WO2016014890A1 - Treatment of multiple myeloma with heterocyclic inhibitors of glutaminase - Google Patents

Treatment of multiple myeloma with heterocyclic inhibitors of glutaminase Download PDF

Info

Publication number
WO2016014890A1
WO2016014890A1 PCT/US2015/041891 US2015041891W WO2016014890A1 WO 2016014890 A1 WO2016014890 A1 WO 2016014890A1 US 2015041891 W US2015041891 W US 2015041891W WO 2016014890 A1 WO2016014890 A1 WO 2016014890A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
arylalkyl
unsubstituted
heteroaryl
alkoxy
Prior art date
Application number
PCT/US2015/041891
Other languages
French (fr)
Inventor
Andrew L. MACKINNON
Mirna L. RODRIGUEZ
Original Assignee
Calithera Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Calithera Biosciences, Inc. filed Critical Calithera Biosciences, Inc.
Publication of WO2016014890A1 publication Critical patent/WO2016014890A1/en

Links

Classifications

    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/424—Oxazoles condensed with heterocyclic ring systems, e.g. clavulanic acid
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Glutamine supports cell survival, growth and proliferation through metabolic and non-metabolic mechanisms.
  • glutamine to lactate also referred to as "glutaminolysis" is a major source of energy in the form of NADPH.
  • the first step in glutaminolysis is the deamination of glutamine to form glutamate and ammonia, which is catalyzed by the glutaminase enzyme (GLS).
  • GLS glutaminase enzyme
  • glutaminase has been theorized to be a potential therapeutic target for the treatment of diseases characterized by actively proliferating cells, such as cancer.
  • the lack of suitable glutaminase inhibitors has made validation of this target impossible until the recent creation of compounds that are specific and capable of being formulated for in vivo use (US 8,604,016).
  • glutaminase inhibitors enter the clinical arena, methods are needed to identify patients that would best benefit from treatment with these compounds.
  • the present invention provides a method for treating multiple myeloma in a patient, comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of a glutaminase inhibitor.
  • the glutaminase inhibitor is a compound of formula I,
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 , CH 2 S, SCH 2 , CH 2 NHCH 2 ,
  • CH CH, or ⁇ -f ⁇ , preferably CH 2 CH 2 , wherein any hydrogen atom of a
  • CH or CH 2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH 2 unit of CH 2 CH 2 , CH 2 CH 2 CH 2 or CH 2 may be replaced by hydroxy;
  • CH CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
  • Y independently for each occurrence, represents H or CH 2 0(CO)R 7 ;
  • R 7 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
  • Z represents H or R 3 (CO);
  • Ri and R 2 each independently represent H, alkyl, alkoxy or hydroxy
  • R 3 independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
  • R 4 and R 5 each independently represent H or substituted or unsubstituted alkyl
  • hydroxyalkyl acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form
  • C(0)R 7 represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form C(0)R 7 ;
  • Rg, R and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl or R 8 and R 9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R 7 , and wherein at least two of
  • the invention relates to methods of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
  • FIG 1 shows the correlation between glutamine-dependence
  • Figure 2 shows single-agent compound 402 treatment of MDA-MB-231 orthotopic xenograft model.
  • Figure 3 shows a combination study with compound 389 and paclitaxel in MDA-MB-231 orthotopic xenograft model.
  • Figure 4 shows results of the median glutaminase:glutamine synthetase expression ratio in various cancer types, including colorectal cancer, renal cancer, lymphoma, melanoma and myeloma.
  • Figure 5 shows that intraperitoneal administration of compound 188 to mice results in reduced tumor size in a HCTl 16 colon carcinoma xenograft model.
  • Figure 6 shows that oral administration of compound 670 to mice results in reduced tumor size in a H2122 lung adenocarcinoma xenograft model.
  • Figure 7 shows that oral administration of compound 670 to mice results in reduced tumor size in a RPMI-8226 multiple myeloma xenograft model.
  • Figure 8 shows that compound 670 synergizes with pomalidomide or dexamethasone to produce an anti-tumor effect in multiple myeloma cells.
  • Figure 9 shows a waterfall plot demonstrating the response variation to CB- 839 treatment in a panel of multiple myeloma tumor cell lines.
  • Figure 10 shows a western blot for pyruvate carboxylase in a panel of multiple myeloma cell lines.
  • Figure 11 shows a correlation between pyruvate carboxylase protein level and cell growth in response to CB-839 treatment relative to no CB-839 treatment/DMSO control.
  • Figure 12 shows pyruvate carboxylase expression values generated from R A-seq data for various multiple myeloma cell lines correlates with pyruvate carboxylase protein levels.
  • the present invention is based on the discovery of a biomarker in multiple myeloma cell lines that indicates sensitivity of the multiple myeloma cells to treatment with a glutaminase inhibitor. As shown in data presented herein, not all multiple myeloma cell lines exhibit the same sensitivity to treatment with a glutaminase inhibitor. Further, this effect is not observed in certain other types of cancer cell lines (data not shown).
  • the present invention provides a method for treating multiple myeloma in a patient, comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of a glutaminase inhibitor.
  • the glutaminase inhibitor is a compound of formula I,
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 , CH 2 S, SCH 2 , CH 2 NHCH 2 ,
  • CH CH, or ⁇ -f ⁇ , preferably CH 2 CH 2 , wherein any hydrogen atom of a
  • CH or CH 2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH 2 unit of CH 2 CH 2 , CH 2 CH 2 CH 2 or CH 2 may be replaced by hydroxy;
  • CH CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
  • Y independently for each occurrence, represents H or CH 2 0(CO)R 7 ;
  • R 7 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
  • Z represents H or R 3 (CO);
  • Ri and R 2 each independently represent H, alkyl, alkoxy or hydroxy
  • R 3 independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
  • R 4 and R 5 each independently represent H or substituted or unsubstituted alkyl
  • hydroxyalkyl acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form
  • C(0)R 7 represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form C(0)R 7 ;
  • Rg, R and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl or R 8 and R 9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R 7 , and wherein at least two of
  • perfluoroalkoxy e.g., trifluoromethoxy
  • alkoxyalkoxy e.g., hydroxyalkyl
  • carboxyalkyl alkoxycarbonylalkyl, formylalkyl, or acylalkyl, including
  • perfluoroacylalkyl e.g., -alkylC(0)CF 3
  • carbamate carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate, sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro, azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester, thioacetate, or
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 , CH 2 S, SCH 2 , or CH 2 NHCH 2 , wherein any hydrogen atom of a CH 2 unit may be replaced by alkyl or alkoxy, and any hydrogen atom of a CH 2 unit of CH 2 CH 2 , CH 2 CH 2 CH 2 or CH 2 may be replaced by hydroxyl.
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2 .
  • L represents CH 2 CH 2 .
  • L is not CH 2 SCH 2 .
  • Y represents H.
  • Z represents R 3 (CO). In certain embodiments wherein Z is R 3 (CO), each occurrence of R 3 is not identical (e.g., the compound of formula I is not symmetrical).
  • Ri and R 2 each represent H.
  • R 3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
  • R 3 represents C(Rg)(R9)(Rio), wherein R 8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, R 9 represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy.
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2 , such as CH 2 CH 2 , CH 2 S or SCH 2
  • Y represents H
  • X represents S
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • each R 3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
  • each occurrence of R 3 is identical.
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2
  • Y represents H
  • X represents S
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • each R 3 represents C(R 8 )(R 9 )(Rio), wherein R 8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl
  • R 9 represents H
  • Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy.
  • each occurrence of R 3 is identical.
  • L represents CH 2 CH 2
  • Y represents H
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • each R 3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
  • each X represents S.
  • each occurrence of R 3 is identical.
  • the two occurrences of R 3 are not identical.
  • L represents CH 2 CH 2
  • Y represents H
  • X represents S
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • each R 3 represents
  • R 8 represents aryl, arylalkyl or heteroaryl
  • R 9 represents H
  • Rio represents hydroxy, hydroxyalkyl or alkoxy.
  • R 8 represents aryl and Rio represents hydroxyalkyl.
  • each occurrence of R 3 is identical.
  • both R 3 groups are not alkyl, such as methyl, or C(R 8 )(R9)(Rio), wherein R 8 , R 9 andRio are each independently hydrogen or alkyl.
  • both R 3 groups are not phenyl or heteroaryl, such as 2-furyl.
  • both R 3 groups are not N(R 4 )(R 5 ) wherein R 4 is aryl, such as phenyl, and R 5 is H.
  • both R 3 groups are not aryl, such as optionally substituted phenyl, aralkyl, such as benzyl, heteroaryl, such as 2-furyl, 2-thienyl or 1,2,4-trizole, substituted or unsubstituted alkyl, such as methyl, chloromethyl, dichloromethyl, n- propyl, n-butyl, t-butyl or hexyl, heterocyclyl, such as pyrimidine-2,4(lH,3H)-dione, or alkoxy, such as methoxy, pentyloxy or ethoxy.
  • both R 3 groups are not N(R 4 )(R 5 ) wherein R 4 is aryl, such as substituted or unsubstituted phenyl (e.g., phenyl, 3-tolyl, 4-tolyl, 4-bromophenyl or 4- nitrophenyl), and R 5 is H.
  • both R 3 groups are not alkyl, such as methyl, ethyl, or propyl, cycloalkyl, such as cyclohexyl, or C(Rg)(R9)(Rio), wherein any of Rg, R9 and Rio together with the C to which they are attached, form any of the foregoing.
  • the compound is not one of the following:
  • the glutaminase inhibitor is a compound of formula la,
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 , CH 2 S, SCH 2 , CH 2 NHCH 2 ,
  • CH CH, or ⁇ -f ⁇ , preferably CH 2 CH 2 , wherein any hydrogen atom of a CH or CH 2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH 2 unit of CH 2 CH 2 , CH 2 CH 2 CH 2 or CH 2 may be replaced by hydroxy;
  • Y independently for each occurrence, represents H or CH 2 0(CO)R 7 ;
  • R 7 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
  • Z represents H or R 3 (CO);
  • Ri and R 2 each independently represent H, alkyl, alkoxy or hydroxy, preferably H;
  • R 3 represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl,
  • acylaminoalkyl alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heteroaryloxyalkyl or
  • R 4 and R 5 each independently represent H or substituted or unsubstituted alkyl
  • hydroxyalkyl acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form C(0)R 7 ;
  • alkyl represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form C(0)R 7 ;
  • R 8 , R9 and R 10 each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl or Rg and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R 7 , and wherein at least two of R 8 , R 9 and R 10 are not H;
  • R 11 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl,
  • heteroaryl heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, or
  • Ri 2 and R13 each independently respresent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
  • heteroaryloxyalkyl wherein any free hydroxyl group may be acylated to form C(0)R 7 , and wherein both of Ri 2 and R13 are not H;
  • Ri 4 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl,
  • heteroaryl heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl.
  • perfluoroalkoxy e.g., trifluoromethylalkoxy
  • alkoxyalkoxy hydroxyalkyl, hydroxyalkylamino, hydroxyalkoxy, amino, aminoalkyl, alkylamino, aminoalkylalkoxy, aminoalkoxy, acylamino, acylaminoalkyl, such as perfluoro acylaminoalkyl (e.g., trifluoromethylacylaminoalkyl), acyloxy, cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy, heteroaryloxyalkyl, heterocyclylaminoalkyl, heterocyclylaminoalkoxy, amido, amidoalkyl, amidine, imine, oxo, carbony
  • carboxyalkyl alkoxycarbonylalkyl, formylalkyl, or acylalkyl, including
  • perfluoroacylalkyl e.g., -alkylC(0)CF 3
  • carbamate carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate, sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro, azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester, thioacetate, or
  • Rn represents substituted or unsubstituted arylalkyl, such as substituted or unsubstituted benzyl.
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 ,
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2, preferably CH 2 CH 2 . In certain embodiments, L is not CH 2 SCH 2 .
  • each Y represents H. In other embodiments, at least one Y is CH 2 0(CO)R 7 .
  • Ri and R 2 each represent H.
  • Z represents R 3 (CO). In certain embodiments wherein Z is R 3 (CO), R 3 and Rn are not identical (e.g., the compound of formula I is not symmetrical).
  • Z represents R 3 (CO) and R 3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
  • Z represents R 3 (CO) and R 3 represents C(Rg)(R9)(Rio), wherein R 8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, Rg represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy.
  • Z represents R 3 (CO) and R 3 represents heteroarylalkyl.
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2 , such as CH 2 CH 2
  • Y represents H
  • X represents S
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • R 3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl
  • Rii represents arylalkyl.
  • R 3 represents
  • L represents CH 2 SCH 2 , CH 2 CH 2 , CH 2 S or SCH 2 , such as CH 2 CH 2
  • Y represents H
  • X represents S
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • R 3 represents C(Rg)(R9)(Rio)
  • R 8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl
  • Rg represents H
  • Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy
  • Rn represents arylalkyl.
  • Rs represents heteroaryl.
  • L represents CH 2 CH 2
  • Y represents H
  • Z represents R 3 (CO)
  • Ri and R 2 each represent H
  • R 3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl
  • Rn represents arylalkyl.
  • R 3 represents
  • L represents CH 2 CH 2
  • Y represents H
  • X represents
  • S, Z represents R 3 (CO), Ri and R 2 each represent H, R 3 represents C(R 8 )(R9)(Rio), wherein R 8 represents aryl, arylalkyl or heteroaryl, R 9 represents H, and Rio represents hydroxy, hydroxyalkyl or alkoxy, and Rn represents arylalkyl.
  • R 8 represents aryl and Rio represents hydroxyalkyl.
  • Rg represents heteroaryl.
  • compounds of the invention may be prodrugs of the compounds of formula I or la, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate, or carboxylic acid present in the parent compound is presented as an ester.
