Modulators of HSP70/DnaK Function and Methods of Use Thereof

Abstract

Compositions and methods for modulating HSP70 function, particularly for the targeted killing of cancer cells, are disclosed.

1 . A method for potentiating the effect of at least one chemotherapeutic agent in the treatment of disease associated with heat shock protein (HSP) 70 activity in a patient in need thereof, said method comprising co-administration of an effective amount of a compound of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts, wherein, R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of —C(R 6 R 7 )—C(R 8 R 9 )—, —CR 10 ═CR 11 —, and —C≡C—, wherein R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R a  and R b  are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkylcarbonyl, and alkyloxycarbonyl; or, optionally, R 1 , R 2 , R 3 , R 4 , R 5 , R a , and R b  are independently substituted with one member of a specific binding pair or a targeting ligand to facilitate targeting of the compound to a target tissue of interest, the compound being co-administered with a chemotherapeutic agent of interest and being effective to inhibit protein function, wherein said protein function is selected from the group consisting of HSP70, HSC70, and DnaK. 
       
     
     
         2 . The method of  claim 1  wherein said disease is cancer and said HSP70 function is selected from the group consisting of modulation of protein aggregation, chaperone protein binding, client protein binding, modulation of cellular stress response, modulation of autophagy, modulation of lysosomal functions, modulation of NFκB activity, modulation of caspase cleavage, modulation of the proteasome system, reduced viability, modulation of anoikis, modulation of formation of detergent-insoluble subcellular complexes, and modulation of vacuolization. 
     
     
         3 . The method of  claim 2 , wherein said compound disrupts HSP70 binding to at least one chaperone protein selected from the group consisting of CHIP, HSP40, and BAG-1M isoform. 
     
     
         4 . The method of  claim 2 , wherein said compound disrupts HSP70 binding to at least one client protein selected from the group consisting of APAF-1, p53, LAMP-2, integrin α5, integrin β1, SV40 T antigen, and HSP90 client proteins. 
     
     
         5 . The method of  claim 1 , wherein Q represents —C≡C—, and R 1 , R 2 , R 4 , R 5 , R a  and R b  each represents hydrogen. 
     
     
         6 . The method of  claim 1 , wherein the compound of formula I is pifithrin-μ. 
     
     
         7 . The method of  claim 1 , wherein said disease is characterized by aberrant cell proliferation. 
     
     
         8 . The method of  claim 7 , wherein said chemotherapeutic agent is an anti-neoplastic agent. 
     
     
         9 . The method of  claim 1 , wherein said chemotherapeutic agent and said compound are administered simultaneously. 
     
     
         10 . The method of  claim 1 , wherein said chemotherapeutic agent and said compound are administered sequentially. 
     
     
         11 . The method of  claim 1 , wherein said chemotherapeutic agent and said compound are administered via a route selected from the group consisting of intravenous, intramuscular, subcutaneous, rectal, intraocular, intrasynovial, transepithelial, transdermal, ophthalmic, sublingual, buccal, topically and inhalation via insufflation aerosol. 
     
     
         12 . The method of  claim 1 , further comprising co-administration of an HSP 90 inhibitor. 
     
     
         13 . A method for treatment of neoplastic disease in vivo comprising co-administration of a chemotherapeutic agent selected from the group consisting of temsirolimus, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), Ethylenimine/Methylmelamine, thriethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), hexamethylmelamine (HMM), altretamine busulfan, dacarbazine (DTIC), methotrexate, trimetrexate, 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine, 2,2′-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, 2-Chlorodeoxyadenosine (cladribine, 2-CdA)), camptothecin, topotecan, irinotecan, paclitaxel, vinblastine (VLB), vincristine, and vinorelbine, Taxotere®, docetaxel, estramustine, estramustine phosphate, etoposide, teniposide, doxorubicin (adriamycin), mitoxantrone, idarubicin, bleomycins; plicamycin (mithramycin), mitomycinC, dactinomycin, L-asparaginase, interferon-alpha, IL-2, G-CSF, GM-CSF, retinoic acid derivatives, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069, E09, RB 6145, SR4233, nicotinamide, 5-bromodeozyuridine, 5-iododeoxyuridine, bromodeoxycytidine, cisplatin, carboplatin, mitoxantrone, hydroxyurea, N-methylhydrazine (MIH), procarbazine, aminoglutethimide, interferon β, interferon γ, interleukin-2, prednisone, dexamethasone, aminoglutethimide, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethynyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, leuprolide, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, Npe6, tin etioporphyrin (SnET2), pheoboride-α, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, bortezomib (Velcade®), epothilone, serratamolide, imatinib mesylate, dasatinib, nilotinib, MK-0457, and Omacetaxine, cetuximab, remicade, herceptin and a compound of the formula 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts, wherein, R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of —C(R 6 R 7 )—C(R 8 R 9 )—, —CR 10 ═CR 11 —, and —C≡C—, wherein R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R a  and R b  are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkylcarbonyl, and alkyloxycarbonyl; or, optionally, R 1 , R 2 , R 3 , R 4 , R 5 , R a , and R b  are independently substituted with one member of a specific binding pair or a targeting ligand to facilitate targeting of the compound to a target tissue of interest, the compound being co-administered with a chemotherapeutic agent of interest and being effective to inhibit heat shock protein (HSP) 70 function. 
       
