transformations (Larock, Wiley). Strategies for introduction and cleavage of protecting groups are readily identified by one skilled in the art and are available in resources such as Protective Groups in Organic Synthesis (Greene and Wutz, Wiley). One ofordinary skill in the art will further recognize that, generally applicable to Fig. 4 and to any and all alternates and variations to the route described in Fig. 4, alternate combinations of reagents, solvents, temperature conditions and reaction times will provide similar chemical outcomes enabling the preparation of Compound-1.

[0037] These composition and treatment methods are relevant to cancers where RSK inhibition has been proposed, including breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, leukemia, and bladder cancer. The presently disclosed compositions and treatment methods also have use in preventing relapse of a disclosed malignancy and ameliorating toxicity caused by other cancer treatments.

[0038] The presently disclosed compositions and treatment methods also have use in veterinary applications for improving the health and well-being of livestock and companion animals by treating any of the foregoing indications that occur in animals.EXAMPLESMaterials and MethodsExample 1. Compound Synthesis: N-desmethyl ruboxistaurin was synthesized according to the following procedures: Step-1: Synthesis of (S)-l-(trityloxy) pent-4-en-2-ol (2):

[0087] To a solution of vinyl magnesium bromide (IM in THF, 840 mL, 0.84 mol) was added copper iodide (4.5 g, 23.62 mmol) at -40°C, under nitrogen atmosphere. After stirring for 20 min at -40°C, Compound-1 (150 g, 0.46 mmol), dissolved in dry THF (750 mL) was added dropwise into the reaction mixture, and the resulting reaction mixture was stirred at -40°C for 2 h. After completion of the reaction, (monitored by TLC), sat. ammonium chloride (1000 mL) was added. Warmed the reaction to RT, while stirring and extracted with ethyl acetate (1000 mL). Separated the organic layer and washed with aq. Ammonia (250 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to obtain Compound-2 as a dark brown sticky material (166 grams, 100% crude yield).1H NMR (400 MHz, CDCIa): 8 7.45-7.42 (m, 6H), 7.32-7.28 (m, 6H), 7.26-7.22 (m, 3H), 5.74-5.71 (m, 1H), 5.09-5.02 (m, 2H), 3.85-3.82 (m, 1H), 3.18 (dd, 7=9.6 Hz, 7=4.0 Hz, 1H), 3.09 (dd, 7=9.2 Hz, 7=6.8 Hz, 1H), 2.27-2.22 (m, 3H).Step-2: Synthesis of (S)-(((2-(allyloxy) pent-4-en-l-yl) oxy) methane trityl) tribenzene (3):

[0088] To a stirred solution of compound-2 (165 g, 0.48 mol) in dry THF (1500 mL) was added potassium tert-butoxide (70.0 g, 0.62 mmol) portion-wise, under nitrogen atmosphere. The resulting reaction contents were heated to 45°C and stirred for 2 h, then cooled to RT, followed by the addition of allyl bromide (145.5 g, 1.22 mol) at RT and continued the stirring for 1 h at RT. After completion of the reaction (monitored by TLC), added sat. ammonium chloride (1500 mL) into the reaction and extracted with ethyl acetate (1500 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to give the crude compound-3, which was further purified by silica gel column chromatography (100-200 mesh), eluting with 0.5-1% ethyl acetate in hexane. The pure fractions were collected and evaporated under reduced pressure to afford the desired compound-3 as a pale yellow semi-solid (106 grams, 58% yield).2H NMR (400 MHz, CDCI3): 67.48-7.44 (m, 6H), 7.31-7.26 (m, 6H), 7.25- 7.20 (m, 3H), 5.95-5.88 (m, 1H), 5.75-5.70 (m, 1H), 5.27 (dd, 7=17.2 Hz, 7=2.0 Hz, 1H), 5.15 (dd, 7=10.4 Hz, 7=2.0 Hz, 1H), 5.03 (dd, 7=17.2 Hz, 7=2.0 Hz, 1H), 4.96 (dt, 7=10.4 Hz, 7=1.2 Hz, 1H), 4.12-4.10 (m, 1H), 4.O4- 4.02 (m, 1H), 3.52-3.49 (m, 1H), 3.17-3.09 (m, 2H), 2.35-2.31 (m, 2H)Step-3: Synthesis of (S)-3-(2-hydroxyethoxy)-4-(trityloxy) butan-l-ol (4):