  • the prodrug is metabolized to the active parent compound in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid).
  • compounds of the invention may be racemic. In certain embodiments, compounds of the invention may be enriched in one
  • a compound of the invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95% or greater ee.
  • compounds of the invention may have more than one stereocenter.
  • compounds of the invention may be enriched in one or more diastereomer.
  • a compound of the invention may have greater than 30% de, 40% de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.
  • the present invention relates to methods of treating or preventing multiple myeloma with a compound of formula I or la, or a
  • the therapeutic preparation may be enriched to provide predominantly one enantiomer of a compound (e.g., of formula I or la).
  • An enantiomerically enriched mixture may comprise, for example, at least 60 mol percent of one enantiomer, or more preferably at least 75, 90, 95, or even 99 mol percent.
  • the compound enriched in one enantiomer is substantially free of the other enantiomer, wherein substantially free means that the substance in question makes up less than 10%>, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% as compared to the amount of the other enantiomer, e.g., in the composition or compound mixture.
  • substantially free means that the substance in question makes up less than 10%>, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% as compared to the amount of the other enantiomer, e.g., in the composition or compound mixture.
  • a composition or compound mixture contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it would be said to contain 98 mol percent of the first enantiomer and only 2% of the second enantiomer.
  • the therapeutic preparation may be enriched to provide predominantly one diastereomer of a compound (e.g., of formula I or la).
  • a diastereomerically enriched mixture may comprise, for example, at least 60 mol percent of one diastereomer, or more preferably at least 75, 90, 95, or even 99 mol percent.
  • the present invention provides a pharmaceutical preparation suitable for use in a human patient in the treatment of multiple myeloma, comprising an effective amount of any of the compounds shown above (e.g., a compound of the invention, such as a compound of formula I or la), and one or more pharmaceutically acceptable excipients.
  • the pharmaceutical preparations may be for use in treating or preventing a condition or disease as described herein.
  • the pharmaceutical preparations have a low enough pyrogen activity to be suitable for use in a human patient.
  • Compounds of any of the above structures may be used in the manufacture of medicaments for the treatment of any diseases or conditions disclosed herein.
  • the invention relates to methods of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
  • the glutaminase inhibitor is a compound of formula I or formula la.
  • Methods of the invention include a step of comparing the level of pyruvate carboxylase in a multiple myeloma patient sample to a reference standard.
  • the reference standard is a level of pyruvate carboxylase in a multiple myeloma cell line that is resistant to a compound of formula (II), also referred to herein as CB-839:
  • the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells.
  • Glutamine plays an important role as a carrier of nitrogen, carbon, and energy.
  • GS glutamine synthetase
  • the conversion of glutamine into glutamate is initiated by the mitochondrial enzyme, glutaminase.
  • glutaminase There are two major forms of the enzyme, K-type and L-type, which are distinguished by their Km values for glutamine and response to glutamate, wherein the Km value, or Michaelis constant, is the concentration of substrate required to reach half the maximal velocity.
  • the L-type also known as “liver-type” or GLS2
  • the K-type also known as "kidney-type” or GLS1 or “KGA”
  • GLS1 an alternative splice form of GLS1 referred to as
  • GAC glutaminase C
  • gene expression analysis of breast cancers has identified five intrinsic subtypes (luminal A, luminal B, basal, HER2+, and normal-like) (Sorlie et al., Proc Natl Acad Sci USA, 2001).
  • glutamine deprivation has an impact on cell growth and viability, basal-like cells appear to be more sensitive to the reduction of exogenous glutamine (Kung et al, PLoS Genetics, 2011).
  • FIG. 1 This supports the concept that glutamine is a very important energy source in basal-like breast cancer cell lines, and suggests that inhibition of the glutaminase enzyme would be beneficial in the treatment of breast cancers comprised of basal-like cells.
  • Figure 1 further supports the correlation that cells dependent on exogenous glutamine are susceptible to the presence of a glutaminase inhibitor in a series of breast cancer cell lines.
  • a xenograft of glutamine dependent cells, MD-MB-231 demonstrated a sensitivity to glutaminase inhibition (Figure 2).
  • the xenograft model showed some variablility in the sensitivity to glutaminase inhibiton; however, the sensitivity could be enhanced further when combined with another agent (Figure 3).
  • Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of cancers selected from colorectal cancer, endocrine cancer, lung cancer, melanoma, mesothelioma, renal cancer and B cell malignancies. Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of multiple myeloma, leukemia (including acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL)), or lymphoma (including Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma and Hodgkin's lymphoma).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • lymphoma including Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma and Hodgkin's lymphoma.
  • Xenograft models of colon cancer, lung cancer and multiple myeloma showed sensitivity to treatment with a glutaminase inhibitor ( Figures 5, 6, and 7), further supporting the concept that certain cancers may prove to be more responsive to treatment with glutaminase inhibitors. It was decided to further examine the dependence of multiple myeloma cells on exogenous glutamine and the sensitivity to glutaminase inhibition.
  • the invention provides a method of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient indicates that the patient may benefit from treatment with a glutaminase inhibitor.
  • the invention provides a method of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
  • the multiple myeloma reference standard is normal tissue from the same patient.
  • the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells.
  • the glutaminase inhibitor is a compound of formula I. In other embodiments, the glutaminase inhibitor is a compound of formula la.
  • the invention provides a method of treating a multiple myeloma patient, the method comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of glutaminase inhibitor.
  • the multiple myeloma reference standard is normal tissue from the same patient.
  • the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells.
  • the glutaminase inhibitor is a compound of formula I. In other embodiments, the glutaminase inhibitor is a compound of formula la.
  • the method of treating or preventing cancer may comprise administering a compound of the invention conjointly with one or more other chemotherapeutic agent(s).
  • Chemotherapeutic agents that may be conjointly administered with compounds of the invention include: ABT-263, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, AZD5363, Bacillus Calmette-Guerin vaccine (beg), bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisp latin, cladribine, clodronate, cobimetinib, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, d
  • fludrocortisone fluorouracil and 5-fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ixabepilone, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, mutamycin, MK-2206, nilutamide, nocodazole, octreotide, oxaliplatin, olaparib, paclitaxel, pamidronate, pentostatin, per
  • chemotherapeutic agents that may be conjointly administered with compounds of the invention include: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine,
  • cyclophosphamide cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,
  • the chemotherapeutic agent conjointly administered with compounds of the invention is a taxane chemotherapeutic agent, such as paclitaxel or docetaxel.
  • the chemotherapeutic agent conjointly administered with compounds of the invention is doxorubicin.
  • a compound of the invention is administered conjointly with a taxane chemotherapeutic agent (e.g., paclitaxel) and doxorubicin.
  • combination therapies have been developed for the treatment of cancer.
  • compounds of the invention may be conjointly administered with a combination therapy.
  • Examples of combination therapies with which compounds of the invention may be conjointly administered are included in Table 1.
  • Table 1 Exemplary combinatorial therapies for the treatment of cancer.
  • BCVPP Carmustine, Cyclophosphamide, Vinblastine,
  • lymphocytic leukemia lymphocytic leukemia
  • PVB Cisplatin Vinblastine, Bleomycin
  • PVDA Prednisone Vincristine, Daunorubicin, Asparaginase
  • VCAP Vincristine Cyclophosphamide
  • Doxorubicin Prednisone
  • the compounds of the invention may be conjointly administered with a chemotherapeutic agent selected from inhibitors of another metabolic enzyme, such as a glucose transporter, hexokinase, pyruvate kinase M2, lactate dehydrogenase 1 or 2, pyruvate dehydrogenase kinase, fatty acid synthase, or glutaminase.
  • a chemotherapeutic agent selected from inhibitors of another metabolic enzyme, such as a glucose transporter, hexokinase, pyruvate kinase M2, lactate dehydrogenase 1 or 2, pyruvate dehydrogenase kinase, fatty acid synthase, or glutaminase.
  • the compounds of the invention may be conjointly administered with an immune -oncology therapeutic, such as an inhibitor of arginase, CTLA-4, indoleamine 2,3-dioxygenase, and PD-1/PD-L1.
  • an immune -oncology therapeutic such as an inhibitor of arginase, CTLA-4, indoleamine 2,3-dioxygenase, and PD-1/PD-L1.
  • the immune -oncology agent is abagovomab, adecatumumab, afutuzumab, anatumomab mafenatox, apolizumab, blinatumomab, catumaxomab, durvalumab, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab, nivolumab, ocaratuzumab, olatatumab, pembrolizumab, pidilizumab, ticilimumab, samalizumab, or tremelimumab.
  • the compounds of the invention may be conjointly administered with an immunomodulatory agent.
  • immunomodulatory agents with which the compounds of the invention may be administered in a combination therapy include granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod, IL-2, IL-7, IL-12, various chemokines, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans, and synthetic small molecules such as apremilast, CC-122, CC-11006, CC-10015, lenalidomide, pomalidomide, and thalidomide.
  • the immunomodulatory agent is a thalidomide analog, such as those disclosed in WO 1999/46258, WO 2008/033567, WO 2010/093434, WO 2010/093605, WO 2011/100380, and WO 2012/097116.
  • the compounds of the invention may be conjointly administered with an anticancer agent selected from an enzyme inhibitor (such as a kinase inhibitor), a mitotic inhibitor, a DNA-modifying agent, and a cytidine analog.
  • an enzyme inhibitor such as a kinase inhibitor
  • anticancer agents with which the compounds of the invention may be administered in a combination therapy include microtubule assembly inhibitors, AKT inhibitors, mTOR inhibitors, MEK inhibitors, RTK inhibitors, ATM inhibitors, ATR inhibitors, PI3K inhibitors, EGFR inhibitors, B-Raf inhibitors, C-kit inhibitors, DNA cross-linking agents, DNA intercalating agents, and cytidine analogs.
  • the anticancer agents include microtubule assembly inhibitors, AKT inhibitors, mTOR inhibitors, MEK inhibitors, RTK inhibitors, ATM inhibitors, ATR inhibitors, PI3K inhibitors, EGFR inhibitors, B-Raf inhibitors, C-kit inhibitors, DNA cross-linking agents, PARP inhibitors, DNA intercalating agents, or cytidine analogs.
  • the anticancer agent is vincristine, carboplatin, cisplatin, gemcitabine, MK2206, everolimus, trametinib, sunitinib, sorafenib, BEZ235, paclitaxel, docetaxel, erlotinib, selumetinib, sirolimus, trametinib, temsirolimus, pazopanib, or GSK1120212.
  • the anticancer agent is vincristine, carboplatin, cisplatin, gemcitabine, MK2206, everolimus, trametinib, sunitinib, sorafenib, BEZ235, paclitaxel, docetaxel, erlotinib, selumetinib, sirolimus, trametinib, temsirolimus, pazopanib, olaparib, or GSK1120212.
  • the compounds of the invention are coadministered with one or more of lenalidomide, pomalidomide, and dexamethasone in the treatment of multiple myeloma.
  • coadministration produces a synergistic effect.
  • acetyl- coA used for lipid synthesis is formed from a mitochondrial pool of pyruvate that is derived from glycolysis. Yet under hypoxic conditions, such as those normally found in a tumor environment, the conversion of pyruvate to acetyl-coA within the mitochondria is downregulated.
  • hypoxic conditions such as those normally found in a tumor environment
  • acetyl-coA within the mitochondria is downregulated.
  • Recent studies from Metallo et al. (2011) and Mullen et al. (2011) revealed that under such hypoxic conditions, cells instead largely switch to using a pathway involving the reductive carboxylation of alpha-ketoglutarate to make acetyl-coA for lipid synthesis.
  • the first step in this pathway involves converting glutamine to glutamate via glutaminase enzymes.
  • glutamate is converting to alpha-ketoglutarate, and the resulting alpha-ketoglutarate is converted to isocitrate in a reductive carboxylation step mediated by the isocitrate dehydrogenase enzymes.
  • a switch to this reductive carboxylation pathway also occurs in some renal carcinoma cell lines that contain either impaired mitochondria or an impaired signal for induction of the enzyme responsible for converting glycolytic pyruvate to acetyl- coA (Mullen et al 2011).
  • mitochondrial respiratory chain inhibitors such as metformin, rotenone, and antimycin (Mullen at al. 2011). Therefore, in some embodiments of this invention, we propose using combinations of mitochondrial respiratory chain inhibitors and glutaminase inhibitors to simultaneously increase cancer cells' dependence on glutaminase-dependent pathways for lipid synthesis while inhibiting those very pathways.
  • glycolytic pathway inhibitors include 2-deoxyglucose, lonidamine, 3-bromopyruvate, imatinib, oxythiamine, rapamycin, and their pharmacological equivalents.
  • Glycolysis can be inhibited indirectly by depleting NAD+ via DNA damage induced by DNA alkylating agents through a pathway activated by poly(ADP-ribose) polymerase (Zong et al. 2004). Therefore, in some embodiments of this invention, we propose using a combination of DNA alkylating agents and glutaminase inhibitors. Cancer cells use the pentose phosphate pathway along with the glycolytic pathway to create metabolic
  • a compound of the invention may be conjointly administered with non-chemical methods of cancer treatment.
  • a compound of the invention may be conjointly administered with radiation therapy.
  • a compound of the invention may be conjointly administered with surgery, with thermoablation, with focused ultrasound therapy, with cryotherapy, or with any combination of these.
  • different compounds of the invention may be conjointly administered with one or more other compounds of the invention.
  • Such combinations may be conjointly administered with other therapeutic agents, such as other agents suitable for the treatment of cancer, immunological or neurological diseases, such as the agents identified above.
  • conjointly administering one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect, such as shown in Figure 8.
  • conjointly administering one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect, such as shown in Figure 8.
  • conjointly administering one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect, such as shown in Figure 8.
  • conjointly administering one or more additional chemotherapeutic agents provides a synergistic effect, such as shown in Figure 8.