     
     
         14 . A method of inhibiting activity of HSP 70, comprising contacting the HSP 70 with a compound of the formula 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts, wherein, R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of —C(R 6 R 7 )—C(R 8 R 9 )—, —CR 10 ═CR 11 —, and —C≡C—, wherein R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R a  and R b  are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkylcarbonyl, and alkyloxycarbonyl; or, optionally, R 1 , R 2 , R 3 , R 4 , R 5 , R a , and R b  are independently substituted with one member of a specific binding pair or a targeting ligand to facilitate targeting of the compound to a target tissue of interest, the compound being co-administered with a chemotherapeutic agent of interest and being effective to inhibit heat shock protein (HSP) 70 function. 
       
     
     
         15 . The method of  claim 14  wherein said HSP70 function is selected from the group consisting of modulation of protein aggregation, chaperone protein binding, client protein binding, modulation of cellular stress response, modulation of autophagy, modulation of lysosomal functions, modulation of caspase cleavage, modulation of the proteasome system, reduced viability, modulation of anoikis, modulation of formation of detergent-insoluble subcellular complexes, modulation of NFκB activity, and modulation of vacuolization. 
     
     
         16 . A method for reducing bacterial load in a sample comprising contacting said sample with a compound of the formula 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts, wherein, R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of —C(R 6 R 7 )—C(R 8 R 9 )—, —CR 10 ═CR 11 —, and —C≡C—, wherein R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R a  and R b  are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, and dialkylamino; or, optionally, R 1 , R 2 , R 3 , R 4 , R 5 , R a , and R b  are independently substituted with one member of a specific binding pair or a targeting ligand, subjecting the sample to heating sufficient to induce a heat shock response, the combination of heat and compound administration being effective to inhibit DnaK activity in said bacteria. 
       
     
     
         17 . The method of  claim 16  further comprising administration of an antimicrobial selected from the group consisting of gentamycin, kanamycin, neomycin, streptomycin, cefazolin, vancomycin, azithromycin, cephalosporin, clarithromycin, erythromycin, spectinomycin, penicillin, amoxicillin, bacitran, and tetracycline. 
     
     
         18 . The method of  claim 13 , further comprising subjecting the patient to heat treatment. 
     
     
         19 . A method for the treatment of neoplastic disease in a patient in need thereof comprising exposing cancer cells to effective amounts of radiation and a compound of the formula 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts, wherein, R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl, hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxyl, alkoxycarbonyl, alkylcarbonyl, alkylcarbonyloxy, guanidino, phosphate, sulfamido and sulfonamido; Q represents a divalent linking moiety selected from the group consisting of —C(R 6 R 7 )—C(R 8 R 9 )—, —CR 10 ═CR 11 —, and —C≡C—, wherein R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  represent a radical selected from the group consisting of hydrogen and optionally substituted alkyl; R a  and R b  are the same or different and represent hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyl, alkoxy, amino, monoalkylamino, and dialkylamino; or, optionally, R 1 , R 2 , R 3 , R 4 , R 5 , R a , and R b  are independently substituted with one member of a specific binding pair or a targeting ligand, said radiation and said compound effectively killing cancer cells. 
       
     
     
         20 . The method of  claim 19 , wherein said radiation is selected from the group consisting of x-rays, gamma rays, neutron radiation, external-beam therapy, internal radiation therapy, implant radiation, brachytherapy, and systemic radiation. 
     
     
         21 . The method of  claim 4 , wherein said HSP90 client protein is selected from the group consisting of EGFR, HER2/ErbB2, AKT. 
     
     
         22 . The method of  claim 19 , optionally further comprising administration of an agent selected from the group consisting of herceptin, cetuximab and remicade. 
     