[0089] To a stirred solution of compound-3 (100 g, 0.26 mol) in MeOH: DCM (1:1) (800 mL) was bubbled ozone gas for 18 h at -45°C. After completion of the reaction (monitored by TLC), it was poured into a solution of sodium borohydride (21.5 g, 0.57 mol) in 0.5 N NaOH solution (370 mL) at 0°C. The resulting reaction mixture was allowed to stir at RT for 16 h. After completion of the reaction (monitored by TLC), quenched with 1 N HCI solution, until pH 6-7. Then resulting solution was extracted with ethyl acetate (750 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to afford crude compound, which was further purified by silica gel column chromatography (100-200mesh), eluting with 20-25% ethyl acetate in hexane. The pure fractions were collected and evaporated to afford the desired compound-4 as a yellow color gummy liquid (58 grams, 57% yield).1H NMR (400 MHz, DMSO-c / g): 67.42-7.40 (m, 6H), 7.34 (t, 1=7.6 Hz, 6H), 7.28-7.24 (m, 3H), 4.60 (t, 1=5.6 Hz 1H), 4.36 (t, 1=5.6 Hz, 1H), 3.60-3.56 (m, 2H), 3.52-3.48 (m, 2H), 3.44-3.41 (m, 3H), 2.99-2.97 (m, 2H), 1.61-1.56 (m, 2H).Step-4: Synthesis of (S)-2-((4-((methylsulfonyl)oxy)- l-(trity loxy)buta n-2-y l)oxy)ethyl methanesulfonate (5):

[0090] To a stirred solution of Compound-4 (60 g, 0.15 mol), in DCM (1000 mL) was added triethyl amine (66 mL, 0.47 mmol) at 0°C and stirred for 15 min, followed by the addition of methane sulfonyl chloride (32.0 mL, 0.41 mmol). The resulting reaction mixture was stirred for 2 h at 0°C, (reaction monitored by TLC), quenched with sat. ammonium chloride solution (600 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum (<25°C) to afford crude compound, which was suspended in a 1:1 mixture of ethyl acetate and heptane (600 mL) and evaporated under vacuum to give solid. The obtained solid compound was suspended in 1:1 mixture of ethyl acetate and heptane (600 mL), stirred for 30 min, filtered, washed the solid with heptane (80 mL) and dried under vacuum to afford compound-5 as a cream color solid (88 grams, 100% crude yield).NMR (400 MHz, DMSO-dg): 8 7.42- 7.39 (m, 5H), 7.37-7.31 (m, 5H), 7.29-7.23 (m, 3H), 7.22-7.18 (m, 2H), 4.34-4.22 (m, 4H), 3.84-3.83 (m, 1H), 3.69-3.64 (m, 2H), 3.17 (s, 3H), 3.13 (s, 3H), 3.09-3.06 (m, 1H), 3.04-3.02 (m, 1H), 1.88-1.85 (m, 2H).Step-5: Synthesis of (12E,32E,7S)-21-methyl-7-((trityloxy)methyl)-22,25-clihydro-llH,21H,31H-6-oxa-1,3(3, l)-diindola-2(3,4)-pyrrolacyclononaphane-22, 25-dione (7):