  • conjointly administering one or more additional chemotherapeutic agents provides a synergistic effect, such as shown in Figure 8.
  • chemotherapeutics agents provides an additive effect.
  • the present invention provides a kit comprising: a) one or more single dosage forms of a compound of the invention; b) one or more single dosage forms of a chemotherapeutic agent as mentioned above; and c) instructions for the administration of the compound of the invention and the chemotherapeutic agent for the treatment of cancer, wherein the cancer is multiple myeloma.
  • the present invention provides a kit comprising:
  • a pharmaceutical formulation e.g., one or more single dosage forms
  • kits further comprises instructions for the administration of the pharmaceutical formulation, e.g., for treating or preventing cancer, such as multiple myeloma.
  • the kit further comprises instructions for the administration of the pharmaceutical formulation comprising a compound of the invention conjointly with a chemotherapeutic agent as mentioned above.
  • the kit further comprises a second pharmaceutical formulation (e.g., as one or more single dosage forms) comprising a chemotherapeutic agent as mentioned above.
  • Protein amounts can be measured using antibodies.
  • Antibodies suitable for use in the methods disclosed herein are commercially available, or can be prepared routinely. Methods for preparing and using antibodies in assays for proteins of interest are conventional, and are described in, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), (Humana Press 1992); Coligan et al, in Current Protocols in Immunology, Sec. 2.4.1 (1992); Kohler & Milstein (1975), Nature 256, 495; Coligan et al, sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Laboratory Pub. 1988).
  • antibodies can be used in methods of the invention.
  • Such antibodies include, for example, polyclonal, monoclonal (mAbs), recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof.
  • the antibodies can be of any isotype, e.g., IgM, various IgG isotypes such as IgGl, IgG2a, etc., and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like.
  • the term "an antibody specific for" a protein means that the antibody recognizes a defined sequence of amino acids, or epitope, in the protein, and binds selectively to the protein and not generally to proteins unintended for binding to the antibody. The parameters required to achieve specific binding can be determined routinely, using conventional methods in the art.
  • antibodies specific for pyruvate carboxylase are immobilized on a surface (e.g., are reactive elements on an array, such as a microarray, or are on another surface, such as used for surface plasmon resonance (SPR)-based technology, such as Biacore), and proteins in the sample are detected by virtue of their ability to bind specifically to the antibodies.
  • proteins in the sample can be immobilized on a surface, and detected by virtue of their ability to bind specifically to the antibodies.
  • immunoassays include immunohistochemical staining, ELISA, Western blot (immunoblot), immunoprecipitation,
  • RIA radioimmunoassay
  • FACS fluorescence-activated cell sorting
  • expression levels of pyruvate carboxylase can be measured by measuring mRNA amounts.
  • the amount of an mRNA encoding a pyruvate carboxylase can be measured using any suitable method. Examples of such methods include, for example, reverse transcriptase-polymerase chain reaction (RT- PCR), including real time PCR, microarray analysis, nanostring, Northern blot analysis, differential hybridization, and ribonuclease protection assay.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • a histological sample is obtained from a subject (e.g., from a tumor biopsy), using any method known in the art, and include, but are not limited to, tissue section, needle biopsy, and the like. Frequently the sample will be a "clinical sample", which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes.
  • the sample can also be derived from supernatants (of cells) or the cells themselves from cell cultures, cells from tissue culture and other media. Protein or mR A is then obtained brom the sample, and used to quantitate the amounts of pyruvate carboxylase.
  • kits for detecting whether a subject having a cancer is likely to be responsive to glutaminase inhibitors may include one or more agents for detecting the amount of expression of a protein of the invention [e.g., the amount of the protein, and/or the amount of a nucleic acid (e.g., an mRNA) encoding the protein].
  • the agents in the kit can encompass, for example, antibodies specific for the proteins, or probes specific for the mRNA that can be used to hybridize to the RNA (or to a cDNA generated from it) or to perform RT-PCR.
  • the kit may also include additional agents suitable for detecting, measuring and/or quantitating the amount of protein or nucleic acid.
  • kits of the invention can be used in experimental applications. A skilled worker will recognize components of kits suitable for carrying out a method of the invention.
  • kits of the invention may comprise instructions for performing the method.
  • Optional elements of a kit of the invention include suitable buffers, containers, or packaging materials.
  • the reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids.
  • the reagents may also be in single use form, e.g., for the performance of an assay for a single subject.
  • acyl is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
  • acylamino is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(0)NH-.
  • acyloxy is art-recognized and refers to a group represented by the general formula hydrocarbylC(0)0-, preferably alkylC(0)0-.
  • alkoxy refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto.
  • Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
  • alkoxyalkyl refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
  • alkenyl refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and “substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those
  • alkyl groups as discussed below, except where stability is prohibitive.
  • substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • alkyl group or “alkane” is a straight chained or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 unless otherwise defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl.
  • a Ci-C 6 straight chained or branched alkyl group is also referred to as a "lower alkyl" group.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having
  • substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF 3 , -CN and the like.
  • Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls, -CF 3 , -CN, and the like.
  • C x _ y when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain.
  • C x _ y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.
  • Co alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal.
  • the terms "C 2 - y alkenyl” and “C 2 _ y alkynyl” refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • alkylamino refers to an amino group substituted with at least one alkyl group.
  • alkylthio refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.
  • alkynyl refers to an aliphatic group containing at least one triple bond and is intended to include both “unsubstituted alkynyls” and “substituted alkynyls”, the latter of which refers to alkynyl moieties having
  • substituents replacing a hydrogen on one or more carbons of the alkynyl group may occur on one or more carbons that are included or not included in one or more triple bonds.
  • substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • amide refers to a group
  • each R independently represent a hydrogen or hydrocarbyl group, or two R 10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by
  • each R 10 independently represents a hydrogen or a hydrocarbyl group, or two R 10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • aminoalkyl refers to an alkyl group substituted with an amino group.
  • aralkyl refers to an alkyl group substituted with an aryl group.
  • aryl as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
  • R 9 and R 10 independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, or R 9 and R 10 taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • carbocycle refers to a saturated or unsaturated ring in which each atom of the ring is carbon.
  • carbocycle includes both aromatic carbocycles and non-aromatic carbocycles.
  • Non- aromatic carbocycles include both cycloalkane rings, in which all carbon atoms are saturated, and cycloalkene rings, which contain at least one double bond.
  • Carbocycle includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings.
  • the term "fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings.
  • an aromatic ring e.g., phenyl
  • a saturated or unsaturated ring e.g., cyclohexane
  • carbocyclic examples include cyclopentane, cyclohexane, bicyclo[2.2.1 Jheptane, 1 ,5-cyclooctadiene, 1 ,2,3,4-tetrahydronaphthalene,
  • Carbocycles may be susbstituted at any one or more positions capable of bearing a hydrogen atom.
  • a “cycloalkyl” group is a cyclic hydrocarbon which is completely saturated.
  • “Cycloalkyl” includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms unless otherwise defined.
  • the second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings.
  • the term “fused cycloalkyl” refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring.
  • the second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings.
  • cycloalkenyl is a cyclic hydrocarbon containing one or more double bonds.
  • Carbocyclylalkyl refers to an alkyl group substituted with a carbocycle group.
  • carbonate is art-recognized and refers to a group -OCO 2 -R 10 , wherein R 10 represents a hydrocarbyl group.
  • carboxy refers to a group represented by the formula -C0 2 H.
  • esters refers to a group -C(0)OR 10 wherein R 10 represents a hydrocarbyl group.
  • ether refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O- heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.
  • heteroalkyl and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.
  • heteroalkyl refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.
  • heteroaryl and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heteroaryl and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
  • heterocyclyl refers to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10- membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • heterocyclyl and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
  • heterocyclylalkyl refers to an alkyl group substituted with a heterocycle group.
  • Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.
  • hydroxyalkyl refers to an alkyl group substituted with a hydroxy group.
  • lower when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer.
  • acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).
  • polycyclyl refers to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings".
  • Each of the rings of the polycycle can be substituted or unsubstituted.
  • each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.
  • sil refers to a silicon moiety with three hydrocarbyl moieties attached thereto.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • sulfate is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.
  • R 9 and R 10 independently represents hydrogen or hydrocarbyl, such as alkyl, or R 9 and R 10 taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • R represents a hydrocarbyl
  • sulfonate is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.
  • sulfone is art-recognized and refers to the group -S(0)2-R 10 , wherein R 10 represents a hydrocarbyl.
  • thioalkyl refers to an alkyl group substituted with a thiol group.
  • thioester refers to a group -C(0)SR 10 or -SC(0)R 10 wherein R 10 represents a hydrocarbyl.
  • thioether is equivalent to an ether, wherein the oxygen is replaced with a sulfur.
  • urea is art-recognized and may be represented by the general formula
  • R 9 and R 10 independently represent hydrogen or a hydrocarbyl, such as alkyl, or either occurrence of R 9 taken together with R 10 and the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • Protecting group refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3 rd Ed., 1999, John Wiley & Sons, NY and Harrison et al, Compendium of Synthetic Organic Methods, Vols. 1- 8, 1971-1996, John Wiley & Sons, NY.
  • nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2- trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups,
  • hydroxylprotecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterified) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.
  • Healthcare providers refers to individuals or organizations that provide healthcare services to a person, community, etc.
  • Examples of “healthcare providers” include doctors, hospitals, continuing care retirement communities, skilled nursing facilities, subacute care facilities, clinics, multispecialty clinics, freestanding ambulatory centers, home health agencies, and HMO's.
  • a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • treating includes prophylactic and/or therapeutic treatments.
  • prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • prodrug is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound of formula I).
  • a common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule.
  • the prodrug is converted by an enzymatic activity of the host animal.
  • esters or carbonates e.g., esters or carbonates of alcohols or carboxylic acids
  • some or all of the compounds of formula I in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.
  • compositions and methods of the present invention may be utilized to treat an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles
  • glycols, glycerol oils such as olive oil, or injectable organic esters.
  • injectable organic esters are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as an eye drop.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally;
  • routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral muco
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients.
  • an active compound such as a compound of the invention
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • Compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.
  • compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the active compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • Ophthalmic formulations eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference.
  • liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatable with such fluids.
  • a preferred route of administration is local
  • administration e.g., topical administration, such as eye drops, or administration via an implant.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical
  • administration usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutan
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or
  • biodegradable devices Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • therapeutically effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention.
  • a larger total dose can be delivered by multiple administrations of the agent.
  • Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
  • compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be any suitable daily dose of the active compound. If desired, the effective daily dose of the active compound may be any suitable daily dose of the active compound.
  • the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • the phrase "conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds).
  • the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.
  • the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.
  • conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) provides improved efficacy relative to each individual administration of the compound of the invention (e.g., compound of formula I or la) or the one or more additional therapeutic agent(s).
  • the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).
  • contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the invention include, but are not limited to, L- arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,
  • contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water- soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT
  • the invention relates to a method for conducting a pharmaceutical business, by manufacturing a formulation of a compound of the invention, or a kit as described herein, and marketing to healthcare providers the benefits of using the formulation or kit for treating or preventing any of the diseases or conditions as described herein.
  • the invention relates to a method for conducting a pharmaceutical business, by providing a distribution network for selling a formulation of a compound of the invention, or kit as described herein, and providing instruction material to patients or physicians for using the formulation for treating or preventing any of the diseases or conditions as described herein.
  • the invention comprises a method for conducting a pharmaceutical business, by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the diseases or conditions as described herein, conducting therapeutic profiling of identified formulations for efficacy and toxicity in animals, and providing a distribution network for selling an identified preparation as having an acceptable therapeutic profile.
  • the method further includes providing a sales group for marketing the preparation to healthcare providers.
  • the invention relates to a method for conducting a pharmaceutical business by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the disease or conditions as described herein, and licensing, to a third party, the rights for further development and sale of the formulation.
  • IC50 is a quantitative measure indicating how much compound is needed to inhibit a given biological activity by half.
  • Example 5 Multiple Myeloma xenograft study.
  • mice subcutaneously with 1 x 10 7 RPMI-8226 myeloma cells per mouse mixed 1 : 1 with Matrigel.
  • Example 6 Treatment of Multiple Myeloma cells with a combination of drugs.
  • MM1S cells panels A & B
  • RPMI-8226 cells panels C & D
  • pomalidomide or a mixture thereof panels A & C) or compound 670, dexamethsone or a mixture thereof (panels B & D) for 72 hours in growth media.
  • cell viability was measured using Cell Titer Glo as per manufacturer's protocol (Promega, Madison, WI). Measured values for compound-treated cells were normalized to DMSO-treated cells and data is reported as a cell survival ratio with a value of 1 (one) corresponding to maximum cell survival and a value of 0 (zero) corresponding to no cell survival. Cell survival ratios for all compound treatments are represented as bar graphs.
  • Combination indices were calculated using the Calcusyn program (biosoft.com) and reported for individual mixtures of compound 670 and pomalidomide [POM] (panels A &C) and individual mixtures of compound 670 and dexamethasone [DEX] (panels B & D). Compound mixtures that produced a synergistic anti-tumor activity are highlighted.
  • Figure 10 shows a western blot for pyruvate carboxylase in multiple myeloma cell lines.
  • Total cell lysates are prepared from cell pellets by sonication and protein concentration is quantitated using standard methods. Lysate proteins are denatured by boiling in SDS-sample buffer and resolved (10 ⁇ g/lane) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins are then transferred to nitrocellulose and probed with antibodies recognizing pyruvate carboxylase or beta- actin followed by HRP-coupled secondary antibodies. Bands are revealed by chemiluminescence. Image J software (NIH) is used to quantify the pyruvate carboxylase band intensity using methods recommended by the software developer.