     
         23 . A compound of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts thereof, wherein R represents a substituent selected from the group consisting of chloro, fluoro, alkyl (C 1 -C 4 ), trifluoromethyl, amino, carboxy, hydroxyl and methoxy; and R′ and R″ are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl (C 1 -C 6 ), hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl and alkylcarbonyloxy; R a  and R b  are the same or different and represent a radical selected from the group of hydrogen, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkoxycarbonyl, alkylcarbonyl and optionally substituted alkyl (C 1 -C 6 ), said alkyl substituent being at least one selected from the group consisting of hydroxyl, thio, alkoxy, alkylthio, halogen, amino, monalkylamino, dialkylamino, guanidino, phosphate, amido, nitro, carboxyl, sulfamido, sulfonamido, alkoxycarbonyl, alkylcarbonyl, and alkylcarbonyloxy. 
       
     
     
         24 . A compound, according to  claim 23 , of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts thereof, wherein R represents a radical selected from the group of chloro, fluoro, amino, carboxy, hydroxy and methoxy. 
       
     
     
         25 . A compound, according to  claim 24 , wherein R represents chloro. 
     
     
         26 . A method for treatment of neoplastic disease in a patient in need thereof, said method comprising administration of an effective amount of compound of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts thereof, wherein R represents a substituent selected from the group consisting of chloro, fluoro, alkyl (C 1 -C 4 ), trifluoromethyl, amino, carboxy, hydroxyl and methoxy; and R′ and R″ are the same or different and represent a radical selected from the group of hydrogen, optionally substituted alkyl (C 1 -C 6 ), hydroxyl, alkoxy, thio, alkylthio, halogen, amino, monoalkylamino, dialkylamino, amido, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl and alkylcarbonyloxy; R a  and R b  are the same or different and represent a radical selected from the group of hydrogen, hydroxyl, alkoxy, amino, monoalkylamino, dialkylamino, carboxy, alkoxycarbonyl, alkylcarbonyl and optionally substituted alkyl (C 1 -C 6 ), said alkyl substituent being at least one selected from the group consisting of hydroxyl, thio, alkoxy, alkylthio, halogen, amino, monalkylamino, dialkylamino, guanidino, phosphate, amido, nitro, carboxyl, sulfamido, sulfonamido, alkoxycarbonyl, alkylcarbonyl, and alkylcarbonyloxy; and wherein the compound is optionally co-administered with at least one chemotherapeutic agent. 
       
     
     
         27 . The method of  claim 26 , wherein the compound is of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts thereof, wherein R represents a radical selected from the group of chloro, fluoro, amino, carboxy, hydroxy and methoxy. 
       
     
     
         28 . The method of  claim 27 , wherein R represents chloro. 
     
     
         29 . The method of  claim 26 , wherein the at least one chemotherapeutic agent is selected from the group consisting of temsirolimus, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), Ethylenimine/Methylmelamine, thriethylenemelamine (TEM), triethylene thiophosphoramide (thiotepa), hexamethylmelamine (HMM), altretamine busulfan, dacarbazine (DTIC), methotrexate, trimetrexate, 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine, 2,2′-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, 2-Chlorodeoxyadenosine (cladribine, 2-CdA)), camptothecin, topotecan, irinotecan, paclitaxel, vinblastine (VLB), vincristine, and vinorelbine, Taxotere®, docetaxel, estramustine, estramustine phosphate, etoposide, teniposide, doxorubicin (adriamycin), mitoxantrone, idarubicin, bleomycins; plicamycin (mithramycin), mitomycinC, dactinomycin, L-asparaginase, interferon-alpha, IL-2, G-CSF, GM-CSF, retinoic acid derivatives, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069, E09, RB 6145, SR4233, nicotinamide, 5-bromodeozyuridine, 5-iododeoxyuridine, bromodeoxycytidine, cisplatin, carboplatin, mitoxantrone, hydroxyurea, N-methylhydrazine (MIH), procarbazine, aminoglutethimide, interferon β, interferon γ, interleukin-2, prednisone, dexamethasone, aminoglutethimide, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol, ethynyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, leuprolide, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, Npe6, tin etioporphyrin (SnET2), pheoboride-α, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, bortezomib (Velcade®), epothilone, serratamolide, imatinib mesylate, dasatinib, nilotinib, and MK-0457. 
     
     
         30 . The method of  claim 29 , wherein the at least one chemotherapeutic agent is temsirolimus. 
     
     
         31 . A compound of the formula: 
       
         
           
           
               
               
           
         
         and pharmaceutically acceptable salts thereof, wherein one of R 1 -R 5  represents halogen and the others represent hydrogen, R a  and R b  are the same or different and each represents a radical selected from the group of hydrogen and optionally substituted alkyl (C 1 -C 6 ), and wherein said halogen is selected from the group consisting of chlorine, bromine, iodine and fluorine, with the proviso that R a  and R b  are not both hydrogen.