[0091] To a stirred solution of Compound-6 (41.5 g, 0.12 mol) in DMF (850 mL) was added cesium carbonate (86.0 g, 0.26 mol) and the reaction mixture was heated to 100°C, then was added Compound- 5 (85.0 g (crude), 0.15 mol) dropwise at same temperature. The resulting reaction mixture was stirred at 100°C for 24 h. After completion of the reaction (monitored by TLC), cooled to 50°C, celite (25 g) was added and stirred for 15 min. The reaction mass was filtered on celite and the filtrate was partitioned between ethyl acetate (800 mL) and water (400 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to afford crude compound, which was further purified by silica gel column chromatography (100-200 mesh), eluting with 25-30% ethyl acetate in hexane. The pure fractions were collected and evaporated to afford the desired compound-7 as a brick red solid (55 grams, 51% yield).2H NMR (400 MHz, DMSO-d6): 6 7.83 (d, 7=7.6 Hz 1H), 7.75 (d, 7=8.0 Hz 1H), 7.49 (d, 7=8.4 Hz 1H), 7.45 (s, 1H), 7.41 (s, 1H), 7.34-7.26 (m, 10H), 7.25-7.22 (m, 6H), 7.18-7.15 (m, 2H), 7.12-7.06 (m, 2H), 4.25-4.24 (m, 1H), 4.17-4.04 (m, 3H), 3.71-3.67 (m, 1H), 3.55-3.50 (m, 1H), 3.31-3.30 (m, 1H), 3.07 (s, 3H), 3.05-3.00 (m, 2H), 2.10-2.07 (m, 1H), 2.02 (m, 1H).Step-6: Synthesis of (12E,32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-llH,31H-6-oxa-l,3(3,l)-diindola- 2(3, 4)-furanacyclononaphane-22, 25-dione (8):

[0092] To a stirred solution of compound-7 (85.0 g, 0.12 mol) in ethanol (850 mL) was added potassium hydroxide (68.0 g, 1.22 mol) and heated to 80°C. The resulting reaction mixture was stirred for 24 h. After completion of the reaction (monitored by TLC), the reaction mixture was evaporated under vacuum to give residue, which was partitioned between DCM (850 mL) and 20% citric acid solution (450 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to afford crude compound-8 as a dark brown solid (62 grams, 74% yield).1H NMR (400 MHz, DMSO-de): 8 7.88 (d, 7=7.6 Hz 1H), 7.82 (d, 7=7.6 Hz 1H), 7.65 (d, 7=2.0 Hz 2H), 7.55 (d, 7=8.0 Hz 1H), 7.41 (d, 7=7.6 Hz 1H), 7.34- 7.26 (m, 12H), 7.25-7.20 (m, 5H), 7.19-7.13 (m, 2H), 4.33-4.28 (m, 1H), 4.20-4.06 (m, 3H), 3.73-3.69 (m, 1H), 3.58-3.54 (m, 1H), 3.09-3.07 (m, 2H), 2.17-2.12 (m, 1H), 2.01-1.97 (m, 1H). (extra protons in the aromatic region, not included)Step-7: Synthesis of (12E, 32E,7S)-7-((trityloxy)methyl)-22,25-dihydro-llH,21H,31H-6-oxa-l, 3(3,1)- diindola-2(3,4)-pyrrolacyclononaphane-22, 25-dione (9):

[0093] To a stirred solution of Compound-8 (95.0 g, 0.25 mol) in DMF (950 mL) was added HMDS (294.0 mL, 2.47 mol), methanol (6.0 mL) and heated to 80°C. The reaction mixture was stirred for 5 h, at 80°C. After completion of the reaction (monitored by TLC), cooled to RT and quenched with IN HCI solution (950 mL) and extracted with DCM (1500 mL). Separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to afford crude compound (84 g), which was further purified by silica gel column chromatography (100-200 mesh), eluting with 20-25% ethyl acetate in hexane. The pure fractions were collected and evaporated under vacuum to afford the desired compound-9 as a purple solid (70 grams, 74% yield).NMR (400 MHz, DMSO-d6): 6 10.91 (s, 1H), 7.81 (d, J=8.0 Hz 1H), 7.73 (d, 7=8.0 Hz 1H), 7.48 (d, 7=8.4 Hz 2H), 7.43 (s, 1H), 7.39 (s, 1H), 7.33-7.23 (m, 12H), 7.23-7.21 (m, 3H), 7.18- 7.14 (m, 2H), 7.11-7.06 (m, 2H), 4.27-4.23 (m, 1H), 4.13-4.00 (m, 3H), 3.70-3.67 (m, 1H), 3.55-3.47 (m, 1H), 3.33-3.26 (m, 1H), 3.02-2.99 (m, 2H), 2.13-2.08 (m, 1H), 2.01-1.98 (m, 1H).Step-8: Synthesis of (12E,32E,7S)-7-(hydroxymethyl)-22,25-dihydro-llH,21H,31H-6-oxa-l, 3(3,1)- diindola-2(3,4)-pyrrolacyclononaphane-22, 25-dione (10):