  • NASH image J software

Abstract

The invention relates to novel heterocyclic compounds and pharmaceutical preparations thereof. The invention further relates to methods of treating or preventing multiple myeloma using the novel heterocyclic compounds of the invention.

Description

Treatment of Multiple Myeloma with Heterocyclic Inhibitors of Glutaminase
Related Applications
This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/028,523, filed July 24, 2014, the contents of which are hereby incorporated by reference.
Background
Glutamine supports cell survival, growth and proliferation through metabolic and non-metabolic mechanisms. In actively proliferating cells, the metabolism of glutamine to lactate, also referred to as "glutaminolysis" is a major source of energy in the form of NADPH. The first step in glutaminolysis is the deamination of glutamine to form glutamate and ammonia, which is catalyzed by the glutaminase enzyme (GLS). Thus, deamination via glutaminase is a control point for glutamine metabolism.
Ever since Warburg's observation that ascites tumor cells exhibited high rates of glucose consumption and lactate secretion in the presence of oxygen (Warburg,
1956), researchers have been exploring how cancer cells utilize metabolic pathways to be able to continue actively proliferating. Several reports have demonstrated how glutamine metabolism supports macromolecular synthesis necessary for cells to replicate (Curthoys, 1995; DeBardinis, 2008).
Thus, glutaminase has been theorized to be a potential therapeutic target for the treatment of diseases characterized by actively proliferating cells, such as cancer. The lack of suitable glutaminase inhibitors has made validation of this target impossible until the recent creation of compounds that are specific and capable of being formulated for in vivo use (US 8,604,016). As glutaminase inhibitors enter the clinical arena, methods are needed to identify patients that would best benefit from treatment with these compounds.
Summary of Invention
The present invention provides a method for treating multiple myeloma in a patient, comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of a glutaminase inhibitor.
In some embodiments the glutaminase inhibitor is a compound of formula I,
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof, wherein:
L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2,
CH=CH, or ^ -f^ , preferably CH2CH2, wherein any hydrogen atom of a
CH or CH2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxy;
X, independently for each occurrence, represents S, O or CH=CH, preferably S or
CH=CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
Y, independently for each occurrence, represents H or CH20(CO)R7 ;
R7, independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
Z represents H or R3(CO);
Ri and R2 each independently represent H, alkyl, alkoxy or hydroxy;
R3, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
heteroaryloxyalkyl or C(Rg)(R9)(Rio), N(R4)(R5) or OR5, wherein any free hydroxyl group may be acylated to form C(0)R7;
R4 and R5 each independently represent H or substituted or unsubstituted alkyl,
hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form
C(0)R7; Re, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7; and
Rg, R and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, or R8 and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein at least two of
Figure imgf000004_0001
In further embodiments, the invention relates to methods of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
Brief Description of the Drawings
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Figure 1 shows the correlation between glutamine-dependence and
antiproliferative effect of compound 670 for a panel of breast tumor cell lines.
Figure 2 shows single-agent compound 402 treatment of MDA-MB-231 orthotopic xenograft model.
Figure 3 shows a combination study with compound 389 and paclitaxel in MDA-MB-231 orthotopic xenograft model. Figure 4 shows results of the median glutaminase:glutamine synthetase expression ratio in various cancer types, including colorectal cancer, renal cancer, lymphoma, melanoma and myeloma.
Figure 5 shows that intraperitoneal administration of compound 188 to mice results in reduced tumor size in a HCTl 16 colon carcinoma xenograft model.
Figure 6 shows that oral administration of compound 670 to mice results in reduced tumor size in a H2122 lung adenocarcinoma xenograft model.
Figure 7 shows that oral administration of compound 670 to mice results in reduced tumor size in a RPMI-8226 multiple myeloma xenograft model.
Figure 8 shows that compound 670 synergizes with pomalidomide or dexamethasone to produce an anti-tumor effect in multiple myeloma cells.
Figure 9 shows a waterfall plot demonstrating the response variation to CB- 839 treatment in a panel of multiple myeloma tumor cell lines.
Figure 10 shows a western blot for pyruvate carboxylase in a panel of multiple myeloma cell lines.
Figure 11 shows a correlation between pyruvate carboxylase protein level and cell growth in response to CB-839 treatment relative to no CB-839 treatment/DMSO control.
Figure 12 shows pyruvate carboxylase expression values generated from R A-seq data for various multiple myeloma cell lines correlates with pyruvate carboxylase protein levels.
Detailed Description of the Invention
The present invention is based on the discovery of a biomarker in multiple myeloma cell lines that indicates sensitivity of the multiple myeloma cells to treatment with a glutaminase inhibitor. As shown in data presented herein, not all multiple myeloma cell lines exhibit the same sensitivity to treatment with a glutaminase inhibitor. Further, this effect is not observed in certain other types of cancer cell lines (data not shown).
The present invention provides a method for treating multiple myeloma in a patient, comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of a glutaminase inhibitor.
In certain embodiments the glutaminase inhibitor is a compound of formula I,
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof, wherein:
L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2,
CH=CH, or ^ -f^ , preferably CH2CH2, wherein any hydrogen atom of a
CH or CH2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxy;
X, independently for each occurrence, represents S, O or CH=CH, preferably S or
CH=CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
Y, independently for each occurrence, represents H or CH20(CO)R7 ;
R7, independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
Z represents H or R3(CO);
Ri and R2 each independently represent H, alkyl, alkoxy or hydroxy;
R3, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
heteroaryloxyalkyl or C(Rg)(R9)(Rio), N(R4)(R5) or OR5, wherein any free hydroxyl group may be acylated to form C(0)R7;
R4 and R5 each independently represent H or substituted or unsubstituted alkyl,
hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form
C(0)R7; Re, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7; and
Rg, R and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, or R8 and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein at least two of
Figure imgf000007_0001
In certain embodiments wherein alkyl, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl are substituted, they are substituted with one or more substituents selected from substituted or unsubstituted alkyl, such as perfluoroalkyl (e.g., trifluoromethyl), alkenyl, alkoxy, alkoxyalkyl, aryl, aralkyl, arylalkoxy, aryloxy, aryloxyalkyl, hydroxyl, halo, alkoxy, such as
perfluoroalkoxy (e.g., trifluoromethoxy), alkoxyalkoxy, hydroxyalkyl,
hydroxyalkylamino, hydroxyalkoxy, amino, aminoalkyl, alkylamino,
aminoalkylalkoxy, aminoalkoxy, acylamino, acylaminoalkyl, such as perfluoro acylaminoalkyl (e.g., trifluoromethylacylaminoalkyl), acyloxy, cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy, heteroaryloxy, heteroaryloxyalkyl, heterocyclylaminoalkyl, heterocyclylaminoalkoxy, amido, amidoalkyl, amidine, imine, oxo, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl, including perfluoroacyl (e.g., C(0)CF3)), carbonylalkyl (such as
carboxyalkyl, alkoxycarbonylalkyl, formylalkyl, or acylalkyl, including
perfluoroacylalkyl (e.g., -alkylC(0)CF3)), carbamate, carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate, sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro, azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester, thioacetate, or
thioformate), phosphoryl, phosphate, phosphonate or phosphinate.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, or CH2NHCH2, wherein any hydrogen atom of a CH2 unit may be replaced by alkyl or alkoxy, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxyl. In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2. In certain embodiments, L represents CH2CH2. In certain embodiments, L is not CH2SCH2.
In certain embodiments, Y represents H.
In certain embodiments, X represents S or CH=CH. In certain embodiments, one or both X represents CH=CH. In certain embodiments, each X represents S. In certain embodiments, one X represents S and the other X represents CH=CH.
In certain embodiments, Z represents R3(CO). In certain embodiments wherein Z is R3(CO), each occurrence of R3 is not identical (e.g., the compound of formula I is not symmetrical).
In certain embodiments, Ri and R2 each represent H.
In certain embodiments, R3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain embodiments, R3 represents C(Rg)(R9)(Rio), wherein R8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2, such as CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and each R3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain such embodiments, each occurrence of R3 is identical.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and each R3 represents C(R8)(R9)(Rio), wherein R8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy. In certain such embodiments, each occurrence of R3 is identical.
In certain embodiments, L represents CH2CH2, Y represents H, X represents S or CH=CH, Z represents R3(CO), Ri and R2 each represent H, and each R3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain such embodiments, each X represents S. In other embodiments, one or both occurrences of X represents CH=CH, such as one occurrence of X represents S and the other occurrence of X represents CH=CH. In certain embodiments of the foregoing, each occurrence of R3 is identical. In other embodiments of the foregoing wherein one occurrence of X represents S and the other occurrence of X represents CH=CH, the two occurrences of R3 are not identical.
In certain embodiments, L represents CH2CH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and each R3 represents
C(Rg)(R9)(Rio), wherein R8 represents aryl, arylalkyl or heteroaryl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl or alkoxy. In certain such embodiments, R8 represents aryl and Rio represents hydroxyalkyl. In certain such embodiments, each occurrence of R3 is identical.
In certain embodiments wherein L represents CH2, CH2CH2CH2 or CH2CH2, X represents O, and Z represents R3(CO), both R3 groups are not alkyl, such as methyl, or C(R8)(R9)(Rio), wherein R8, R9 andRio are each independently hydrogen or alkyl.
In certain embodiments wherein L represents CH2CH2, X represents S, and Z represents R3(CO), both R3 groups are not phenyl or heteroaryl, such as 2-furyl.
In certain embodiments wherein L represents CH2CH2, X represents O, and Z represents R3(CO), both R3 groups are not N(R4)(R5) wherein R4 is aryl, such as phenyl, and R5 is H.
In certain embodiments wherein L represents CH2SCH2, X represents S, and Z represents R3(CO), both R3 groups are not aryl, such as optionally substituted phenyl, aralkyl, such as benzyl, heteroaryl, such as 2-furyl, 2-thienyl or 1,2,4-trizole, substituted or unsubstituted alkyl, such as methyl, chloromethyl, dichloromethyl, n- propyl, n-butyl, t-butyl or hexyl, heterocyclyl, such as pyrimidine-2,4(lH,3H)-dione, or alkoxy, such as methoxy, pentyloxy or ethoxy.
In certain embodiments wherein L represents CH2SCH2, X represents S, and Z represents R3(CO), both R3 groups are not N(R4)(R5) wherein R4 is aryl, such as substituted or unsubstituted phenyl (e.g., phenyl, 3-tolyl, 4-tolyl, 4-bromophenyl or 4- nitrophenyl), and R5 is H.
In certain embodiments wherein L represents CH2CH2CH2, X represents S, and Z represents R3(CO), both R3 groups are not alkyl, such as methyl, ethyl, or propyl, cycloalkyl, such as cyclohexyl, or C(Rg)(R9)(Rio), wherein any of Rg, R9 and Rio together with the C to which they are attached, form any of the foregoing.
In certain embodiments, the compound is not one of the following:
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
' NHC(0)NHPh
Figure imgf000012_0002
In further embodiments of the methods of the invention, the glutaminase inhibitor is a compound of formula la,
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof, wherein:
L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2,
CH=CH, or ^ -f^ , preferably CH2CH2, wherein any hydrogen atom of a CH or CH2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxy;
X represents S, O or CH=CH, preferably S or CH=CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
Y, independently for each occurrence, represents H or CH20(CO)R7 ;
R7, independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
Z represents H or R3(CO);
Ri and R2 each independently represent H, alkyl, alkoxy or hydroxy, preferably H;
R3 represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl,
acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heteroaryloxyalkyl or
C(Rg)(R9)(Rio), N(R4)(R5) or OR5, wherein any free hydroxyl group may be acylated to form C(0)R7;
R4 and R5 each independently represent H or substituted or unsubstituted alkyl,
hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7;
5, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7; and
R8, R9 and R10 each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, or Rg and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein at least two of R8, R9 and R10 are not H;
R11 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, or
C(Ri2)(Ri3)(Ri4), N(Rzt)(R14) or OR14, wherein any free hydroxyl group may be acylated to form C(0)R7;
Ri2 and R13 each independently respresent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein both of Ri2 and R13 are not H; and
Ri4 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl,
heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl.
In certain embodiments wherein alkyl, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl are substituted, they are substituted with one or more substituents selected from substituted or unsubstituted alkyl, such as perfluoroalkyl (e.g., trifluoromethyl), alkenyl, alkoxy, alkoxyalkyl, aryl, aralkyl, arylalkoxy, aryloxy, aryloxyalkyl, hydroxyl, halo, alkoxy, such as
perfluoroalkoxy (e.g., trifluoromethylalkoxy), alkoxyalkoxy, hydroxyalkyl, hydroxyalkylamino, hydroxyalkoxy, amino, aminoalkyl, alkylamino, aminoalkylalkoxy, aminoalkoxy, acylamino, acylaminoalkyl, such as perfluoro acylaminoalkyl (e.g., trifluoromethylacylaminoalkyl), acyloxy, cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy, heteroaryloxy, heteroaryloxyalkyl, heterocyclylaminoalkyl, heterocyclylaminoalkoxy, amido, amidoalkyl, amidine, imine, oxo, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl, including perfluoroacyl (e.g., C(0)CF3)), carbonylalkyl (such as
carboxyalkyl, alkoxycarbonylalkyl, formylalkyl, or acylalkyl, including
perfluoroacylalkyl (e.g., -alkylC(0)CF3)), carbamate, carbamatealkyl, urea, ureaalkyl, sulfate, sulfonate, sulfamoyl, sulfone, sulfonamide, sulfonamidealkyl, cyano, nitro, azido, sulfhydryl, alkylthio, thiocarbonyl (such as thioester, thioacetate, or
thioformate), phosphoryl, phosphate, phosphonate or phosphinate.
In certain embodiments, Rn represents substituted or unsubstituted arylalkyl, such as substituted or unsubstituted benzyl.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2,
CH2S, SCH2, or CH2NHCH2, wherein any hydrogen atom of a CH2 unit may be replaced by alkyl or alkoxy, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxyl. In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2, preferably CH2CH2. In certain embodiments, L is not CH2SCH2.
In certain embodiments, each Y represents H. In other embodiments, at least one Y is CH20(CO)R7.
In certain embodiments, X represents S or CH=CH. In certain embodiments, X represents S.
In certain embodiments, Ri and R2 each represent H.
In certain embodiments, Z represents R3(CO). In certain embodiments wherein Z is R3(CO), R3 and Rn are not identical (e.g., the compound of formula I is not symmetrical).
In certain embodiments, Z represents R3(CO) and R3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl. In certain embodiments, Z represents R3(CO) and R3 represents C(Rg)(R9)(Rio), wherein R8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, Rg represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy. In certain embodiments, Z represents R3(CO) and R3 represents heteroarylalkyl.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2, such as CH2CH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, R3 represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl, and Rii represents arylalkyl. In certain such embodiments, R3 represents
heteroarylalkyl.
In certain embodiments, L represents CH2SCH2, CH2CH2, CH2S or SCH2, such as CH2CH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and R3 represents C(Rg)(R9)(Rio), wherein R8 represents aryl, arylalkyl, heteroaryl or heteroaralkyl, such as aryl, arylalkyl or heteroaryl, Rg represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, such as hydroxy, hydroxyalkyl or alkoxy, and Rn represents arylalkyl. In certain such embodiments, Rs represents heteroaryl.
In certain embodiments, L represents CH2CH2, Y represents H, X represents S or CH=CH, such as S, Z represents R3(CO), Ri and R2 each represent H, R3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl, and Rn represents arylalkyl. In certain such embodiments, R3 represents
heteroarylalkyl.
In certain embodiments, L represents CH2CH2, Y represents H, X represents
S, Z represents R3(CO), Ri and R2 each represent H, R3 represents C(R8)(R9)(Rio), wherein R8 represents aryl, arylalkyl or heteroaryl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl or alkoxy, and Rn represents arylalkyl. In certain such embodiments, R8 represents aryl and Rio represents hydroxyalkyl. In certain other embodiments, Rg represents heteroaryl.
In certain embodiments, compounds of the invention may be prodrugs of the compounds of formula I or la, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate, or carboxylic acid present in the parent compound is presented as an ester. In certain such embodiments, the prodrug is metabolized to the active parent compound in vivo (e.g., the ester is hydrolyzed to the corresponding hydroxyl, or carboxylic acid).
In certain embodiments, compounds of the invention may be racemic. In certain embodiments, compounds of the invention may be enriched in one
enantiomer. For example, a compound of the invention may have greater than 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95% or greater ee. In certain embodiments, compounds of the invention may have more than one stereocenter. In certain such embodiments, compounds of the invention may be enriched in one or more diastereomer. For example, a compound of the invention may have greater than 30% de, 40% de, 50% de, 60% de, 70% de, 80% de, 90% de, or even 95% or greater de.
In certain embodiments, the present invention relates to methods of treating or preventing multiple myeloma with a compound of formula I or la, or a
pharmaceutically acceptable salt thereof. In certain embodiments, the therapeutic preparation may be enriched to provide predominantly one enantiomer of a compound (e.g., of formula I or la). An enantiomerically enriched mixture may comprise, for example, at least 60 mol percent of one enantiomer, or more preferably at least 75, 90, 95, or even 99 mol percent. In certain embodiments, the compound enriched in one enantiomer is substantially free of the other enantiomer, wherein substantially free means that the substance in question makes up less than 10%>, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% as compared to the amount of the other enantiomer, e.g., in the composition or compound mixture. For example, if a composition or compound mixture contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it would be said to contain 98 mol percent of the first enantiomer and only 2% of the second enantiomer.
In certain embodiments, the therapeutic preparation may be enriched to provide predominantly one diastereomer of a compound (e.g., of formula I or la). A diastereomerically enriched mixture may comprise, for example, at least 60 mol percent of one diastereomer, or more preferably at least 75, 90, 95, or even 99 mol percent.
In certain embodiments, the present invention provides a pharmaceutical preparation suitable for use in a human patient in the treatment of multiple myeloma, comprising an effective amount of any of the compounds shown above (e.g., a compound of the invention, such as a compound of formula I or la), and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical preparations may be for use in treating or preventing a condition or disease as described herein. In certain embodiments, the pharmaceutical preparations have a low enough pyrogen activity to be suitable for use in a human patient. Compounds of any of the above structures may be used in the manufacture of medicaments for the treatment of any diseases or conditions disclosed herein.
In further embodiments, the invention relates to methods of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
In certain embodiments, the glutaminase inhibitor is a compound of formula I or formula la.
Methods of the invention include a step of comparing the level of pyruvate carboxylase in a multiple myeloma patient sample to a reference standard. In certain embodiments, the reference standard is a level of pyruvate carboxylase in a multiple myeloma cell line that is resistant to a compound of formula (II), also referred to herein as CB-839:
Figure imgf000018_0001
In certain embodiments, the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells.
Uses of enzyme inhibitors
Glutamine plays an important role as a carrier of nitrogen, carbon, and energy.
It is used for hepatic urea synthesis, for renal ammoniagenesis, for gluconeogenesis, and as respiratory fuel for many cells. Cells get their glutamine by either synthesizing it internally via an enzyme called glutamine synthetase (GS) or exogenously from the environment.
The conversion of glutamine into glutamate is initiated by the mitochondrial enzyme, glutaminase. There are two major forms of the enzyme, K-type and L-type, which are distinguished by their Km values for glutamine and response to glutamate, wherein the Km value, or Michaelis constant, is the concentration of substrate required to reach half the maximal velocity. The L-type, also known as "liver-type" or GLS2, has a high Km for glutamine and is glutamate resistant. The K-type, also known as "kidney-type" or GLS1 or "KGA", has a low Km for glutamine and is inhibited by glutamate. An alternative splice form of GLS1, referred to as
glutaminase C or "GAC", has recently been identified.
In addition to serving as the basic building blocks of protein synthesis, amino acids have been shown to contribute to many processes critical for growing and dividing cells, and this is particularly true for cancer cells. Nearly all definitions of cancer include reference to dysregulated proliferation. Numerous studies on glutamine metabolism in cancer indicate that many tumors are avid glutamine consumers (Souba, Ann. Surg., 1993; Collins et al, J. Cell. Physiol, 1998; Medina, J. Nutr., 2001; Shanware et al, J. Mol. Med., 2011), and this includes, but not limited to breast cancer, renal cell carcinoma, glioma, pancreatic ductal adenocarcinoma and non-small cell lung cancer. Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of these cancers.
While many cancer cells depend on exogenous glutamine for survival, the degree of glutamine dependence among tumor cell subtypes may make a population of cells more susceptible to the reduction of glutamine. As an example, gene expression analysis of breast cancers has identified five intrinsic subtypes (luminal A, luminal B, basal, HER2+, and normal-like) (Sorlie et al., Proc Natl Acad Sci USA, 2001). Although glutamine deprivation has an impact on cell growth and viability, basal-like cells appear to be more sensitive to the reduction of exogenous glutamine (Kung et al, PLoS Genetics, 2011). This supports the concept that glutamine is a very important energy source in basal-like breast cancer cell lines, and suggests that inhibition of the glutaminase enzyme would be beneficial in the treatment of breast cancers comprised of basal-like cells. Figure 1 further supports the correlation that cells dependent on exogenous glutamine are susceptible to the presence of a glutaminase inhibitor in a series of breast cancer cell lines. A xenograft of glutamine dependent cells, MD-MB-231, demonstrated a sensitivity to glutaminase inhibition (Figure 2). Interestingly, the xenograft model showed some variablility in the sensitivity to glutaminase inhibiton; however, the sensitivity could be enhanced further when combined with another agent (Figure 3).
Enzyme expression levels can be determined in multiple manners, and quantitation is relative, based on a specific standard for each assay. The results can be used to provide a genetic profile, where the levels of certain genes, mRNAs or resulting expression products form a signature pattern that can used to characterize cell types. Kung et al, demonstrated that the basal-like breast cancer cells that showed glutamine dependency exhibited a genetic profile in which GLS expression was relatively high and GS expression was relatively low. Furthermore, the expression level of GLS2 was relatively low. Analysis of primary breast tumors mRNA expression dataset (The Cancer Genome Atlas; N=756) support that basal-type cells generally have high GLS expression relative to GS expression.
This led to the hypothesis that the high GLS expression and low GS expression profile may serve as a genetic signature to identify other cancers that may be particularly dependent on exogenous glutamine, and therefore susceptible to glutaminase inhibition. Upon analysis of a vast number of primary human cancers from a commercial database, several cancers exhibited high GLS to low GS expression patterns. In addition to the breast cancers previously noted, colorectal cancer, lung cancer, melanoma, mesothelioma, renal cancer and B cell malignancies had notably high GLS/GS ratios (Figure 4). Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of cancers selected from colorectal cancer, endocrine cancer, lung cancer, melanoma, mesothelioma, renal cancer and B cell malignancies. Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of multiple myeloma, leukemia (including acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL)), or lymphoma (including Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma and Hodgkin's lymphoma).
Xenograft models of colon cancer, lung cancer and multiple myeloma showed sensitivity to treatment with a glutaminase inhibitor (Figures 5, 6, and 7), further supporting the concept that certain cancers may prove to be more responsive to treatment with glutaminase inhibitors. It was decided to further examine the dependence of multiple myeloma cells on exogenous glutamine and the sensitivity to glutaminase inhibition.
Similar to observations in breast cancer cell lines, multiple myeloma cell lines demonstrated varied sensitivity to glutaminase inhibition (Figure 9).
Given this variability in a multiple myeloma's sensitivity to glutaminase inhibition, identifying the multiple myeloma patients that would most likely benefit from treatment with a glutaminase inhibitor would be advantageous. Several groups have suggested that glucose can also serve as a source of carbon via the conversion of pyruvate into the TCA cycle intermediate oxaloacetate by the enzyme pyruvate carboxylase (PC). The expression levels of PC across a panel of multiple myeloma cell lines with varying sensitivity to glutaminase inhibiton was examined (Figure 10). Strikingly, PC expression was higher in multiple myeloma cell lines resistant to glutaminase inhibition, while PC expression was low across most sensitive cell lines. Quantification of PC protein levels further supported this correlation to sensitivity to glutaminase inhibition (Figure 11). The correlation is maintained using alternative means of measuring PC expression levels (Figure 12).
In certain embodiments, the invention provides a method of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient indicates that the patient may benefit from treatment with a glutaminase inhibitor. In certain embodiments, the invention provides a method of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor. In some embodiments, the multiple myeloma reference standard is normal tissue from the same patient. In certain embodiments, the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells. In certain embodiments the glutaminase inhibitor is a compound of formula I. In other embodiments, the glutaminase inhibitor is a compound of formula la.
In certain embodiments, the invention provides a method of treating a multiple myeloma patient, the method comprising determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells, comparing the level of pyruvate carboxylase in the patient sample to a reference standard, and if the the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of glutaminase inhibitor. In certain embodiments, the multiple myeloma reference standard is normal tissue from the same patient. In certain embodiments, the multiple myeloma cell line of the reference standard is KMS-28PE or KMS-11 cells. In certain embodiments the glutaminase inhibitor is a compound of formula I. In other embodiments, the glutaminase inhibitor is a compound of formula la.
In some embodiments, the method of treating or preventing cancer, such as multiple myeloma, may comprise administering a compound of the invention conjointly with one or more other chemotherapeutic agent(s). Chemotherapeutic agents that may be conjointly administered with compounds of the invention include: ABT-263, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, AZD5363, Bacillus Calmette-Guerin vaccine (beg), bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisp latin, cladribine, clodronate, cobimetinib, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, eribulin, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil and 5-fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ixabepilone, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, mutamycin, MK-2206, nilutamide, nocodazole, octreotide, oxaliplatin, olaparib, paclitaxel, pamidronate, pentostatin, perifosine, PF-04691502, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, romidepsin, rucaparib, selumetinib, sorafenib, streptozocin, sunitinib, suramin, talazoparib, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trametinib, trastuzumab, tretinoin, veliparib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat (SAHA). For example, chemotherapeutic agents that may be conjointly administered with compounds of the invention include: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine,
cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,
daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, perifosine, plicamycin, pomalidomide, porfmier, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. In other embodiments, chemotherapeutic agents that may be conjointly administered with compounds of the invention include: ABT-263, dexamethasone, 5 -fluorouracil, PF-04691502, romidepsin, and vorinostat (SAHA). In certain embodiments of the methods of the invention described herein, the chemotherapeutic agent conjointly administered with compounds of the invention is a taxane chemotherapeutic agent, such as paclitaxel or docetaxel. In certain embodiments of the methods of the invention described herein, the chemotherapeutic agent conjointly administered with compounds of the invention is doxorubicin. In certain embodiments of the methods of the invention described herein, a compound of the invention is administered conjointly with a taxane chemotherapeutic agent (e.g., paclitaxel) and doxorubicin.
Many combination therapies have been developed for the treatment of cancer. In certain embodiments, compounds of the invention may be conjointly administered with a combination therapy. Examples of combination therapies with which compounds of the invention may be conjointly administered are included in Table 1. Table 1 : Exemplary combinatorial therapies for the treatment of cancer.