[0094] To a stirred solution of compound-9 (70.0 g, 0.18 mol) in ethanol (700 mL) was added 6N HCI (700 ml) at RT. The resulting reaction contents were heated to 80°C for 3 h. After completion of the reaction (monitored by TLC), cooled to RT, stirred for 1 h, filtered the resulting solid and washed with water (350 mL), dried under vacuum at 45°C to afford Compound-10 as a purple solid (40 grams, 88% crude yield).NMR (400 MHz, DMSO-c / 6): 6 10.92 (s, 1H), 7.82 (d, J=7.6 Hz 1H), 7.78 (d, J=7.6 Hz 1H), 7.53 (d, J=8.0 Hz, 1H), 7.51 (s, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.45 (s, 1H), 7.25-7.22 (m, 2H), 7.13-7.10 (m, 2H), 4.69 (t, J=5.2 Hz 1H), 4.35-4.33 (m, 1H), 4.24-4.15 (m, 3H), 3.91-3.87 (m, 1H), 3.65-3.60 (m, 1H), 3.53-3.49 (m, 1H), 3.43- 3.39 (m, 1H), 2.09-2.07 (m, 1H), 1.98-1.97 (m, 1H).Step-9: Synthesis of ((12E,32E,7S)-22,25-dioxo-22,25-dihydro-llH,21H,31H-6-oxa-l,3(3,l)-diindola-2(3,4)-pyrrolacyclononaphane-7-yl)methyl methanesulfonate (11):

[0095] To a stirred solution of compound-10 (39.0 g, 0.09 mol) in THF (400 mL) was added pyridine (33.2 mL, 0.39 mol) at RT, stirred for 20 min, then methane sulfonic anhydride (46.0 g, 0.26 mol) was added into the reaction at RT. The resulting reaction mixture was stirred for 4 h. After completion of the reaction (monitored by TLC), partitioned the reaction between ethyl acetate (100 mL) and water (50 mL), separated the organic layer, dried over sodium sulphate, filtered and evaporated under vacuum to give crude compound (37.0 g), which was further purified by silica gel column chromatography (100-200 mesh), eluting with DCM. The pure fractions were collected and evaporated under reduced pressure to afford the desired compound-11 as a purple color solid (30 grams, 65% yield). *H NMR (400 MHz, DMSO-dg): 8 10.92 (s, 1H), 7.83 (d, J=7.6 Hz 1H), 7.78 (d, J=7.6 Hz 1H), 7.54 (d, J=8.4 Hz, 1H), 7.52 (s, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.46 (s, 1H), 7.22-7.17 (m, 2H), 7.14-7.10 (m, 2H), 4.44-4.38 (m, 2H), 4.22-4.14 (m, 4H), 3.93-3.90 (m, 1H), 3.66-3.61 (m, 1H), 3.17 (s, 3H), 2.19-2.14 (m, 1H), 2.03-1.98 (m, 1H).Step-10: Synthesis of (12E, 32E,7S)-7-((methylamino)methyl)-22,25-dihydro-llH,21H,31H-6-oxa-l, 3(3,1)- diindola-2(3,4)-pyrrolacyclononaphane-22, 25-dione hydrochloride (Compound-1):