Figure imgf000023_0001
Name Therapeutic agents
ACe Cyclophosphamide, Doxorubicin
AD Doxorubicin, Dacarbazine
AP Doxorubicin, Cisplatin
ARAC-DNR Cytarabine, Daunorubicin
B-CAVe Bleomycin, Lomustine, Doxorubicin, Vinblastine
BCVPP Carmustine, Cyclophosphamide, Vinblastine,
Procarbazine, Prednisone
BEACOPP Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide,
Vincristine, Procarbazine, Prednisone, Filgrastim
BEP Bleomycin, Etoposide, Cisplatin
BIP Bleomycin, Cisplatin, Ifosfamide, Mesna
BOMP Bleomycin, Vincristine, Cisplatin, Mitomycin
CA Cytarabine, Asparaginase
CABO Cisplatin, Methotrexate, Bleomycin, Vincristine
CAF Cyclophosphamide, Doxorubicin, Fluorouracil
CAL-G Cyclophosphamide, Daunorubicin, Vincristine,
Prednisone, Asparaginase
CAMP Cyclophosphamide, Doxorubicin, Methotrexate,
Procarbazine
CAP Cyclophosphamide, Doxorubicin, Cisplatin
CaT Carboplatin, Paclitaxel
CAV Cyclophosphamide, Doxorubicin, Vincristine
CAVE ADD CAV and Etoposide
CA-VP16 Cyclophosphamide, Doxorubicin, Etoposide
CC Cyclophosphamide, Carboplatin
CDDP/VP-16 Cisplatin, Etoposide
CEF Cyclophosphamide, Epirubicin, Fluorouracil
CEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or
without/ Bleomycin
CEV Cyclophosphamide, Etoposide, Vincristine
CF Cisplatin, Fluorouracil or Carboplatin Fluorouracil
CHAP Cyclophosphamide or Cyclophosphamide, Altretamine, Name Therapeutic agents
Doxorubicin, Cisplatin
ChlVPP Chlorambucil, Vinblastine, Procarbazine, Prednisone
CHOP Cyclophosphamide, Doxorubicin, Vincristine, Prednisone
CHOP-BLEO Add Bleomycin to CHOP
CISCA Cyclophosphamide, Doxorubicin, Cisplatin
CLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin
CMF Methotrexate, Fluorouracil, Cyclophosphamide
CMFP Cyclophosphamide, Methotrexate, Fluorouracil,
Prednisone
CMFVP Cyclophosphamide, Methotrexate, Fluorouracil,
Vincristine, Prednisone
CMV Cisplatin, Methotrexate, Vinblastine
CNF Cyclophosphamide, Mitoxantrone, Fluorouracil
CNOP Cyclophosphamide, Mitoxantrone, Vincristine, Prednisone
COB Cisplatin, Vincristine, Bleomycin
CODE Cisplatin, Vincristine, Doxorubicin, Etoposide
COMLA Cyclophosphamide, Vincristine, Methotrexate,
Leucovorin, Cytarabine
COMP Cyclophosphamide, Vincristine, Methotrexate, Prednisone
Cooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil,
Vincristine, Prednisone
COP Cyclophosphamide, Vincristine, Prednisone
COPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide
COPP Cyclophosphamide, Vincristine, Procarbazine, Prednisone
CP(Chronic Chlorambucil, Prednisone
lymphocytic leukemia)
CP (Ovarian Cancer) Cyclophosphamide, Cisplatin
CT Cisplatin, Paclitaxel
CVD Cisplatin, Vinblastine, Dacarbazine
CVI Carboplatin, Etoposide, Ifosfamide, Mesna
CVP Cyclophosphamide, Vincristine, Prednisome
CVPP Lomustine, Procarbazine, Prednisone Name Therapeutic agents
CYVADIC Cyclophosphamide, Vincristine, Doxorubicin,
Dacarbazine
DA Daunorubicin, Cytarabine
DAT Daunorubicin, Cytarabine, Thioguanine
DAV Daunorubicin, Cytarabine, Etoposide
DCT Daunorubicin, Cytarabine, Thioguanine
DHAP Cisplatin, Cytarabine, Dexamethasone
DI Doxorubicin, Ifosfamide
DTIC/Tamoxifen Dacarbazine, Tamoxifen
DVP Daunorubicin, Vincristine, Prednisone
EAP Etoposide, Doxorubicin, Cisplatin
EC Etoposide, Carboplatin
EFP Etoposie, Fluorouracil, Cisplatin
ELF Etoposide, Leucovorin, Fluorouracil
EM A 86 Mitoxantrone, Etoposide, Cytarabine
EP Etoposide, Cisplatin
EVA Etoposide, Vinblastine
FAC Fluorouracil, Doxorubicin, Cyclophosphamide
FAM Fluorouracil, Doxorubicin, Mitomycin
FAMTX Methotrexate, Leucovorin, Doxorubicin
FAP Fluorouracil, Doxorubicin, Cisplatin
F-CL Fluorouracil, Leucovorin
FEC Fluorouracil, Cyclophosphamide, Epirubicin
FED Fluorouracil, Etoposide, Cisplatin
FL Flutamide, Leuprolide
FZ Flutamide, Goserelin acetate implant
HDMTX Methotrexate, Leucovorin
Hexa-CAF Altretamine, Cyclophosphamide, Methotrexate,
Fluorouracil
ICE-T Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna
IDMTX/6-MP Methotrexate, Mercaptopurine, Leucovorin Name Therapeutic agents
IE Ifosfamide, Etoposie, Mesna
IfoVP Ifosfamide, Etoposide, Mesna
IPA Ifosfamide, Cisplatin, Doxorubicin
M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone,
Melphalan
MAC-III Methotrexate, Leucovorin, Dactinomycin,
Cyclophosphamide
MACC Methotrexate, Doxorubicin, Cyclophosphamide,
Lomustine
MACOP-B Methotrexate, Leucovorin, Doxorubicin,
Cyclophosphamide, Vincristine, Bleomycin, Prednisone
MAID Mesna, Doxorubicin, Ifosfamide, Dacarbazine
m-BACOD Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine,
Dexamethasone, Methotrexate, Leucovorin
MBC Methotrexate, Bleomycin, Cisplatin
MC Mitoxantrone, Cytarabine
MF Methotrexate, Fluorouracil, Leucovorin
MICE Ifosfamide, Carboplatin, Etoposide, Mesna
MINE Mesna, Ifosfamide, Mitoxantrone, Etoposide
mini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan
MOBP Bleomycin, Vincristine, Cisplatin, Mitomycin
MOP Mechlorethamine, Vincristine, Procarbazine
MOPP Mechlorethamine, Vincristine, Procarbazine, Prednisone
MOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,
Doxorubicin, Bleomycin, Vinblastine
MP (multiple Melphalan, Prednisone
myeloma)
MP (prostate cancer) Mitoxantrone, Prednisone
MTX/6-MO Methotrexate, Mercaptopurine
MTX/6-MP/VP Methotrexate, Mercaptopurine, Vincristine, Prednisone
MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin, Doxorubicin
MV (breast cancer) Mitomycin, Vinblastine Name Therapeutic agents
MV (acute myelocytic Mitoxantrone, Etoposide
leukemia)
M-VAC Methotrexate Vinblastine, Doxorubicin, Cisplatin
MVP Mitomycin Vinblastine, Cisplatin
MVPP Mechlorethamme, Vinblastine, Procarbazine, Prednisone
NFL Mitoxantrone, Fluorouracil, Leucovorin
NOVP Mitoxantrone, Vinblastine, Vincristine
OPA Vincristine, Prednisone, Doxorubicin
OPPA Add Procarbazine to OPA.
PAC Cisplatin, Doxorubicin
PAC-I Cisplatin, Doxorubicin, Cyclophosphamide
PA-CI Cisplatin, Doxorubicin
PC Paclitaxel, Carboplatin or Paclitaxel, Cisplatin
PCV Lomustine, Procarbazine, Vincristine
PE Paclitaxel, Estramustine
PFL Cisplatin, Fluorouracil, Leucovorin
POC Prednisone, Vincristine, Lomustine
ProMACE Prednisone, Methotrexate, Leucovorin, Doxorubicin,
Cyclophosphamide, Etoposide
ProMACE/cytaBOM Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,
Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin, Cotrimoxazole
PRoMACE/MOPP Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,
Mechlorethamme, Vincristine, Procarbazine, Methotrexate, Leucovorin
Pt/VM Cisplatin, Teniposide
PVA Prednisone, Vincristine, Asparaginase
PVB Cisplatin, Vinblastine, Bleomycin
PVDA Prednisone, Vincristine, Daunorubicin, Asparaginase
SMF Streptozocin, Mitomycin, Fluorouracil
TAD Mechlorethamme, Doxorubicin, Vinblastine, Vincristine,
Bleomycin, Etoposide, Prednisone Name Therapeutic agents
TCF Paclitaxel, Cisplatin, Fluorouracil
TIP Paclitaxel, Ifosfamide, Mesna, Cisplatin
TTT Methotrexate, Cytarabine, Hydrocortisone
Topo/CTX Cyclophosphamide, Topotecan, Mesna
VAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin,
Bleomycin
VAC Vincristine, Dactinomycin, Cyclophosphamide
VACAdr Vincristine, Cyclophosphamide, Doxorubicin,
Dactinomycin, Vincristine
VAD Vincristine, Doxorubicin, Dexamethasone
VATH Vinblastine, Doxorubicin, Thiotepa, Flouxymesterone
VBAP Vincristine, Carmustine, Doxorubicin, Prednisone
VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide,
Prednisone
VC Vinorelbine, Cisplatin
VCAP Vincristine, Cyclophosphamide, Doxorubicin, Prednisone
VD Vinorelbine, Doxorubicin
VelP Vinblastine, Cisplatin, Ifosfamide, Mesna
VIP Etoposide, Cisplatin, Ifosfamide, Mesna
VM Mitomycin, Vinblastine
VMCP Vincristine, Melphalan, Cyclophosphamide, Prednisone
VP Etoposide, Cisplatin
V-TAD Etoposide, Thioguanine, Daunorubicin, Cytarabine
5 + 2 Cytarabine, Daunorubicin, Mitoxantrone
7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or
Mitoxantrone
"8 in 1" Methylprednisolone, Vincristine, Lomustine,
Procarbazine, Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine
In certain embodiments, the compounds of the invention may be conjointly administered with a chemotherapeutic agent selected from inhibitors of another metabolic enzyme, such as a glucose transporter, hexokinase, pyruvate kinase M2, lactate dehydrogenase 1 or 2, pyruvate dehydrogenase kinase, fatty acid synthase, or glutaminase.
In some embodiments, the compounds of the invention may be conjointly administered with an immune -oncology therapeutic, such as an inhibitor of arginase, CTLA-4, indoleamine 2,3-dioxygenase, and PD-1/PD-L1. In certain embodiments, the immune -oncology agent is abagovomab, adecatumumab, afutuzumab, anatumomab mafenatox, apolizumab, blinatumomab, catumaxomab, durvalumab, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab, nivolumab, ocaratuzumab, olatatumab, pembrolizumab, pidilizumab, ticilimumab, samalizumab, or tremelimumab.
In certain embodiments, the compounds of the invention may be conjointly administered with an immunomodulatory agent. Examples of immunomodulatory agents with which the compounds of the invention may be administered in a combination therapy include granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod, IL-2, IL-7, IL-12, various chemokines, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans, and synthetic small molecules such as apremilast, CC-122, CC-11006, CC-10015, lenalidomide, pomalidomide, and thalidomide. In certain embodiments, the immunomodulatory agent is a thalidomide analog, such as those disclosed in WO 1999/46258, WO 2008/033567, WO 2010/093434, WO 2010/093605, WO 2011/100380, and WO 2012/097116.
In certain embodiments, the compounds of the invention may be conjointly administered with an anticancer agent selected from an enzyme inhibitor (such as a kinase inhibitor), a mitotic inhibitor, a DNA-modifying agent, and a cytidine analog. Examples of anticancer agents with which the compounds of the invention may be administered in a combination therapy include microtubule assembly inhibitors, AKT inhibitors, mTOR inhibitors, MEK inhibitors, RTK inhibitors, ATM inhibitors, ATR inhibitors, PI3K inhibitors, EGFR inhibitors, B-Raf inhibitors, C-kit inhibitors, DNA cross-linking agents, DNA intercalating agents, and cytidine analogs. In certain embodiments, the anticancer agents include microtubule assembly inhibitors, AKT inhibitors, mTOR inhibitors, MEK inhibitors, RTK inhibitors, ATM inhibitors, ATR inhibitors, PI3K inhibitors, EGFR inhibitors, B-Raf inhibitors, C-kit inhibitors, DNA cross-linking agents, PARP inhibitors, DNA intercalating agents, or cytidine analogs. In certain embodiments, the anticancer agent is vincristine, carboplatin, cisplatin, gemcitabine, MK2206, everolimus, trametinib, sunitinib, sorafenib, BEZ235, paclitaxel, docetaxel, erlotinib, selumetinib, sirolimus, trametinib, temsirolimus, pazopanib, or GSK1120212. In certain embodiments, the anticancer agent is vincristine, carboplatin, cisplatin, gemcitabine, MK2206, everolimus, trametinib, sunitinib, sorafenib, BEZ235, paclitaxel, docetaxel, erlotinib, selumetinib, sirolimus, trametinib, temsirolimus, pazopanib, olaparib, or GSK1120212.
In certain embodiments, the compounds of the invention are coadministered with one or more of lenalidomide, pomalidomide, and dexamethasone in the treatment of multiple myeloma. In certain embodiments, coadministration produces a synergistic effect.
The proliferation of cancer cells requires lipid synthesis. Normally, acetyl- coA used for lipid synthesis is formed from a mitochondrial pool of pyruvate that is derived from glycolysis. Yet under hypoxic conditions, such as those normally found in a tumor environment, the conversion of pyruvate to acetyl-coA within the mitochondria is downregulated. Recent studies from Metallo et al. (2011) and Mullen et al. (2011) revealed that under such hypoxic conditions, cells instead largely switch to using a pathway involving the reductive carboxylation of alpha-ketoglutarate to make acetyl-coA for lipid synthesis. The first step in this pathway involves converting glutamine to glutamate via glutaminase enzymes. Subsequently, glutamate is converting to alpha-ketoglutarate, and the resulting alpha-ketoglutarate is converted to isocitrate in a reductive carboxylation step mediated by the isocitrate dehydrogenase enzymes. A switch to this reductive carboxylation pathway also occurs in some renal carcinoma cell lines that contain either impaired mitochondria or an impaired signal for induction of the enzyme responsible for converting glycolytic pyruvate to acetyl- coA (Mullen et al 2011). A similar switch occurs in cells exposed to mitochondrial respiratory chain inhibitors such as metformin, rotenone, and antimycin (Mullen at al. 2011). Therefore, in some embodiments of this invention, we propose using combinations of mitochondrial respiratory chain inhibitors and glutaminase inhibitors to simultaneously increase cancer cells' dependence on glutaminase-dependent pathways for lipid synthesis while inhibiting those very pathways.
The increased dependence on glycolysis in tumor cells is likely because the hypoxic tumor environment impairs mitochondrial respiration. Furthermore, depletion of glucose induces apoptosis in cells transformed with the MYC oncogene. These findings suggest that inhibiting glycolysis would have a therapeutic value in preventing cancer cell proliferation. There are currently many documented glycolytic inhibitors (Pelicano et al. 2006). However, as pointed out by Zhao et al. (2012), "available glycolytic inhibitors are generally not very potent, and high doses are required, which may cause high levels of systemic toxicity." Since cancer cells typically use both glucose and glutamine at higher levels than normal cells, impairing utilization of each of those metabolites will likely have a synergistic effect.
Therefore, in some embodiments of this invention, we propose using combinations of glycolytic pathway inhibitors and glutaminase inhibitors. Such glycolytic inhibitors include 2-deoxyglucose, lonidamine, 3-bromopyruvate, imatinib, oxythiamine, rapamycin, and their pharmacological equivalents. Glycolysis can be inhibited indirectly by depleting NAD+ via DNA damage induced by DNA alkylating agents through a pathway activated by poly(ADP-ribose) polymerase (Zong et al. 2004). Therefore, in some embodiments of this invention, we propose using a combination of DNA alkylating agents and glutaminase inhibitors. Cancer cells use the pentose phosphate pathway along with the glycolytic pathway to create metabolic
intermediates derived from glucose. Therefore, in some embodiments of this invention, we propose using a combination of pentose phosphate inhibitors such as 6- aminonicotinamide along with glutaminase inhibitors.
In certain embodiments, a compound of the invention may be conjointly administered with non-chemical methods of cancer treatment. In certain
embodiments, a compound of the invention may be conjointly administered with radiation therapy. In certain embodiments, a compound of the invention may be conjointly administered with surgery, with thermoablation, with focused ultrasound therapy, with cryotherapy, or with any combination of these.
In certain embodiments, different compounds of the invention may be conjointly administered with one or more other compounds of the invention.
Moreover, such combinations may be conjointly administered with other therapeutic agents, such as other agents suitable for the treatment of cancer, immunological or neurological diseases, such as the agents identified above. In certain embodiments, conjointly administering one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect, such as shown in Figure 8. In certain embodiments, conjointly administering one or more additional
chemotherapeutics agents provides an additive effect.
In certain embodiments, the present invention provides a kit comprising: a) one or more single dosage forms of a compound of the invention; b) one or more single dosage forms of a chemotherapeutic agent as mentioned above; and c) instructions for the administration of the compound of the invention and the chemotherapeutic agent for the treatment of cancer, wherein the cancer is multiple myeloma.
The present invention provides a kit comprising:
a) a pharmaceutical formulation (e.g., one or more single dosage forms)
comprising a compound of the invention; and
b) instructions for the administration of the pharmaceutical formulation, e.g., for treating or preventing cancer, such as multiple myeloma. In certain embodiments, the kit further comprises instructions for the administration of the pharmaceutical formulation comprising a compound of the invention conjointly with a chemotherapeutic agent as mentioned above. In certain embodiments, the kit further comprises a second pharmaceutical formulation (e.g., as one or more single dosage forms) comprising a chemotherapeutic agent as mentioned above.
Protein amounts can be measured using antibodies. Antibodies suitable for use in the methods disclosed herein are commercially available, or can be prepared routinely. Methods for preparing and using antibodies in assays for proteins of interest are conventional, and are described in, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), (Humana Press 1992); Coligan et al, in Current Protocols in Immunology, Sec. 2.4.1 (1992); Kohler & Milstein (1975), Nature 256, 495; Coligan et al, sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Laboratory Pub. 1988).
Any of a variety of antibodies can be used in methods of the invention. Such antibodies include, for example, polyclonal, monoclonal (mAbs), recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof. The antibodies can be of any isotype, e.g., IgM, various IgG isotypes such as IgGl, IgG2a, etc., and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like. The term "an antibody specific for" a protein means that the antibody recognizes a defined sequence of amino acids, or epitope, in the protein, and binds selectively to the protein and not generally to proteins unintended for binding to the antibody. The parameters required to achieve specific binding can be determined routinely, using conventional methods in the art.
In some embodiments of the invention, antibodies specific for pyruvate carboxylase are immobilized on a surface (e.g., are reactive elements on an array, such as a microarray, or are on another surface, such as used for surface plasmon resonance (SPR)-based technology, such as Biacore), and proteins in the sample are detected by virtue of their ability to bind specifically to the antibodies. Alternatively, proteins in the sample can be immobilized on a surface, and detected by virtue of their ability to bind specifically to the antibodies. Methods of preparing the surfaces and performing the analyses, including conditions effective for specific binding, are conventional and well-known in the art.
Among the many types of suitable immunoassays are immunohistochemical staining, ELISA, Western blot (immunoblot), immunoprecipitation,
radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), etc. Assays used in methods of the invention can be based on colorimetric readouts, fluorescent readouts, mass spectroscopy, visual inspection, etc.
As mentioned above, expression levels of pyruvate carboxylase can be measured by measuring mRNA amounts. The amount of an mRNA encoding a pyruvate carboxylase can be measured using any suitable method. Examples of such methods include, for example, reverse transcriptase-polymerase chain reaction (RT- PCR), including real time PCR, microarray analysis, nanostring, Northern blot analysis, differential hybridization, and ribonuclease protection assay. Such methods are well-known in the art and are described in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, current edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & sons, New York, N.Y.
In some embodiments of the invention, a histological sample is obtained from a subject (e.g., from a tumor biopsy), using any method known in the art, and include, but are not limited to, tissue section, needle biopsy, and the like. Frequently the sample will be a "clinical sample", which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes. The sample can also be derived from supernatants (of cells) or the cells themselves from cell cultures, cells from tissue culture and other media. Protein or mR A is then obtained brom the sample, and used to quantitate the amounts of pyruvate carboxylase.
The disclosure also provides kits for detecting whether a subject having a cancer is likely to be responsive to glutaminase inhibitors. The kit may include one or more agents for detecting the amount of expression of a protein of the invention [e.g., the amount of the protein, and/or the amount of a nucleic acid (e.g., an mRNA) encoding the protein]. The agents in the kit can encompass, for example, antibodies specific for the proteins, or probes specific for the mRNA that can be used to hybridize to the RNA (or to a cDNA generated from it) or to perform RT-PCR. The kit may also include additional agents suitable for detecting, measuring and/or quantitating the amount of protein or nucleic acid. Among other uses, kits of the invention can be used in experimental applications. A skilled worker will recognize components of kits suitable for carrying out a method of the invention.
Optionally, a kit of the invention may comprise instructions for performing the method. Optional elements of a kit of the invention include suitable buffers, containers, or packaging materials. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., for the performance of an assay for a single subject.
Definitions
The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(0)NH-.
The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(0)0-, preferably alkylC(0)0-.
The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
The term "alkenyl", as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those
contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
An "alkyl" group or "alkane" is a straight chained or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 unless otherwise defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A Ci-C6 straight chained or branched alkyl group is also referred to as a "lower alkyl" group.
Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, if not otherwise specified, can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls, -CF3, -CN, and the like.
The term "Cx_y" when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "Cx_yalkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc. Co alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. The terms "C2-yalkenyl" and "C2_yalkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
The term "alkylamino", as used herein, refers to an amino group substituted with at least one alkyl group.
The term "alkylthio", as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.
The term "alkynyl", as used herein, refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having
substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
The term "amide", as used herein, refers to a group
Figure imgf000037_0001
wherein each R independently represent a hydrogen or hydrocarbyl group, or two R10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by
Figure imgf000038_0001
wherein each R10 independently represents a hydrogen or a hydrocarbyl group, or two R10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
The term "aminoalkyl", as used herein, refers to an alkyl group substituted with an amino group.
The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group.
The term "aryl" as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
The term "carbamate" is art-recognized and refers to a group
O O
^O^N'Rl° or ^N^0'R1°
R9 R9
wherein R9 and R10 independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, or R9 and R10 taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
The terms "carbocycle", and "carbocyclic", as used herein, refers to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic carbocycles and non-aromatic carbocycles. Non- aromatic carbocycles include both cycloalkane rings, in which all carbon atoms are saturated, and cycloalkene rings, which contain at least one double bond.
"Carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane,
cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of
carbocyclic. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1 Jheptane, 1 ,5-cyclooctadiene, 1 ,2,3,4-tetrahydronaphthalene,
bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. "Carbocycles" may be susbstituted at any one or more positions capable of bearing a hydrogen atom.
A "cycloalkyl" group is a cyclic hydrocarbon which is completely saturated. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms unless otherwise defined. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. A
"cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.
The term "carbocyclylalkyl", as used herein, refers to an alkyl group substituted with a carbocycle group.
The term "carbonate" is art-recognized and refers to a group -OCO2-R10, wherein R10 represents a hydrocarbyl group. The term "carboxy", as used herein, refers to a group represented by the formula -C02H.
The term "ester", as used herein, refers to a group -C(0)OR10 wherein R10 represents a hydrocarbyl group.
The term "ether", as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O- heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.
The terms "halo" and "halogen" as used herein means halogen and includes chloro, fluoro, bromo, and iodo.
The terms "hetaralkyl" and "heteroaralkyl", as used herein, refers to an alkyl group substituted with a hetaryl group.
The term "heteroalkyl", as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.
The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10- membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
The term "heterocyclylalkyl", as used herein, refers to an alkyl group substituted with a heterocycle group.
The term "hydrocarbyl", as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxy ethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.
The term "hydroxyalkyl", as used herein, refers to an alkyl group substituted with a hydroxy group.
The term "lower" when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer. A "lower alkyl", for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).
The terms "polycyclyl", "polycycle", and "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.
The term "silyl" refers to a silicon moiety with three hydrocarbyl moieties attached thereto.
The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.
The term "sulfate" is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.
The term "sulfonamide" is art-recognized and refers to the group represented by the general formulae
Figure imgf000043_0001
wherein R9 and R10 independently represents hydrogen or hydrocarbyl, such as alkyl, or R9 and R10 taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
10
The term "sulfoxide" is art-recognized and refers to the group -S(0)-R
, 10
wherein R represents a hydrocarbyl.
The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.
The term "sulfone" is art-recognized and refers to the group -S(0)2-R10, wherein R10 represents a hydrocarbyl.
The term "thioalkyl", as used herein, refers to an alkyl group substituted with a thiol group.
The term "thioester", as used herein, refers to a group -C(0)SR10 or -SC(0)R10 wherein R10 represents a hydrocarbyl.
The term "thioether", as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.
The term "urea" is art-recognized and may be represented by the general formula
R9 R9
wherein R9 and R10 independently represent hydrogen or a hydrocarbyl, such as alkyl, or either occurrence of R9 taken together with R10 and the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
"Protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al, Compendium of Synthetic Organic Methods, Vols. 1- 8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2- trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups,
allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro- veratryloxycarbonyl ("NVOC") and the like. Representative hydroxylprotecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterified) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.
The term "healthcare providers" refers to individuals or organizations that provide healthcare services to a person, community, etc. Examples of "healthcare providers" include doctors, hospitals, continuing care retirement communities, skilled nursing facilities, subacute care facilities, clinics, multispecialty clinics, freestanding ambulatory centers, home health agencies, and HMO's.
As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
The term "treating" includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
The term "prodrug" is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound of formula I). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds of formula I in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.
Pharmaceutical Compositions
The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred
embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.
A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally;
intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.
To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.
Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatable with such fluids. A preferred route of administration is local
administration (e.g., topical administration, such as eye drops, or administration via an implant).
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical
administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Methods of introduction may also be provided by rechargeable or
biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By "therapeutically effective amount" is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
In general, a suitable daily dose of an active compound used in the
compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
If desired, the effective daily dose of the active compound may be
administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.
The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.
In certain embodiments, conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) (e.g., one or more additional chemotherapeutic agent(s)) provides improved efficacy relative to each individual administration of the compound of the invention (e.g., compound of formula I or la) or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).
This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain
embodiments, contemplated salts of the invention include, but are not limited to, L- arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,
ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically acceptable antioxidants include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
In certain embodiments, the invention relates to a method for conducting a pharmaceutical business, by manufacturing a formulation of a compound of the invention, or a kit as described herein, and marketing to healthcare providers the benefits of using the formulation or kit for treating or preventing any of the diseases or conditions as described herein.
In certain embodiments, the invention relates to a method for conducting a pharmaceutical business, by providing a distribution network for selling a formulation of a compound of the invention, or kit as described herein, and providing instruction material to patients or physicians for using the formulation for treating or preventing any of the diseases or conditions as described herein.