[0096] To a stirred solution of compound-11 (10.0 g, 0.019 mol) in THF (400 mL) was added 2M Methyl amine in THF (400 mL) at -40°C, in an auto-clave. Gradually heated the reaction to 70°C and stirred for 24 h. After completion of the reaction (monitored by TLC), evaporated under vacuum to obtain crude compound (12.0 g). This batch was combined with 4 additional batches of the same scale giving 60.0 g of crude product. The 60 grams were purified by silica gel column chromatography (230-400 mesh, 2% MeOH / DCM). The pure fractions were collected and concentrated to give the desired compound-1 free- base (22.0 g) as a red solid. The free-base was suspended in diethyl ether (220 mL) and cooled to 0°C. Ethanolic HCI (33 mL) was added at at 0°C. The resulting suspension was stirred at 0°C for 30 min, filtered, washed with diethyl ether (50 mL) and dried under vacuum at 40°C for 1 h to afford Compound-1 as a brick red color solid (16.9 grams, 36% yield).1H NMR (400 MHz, DMSO-d6): 6 10.93 (s, 1H, exchanged in D2O), 8.72-8.71 (m, 2H, exchanged in D2O), 7.81 (t, 7=8.0 Hz, 2H), 7.55 (d, 7=8.0 Hz 1H), 7.49 (s, 2H), 7.47 (d, 7=8.4 Hz, 1H), 7.23 (t, 7=7.2 Hz, 2H), 7.14 (t, 7=7.2 Hz, 2H), 4.46-4.41 (m, 1H), 4.33-4.25 (m, 2H), 4.15- 4.10 (m, 1H), 3.86-3.84 (m, 1H), 3.73-3.71 (m, 1H), 3.62 (t, 7=9.2 Hz, 1H), 3.27-3.24 (m, 1H), 3.01-2.98 (m, 1H), 2.53 (t, 7=5.6 Hz, 3H), 2.22-2.20 (m, 1H), 2.06-2.03 (m, 1H).Example 2. Kinase Assay:

[0097] N-desmethyl ruboxistaurin was synthesized as in Example 1. Reaction conditions included a base reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCI2, 1 mM EGTA, 0.02% Brij-35, 0.02 mg / ml BSA, 0.1 mM NajVC , 2 mM DTT, and 1% DMSO. N-desmethyl ruboxistaurin, stored as 10 mM DMSO stocks at -80°C, were dissolved in 100% DMSO to specific concentrations and subjected to a three-fold serial dilution starting at 10 pM. Substrate was prepared in reaction buffer, cofactors were added, andRSK1 was introduced into the substrate solution. Reactions were carried out at 20 pM ATP. The compound was then added in 100% DMSO, followed by a 20-minute incubation at room temperature. The reaction was initiated by adding 33P-ATP (specific activity 10 pCi / pL), and incubation continued for 2 hours at room temperature. Radioactivity was detected via the filter-binding method. Recorded data included raw readings, percent enzyme activity relative to DMSO controls, and curve fits, with the latter performed when enzyme activities at the highest compound concentrations were less than 65% (Fig. 5). The assay was validated with staurosporine as a control compound. In Table 1 below, activities for RSK1, RSK2, RSK3, and RSK4 were evaluated in the same manner as described above, at a 1 pM concentration of N-desmethyl ruboxistaurin. Only 3% of activity remained of RSK1 at 1 pM, and even less activity remained for RSK2, RSK3, and RSK4, indicating potent inhibition.Table 1

Claims

CLAIMSWhat is claimed is:

1. N-desmethyl ruboxistaurin or a pharmaceutically acceptable salt thereof, for use in treating a disorder characterized by aberrant signaling of RSK, wherein the use is characterized by administering to a subject in need thereof a therapeutically effective dose of the N-desmethyl ruboxistaurin or a pharmaceutically acceptable salt thereof.

2. The N-desmethyl ruboxistaurin or pharmaceutically acceptable salt thereof for use according to Claim 1, wherein the subject has cancer or a history of cancer.

3. The N-desmethyl ruboxistaurin or pharmaceutically acceptable salt thereof for use according to Claim 2, wherein the disease / disorder is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, melanoma, osteosarcoma, myeloproliferative neoplasms, leukemia, or bladder cancer.

4. The N-desmethyl ruboxistaurin or pharmaceutically acceptable salt thereof for use according to Claim 2, wherein the subject has a cancer with evidence of dysregulated RSK activity identified by a companion diagnostic.

5. The N-desmethyl ruboxistaurin or pharmaceutically acceptable salt thereof for use according to any one of the above Claims, wherein N-desmethyl ruboxistaurin, or a pharmaceutically acceptable salt thereof is administered in an amount of about 32 to about 320 mg once daily, or about 16 to about 160 mg twice daily.

6. The N-desmethyl ruboxistaurin or pharmaceutically acceptable salt thereof for use according to any one of the above Claims, wherein N-desmethyl ruboxistaurin, or a pharmaceutically acceptable salt thereof is administered in combination with other cancer therapies.

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