In certain embodiments, the invention comprises a method for conducting a pharmaceutical business, by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the diseases or conditions as described herein, conducting therapeutic profiling of identified formulations for efficacy and toxicity in animals, and providing a distribution network for selling an identified preparation as having an acceptable therapeutic profile. In certain embodiments, the method further includes providing a sales group for marketing the preparation to healthcare providers.
In certain embodiments, the invention relates to a method for conducting a pharmaceutical business by determining an appropriate formulation and dosage of a compound of the invention for treating or preventing any of the disease or conditions as described herein, and licensing, to a third party, the rights for further development and sale of the formulation.
Examples
The synthesis of exemplary compounds of the invention is described in U.S. Patent No. 8,604,016, which is incorporated herein by reference. Also described in U.S. Patent No. 8,604,016 are protocols for various assays including recombinant enzyme assays and assays surveying cell proliferation, solubility, and Caco-2 permeability using the compounds of the invention. Certain data are found in Table 3, below.
IC50 is a quantitative measure indicating how much compound is needed to inhibit a given biological activity by half.
Table 3:
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Further experimental data pertaining to use of the compounds of the invention in anti-proliferation studies, metabolite studies, and treatment of cancer xenografts can be found at Examples 5-16 of U.S. Patent Application Publication No.
2015/0004134, which is hereby incorporated by reference. Example 5: Multiple Myeloma xenograft study.
Female CB.17 SCID mice (n=20) age 8-12 weeks were implanted
subcutaneously with 1 x 107 RPMI-8226 myeloma cells per mouse mixed 1 : 1 with Matrigel. Mice were randomized into the following two groups of n=10 mice/group: 1) Vehicle control (25% Hydroxypropyl-P-cyclodextrin) and 2) Compound 670 dosed at orally at 200 mg/kg (formulated at 20 mg/mL in 25% ΗΡ-β-CD). For both groups, dosing was initiated when tumors reached a volume of 100- 150mm3 and continued orally BID for 28 days. Tumors were measured with calipers two times per week and tumor volume calculated using the formula tumor volume (mm3) = (a x b2/2) where 'b' is the smallest diameter and 'a' is the largest perpendicular diameter. **P-value < 0.01 (Two-sided T-test). Results are shown in Figure 17.
Example 6: Treatment of Multiple Myeloma cells with a combination of drugs.
As shown in Figure 18, MM1S cells (panels A & B) and RPMI-8226 cells (panels C & D) were treated with a dose titration of either compound 670,
pomalidomide or a mixture thereof (panels A & C) or compound 670, dexamethsone or a mixture thereof (panels B & D) for 72 hours in growth media. At the end of the incubation, cell viability was measured using Cell Titer Glo as per manufacturer's protocol (Promega, Madison, WI). Measured values for compound-treated cells were normalized to DMSO-treated cells and data is reported as a cell survival ratio with a value of 1 (one) corresponding to maximum cell survival and a value of 0 (zero) corresponding to no cell survival. Cell survival ratios for all compound treatments are represented as bar graphs. Combination indices were calculated using the Calcusyn program (biosoft.com) and reported for individual mixtures of compound 670 and pomalidomide [POM] (panels A &C) and individual mixtures of compound 670 and dexamethasone [DEX] (panels B & D). Compound mixtures that produced a synergistic anti-tumor activity are highlighted.
Example 7: SDS-PAGE and Western Blotting
Figure 10 shows a western blot for pyruvate carboxylase in multiple myeloma cell lines. Total cell lysates are prepared from cell pellets by sonication and protein concentration is quantitated using standard methods. Lysate proteins are denatured by boiling in SDS-sample buffer and resolved (10 μg/lane) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins are then transferred to nitrocellulose and probed with antibodies recognizing pyruvate carboxylase or beta- actin followed by HRP-coupled secondary antibodies. Bands are revealed by chemiluminescence. Image J software (NIH) is used to quantify the pyruvate carboxylase band intensity using methods recommended by the software developer.
Example 8: Cell viability assay
Cells were treated with a dose range of CB-839 for 72 h at 37°C with 5% C02. Anti-proliferative effects were determined by measuring ATP levels using Cell Titer Glo reagent (Promega) following the manufactures' instructions. The Cell Titer Glo signal was measured on the day of compound addition and compared to the measurements taken at 72 h to determine effects on cell growth and survival during the assay period. Inhibition curves were fitted (GraphPad Prism) to a four-parameter dose response equation of the form: % activity = Bottom + (Top- Bottom)/(l+10A((LogIC50-X)*HillSlope)). Results are shown in Figure 11.
Whole Transcriptome Sequencing (RNA-Seq)
Whole transcriptome sequencing on cell pellets is performed by Expression Analysis (Durham, NC). Normalized expression values are determined using the RSEM (RNA-Seq by Expectation Maximization) software package. Normalized expression level is shown in Figure 12. Incorporation by Reference
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. The compounds, synthetic methods, and experimental protocols and results of U.S. Patent Number 8,604,016, filed November 19, 2012 and issued December 10, 2013, are hereby incorporated by reference.
Equivalents While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations .

Claims

Claims:
1. A method for treating multiple myeloma in a patient, the method comprising: a) determining a level of pyruvate carboxylase in a patient sample comprising multiple myeloma cancer cells;
b) comparing the level of pyruvate carboxylase in the patient sample to a reference standard; and
c) if the level of pyruvate carboxylase in the patient sample is lower than the reference standard, then administering to the patient an effective amount of a glutaminase inhibitor.
2. A method of identifying a multiple myeloma patient that may benefit from treatment with a glutaminase inhibitor, comprising determining the level of pyruvate carboxylase in multiple myeloma cancer cells of the patient compared to a reference standard, wherein a lower level in the cancer cells of the patient as compared to the standard indicates that the patient may benefit from treatment with a glutaminase inhibitor.
3. The method of claim 1 or claim 2, wherein the glutaminase inhibitor is a compound of formula I,
Figure imgf000063_0001
or a pharmaceutically acceptable salt thereof, wherein:
L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2,
CH=CH, or ^ A -f^, , wherein any hydrogen atom of a CH or CH2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxy;
X, independently for each occurrence, represents S, O or CH=CH, wherein any
hydrogen atom of a CH unit may be replaced by alkyl;
Y, independently for each occurrence, represents H or CH20(CO)R7; R7, independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, or
heterocyclylalkoxy;
Z represents H or R3(CO);
Ri and R2 each independently represent H, alkyl, alkoxy or hydroxy;
R3, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy,
heteroaryloxyalkyl or C(Rg)(R9)(Rio), N(R )(R5) or OR5, wherein any free hydroxyl group may be acylated to form C(0)R7;
R4 and R5 each independently represent H or substituted or unsubstituted alkyl,
hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7;
5, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7; and
Rg, R9 and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, or R8 and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein at least two of
Figure imgf000064_0001
4. The method of claim 3, wherein L represents CH2SCH2, CH2CH2, CH2S or SCH2.
5. The method of claim 3, wherein L represents CH2CH2.
6. The method of any one of claims 3-5, wherein Y represents H.
7. The method of any one of claims 3-6, wherein X, independently for each occurrence, represents S or CH=CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl.
8. The method of any one of claims 3-7, wherein Z represents R3(CO).
9. The method of claim 8, wherein each occurrence of R3 is not identical.
10. The method of any one of claims 3-9, wherein Ri and R2 each represent H.
11. The method of any one of claims 3-10, wherein R3, independently for each occurrence, represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
12. The method of any one of claims 3-11, wherein R3, independently for each occurrence, represents C(Rg)(R9)(Rio), wherein R8 represents substituted or unsubstituted aryl, arylalkyl, heteroaryl or heteroaralkyl, R9 represents H, and R10 represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl.
13. The method of claim 12, wherein R8 represents substituted or unsubstituted aryl, arylalkyl, or heteroaryl.
14. The method of claim 12 or 13, wherein Rio represents hydroxy, hydroxyalkyl, or alkoxy.
15. The method of claim 3, wherein L represents CH2SCH2, CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and R3, independently for each occurrence, represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
16. The method of claim 15, wherein each occurrence of R3 is identical.
17. The method of claim 3, wherein L represents CH2SCH2, CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, and R3, independently for each occurrence, represents C(Rg)(R9)(Rio), wherein R8 represents substituted or unsubstituted aryl, arylalkyl, heteroaryl or heteroaralkyl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl.
18. The method of claim 17, wherein L represents CH2CH2.
19. The method of claim 17 or 18, wherein R8 represents substituted or unsubstituted aryl, arylalkyl or heteroaryl.
20. The method of claim 19, wherein R8 represents substituted or unsubstituted aryl.
21. The method of any of claims 17-20, wherein Rio represents hydroxy, hydroxyalkyl or alkoxy.
22. The method of claim 21, wherein R10 represents hydroxyalkyl.
23. The method of any one of claims 17-22, wherein each occurrence of R3 is identical.
24. The method of claim 3, wherein L represents CH2CH2, Y represents H, X, independently for each occurrence, represents S or CH=CH, Z represents R3(CO), Ri and R2 each represent H, and R3, independently for each occurrence, represents arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
25. The method of claim 24, wherein each occurrence of R3 is identical.
26. The method of claim 1 or claim 2, wherein the glutaminase inhibitor is a compound of formula la,
Figure imgf000067_0001
or a pharmaceutically acceptable salt thereof, wherein:
L represents CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2,
CH=CH, or ^ , preferably CH2CH2, wherein any hydrogen atom of a
CH or CH2 unit may be replaced by alkyl or alkoxy, any hydrogen of an NH unit may be replaced by alkyl, and any hydrogen atom of a CH2 unit of CH2CH2, CH2CH2CH2 or CH2 may be replaced by hydroxy;
X represents S, O or CH=CH, preferably S or CH=CH, wherein any hydrogen atom of a CH unit may be replaced by alkyl;
Y, independently for each occurrence, represents H or CH20(CO)R7 ;
R7, independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl, or heterocyclylalkoxy;
Z represents H or R3(CO);
Ri and R2 each independently represent H, alkyl, alkoxy or hydroxy, preferably H;
R3 represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl,
acylaminoalkyl, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heteroaryloxyalkyl or
C(R8)(R9)(Rio), N(R )(R5) or OR6, wherein any free hydroxyl group may be acylated to form C(0)R7;
R4 and R5 each independently represent H or substituted or unsubstituted alkyl,
hydroxyalkyl, acyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form
C(0)R7; Re, independently for each occurrence, represents substituted or unsubstituted alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7; and
Rg, R9 and Rio each independently represent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, or R8 and R9 together with the carbon to which they are attached, form a carbocyclic or heterocyclic ring system, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein at least two of
Figure imgf000068_0001
R11 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl, or
C(Ri2)(Ri3)(Ri4), N(R4)(Ri4) or OR14, wherein any free hydroxyl group may be acylated to form C(0)R7;
Ri2 and R13 each independently respresent H or substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acylamino, aminoalkyl, acylaminoalkyl, alkoxycarbonyl, alkoxycarbonylamino, alkenyl, alkoxy, alkoxyalkyl, aryl, arylalkyl, aryloxy, aryloxyalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or
heteroaryloxyalkyl, wherein any free hydroxyl group may be acylated to form C(0)R7, and wherein both of Ri2 and R13 are not H; and
Ri4 represents substituted or unsubstituted aryl, arylalkyl, aryloxy, aryloxyalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, or heteroaryloxyalkyl.
27. The method of claim 26, wherein Rn represents substituted or unsubstituted arylalkyl.
28. The method of claim 27, wherein Rn represents substituted or unsubstituted benzyl.
29. The method of claim any one of claims 26-28, wherein L represents
CH2SCH2, CH2CH2, CH2S or SCH2.
30. The method of claim 29, wherein L represents CH2CH2.
31. The method of any of claims 26-30, wherein each Y represents H.
32. The method of any of claims 26-31 , wherein X represents S or CH=CH.
33. The method of claim 32, wherein X represents S.
34. The method of any of claims 26-33, wherein Z represents R3(CO).
35. The method of claim 34, wherein R3 and Rn are not identical.
36. The method of any of claims 26-35, wherein Ri and R2 each represent H.
37. The method of claim 34, wherein R3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl.
38. The method of claim 37, wherein R3 represents substituted or unsubstituted heteroarylalkyl.
39. The method of claim 34, wherein R3 represents C(Rg)(R9)(Rio), wherein R8 represents substituted or unsubstituted aryl, arylalkyl, heteroaryl or heteroaralkyl, R9 represents H, and Rio represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl.
40. The method of claim 39, wherein R8 represents substituted or unsubstituted aryl, arylalkyl, or heteroaryl.
41. The method of claim 39 or 40, wherein Rio represents hydroxy, hydroxyalkyl, or alkoxy.
42. The method of claim 26, wherein L represents CH2SCH2, CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, R3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl, and Rn represents substituted or unsubstituted arylalkyl.
43. The method of claim 42, wherein R3 represents substituted or unsubstituted heteroarylalkyl.
44. The method of claim 26, wherein L represents CH2SCH2, CH2CH2, CH2S or SCH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, R3 represents C(Rg)(R9)(Rio), wherein R8 represents substituted or unsubstituted aryl, arylalkyl, heteroaryl or heteroaralkyl, R9 represents H, R10 represents hydroxy, hydroxyalkyl, alkoxy or alkoxyalkyl, and Rn represents substituted or unsubstituted arylalkyl.
45. The method of claim 44, wherein R8 represents substituted or unsubstituted aryl, arylalkyl or heteroaryl.
46. The method of claim 45, wherein Rg represents heteroaryl.
47. The method of any of claims 44-46, wherein Rio represents hydroxy, hydroxyalkyl or alkoxy.
48. The method of claim 26, wherein L represents CH2CH2, Y represents H, X represents S or CH=CH, Z represents R3(CO), Ri and R2 each represent H, R3 represents substituted or unsubstituted arylalkyl, heteroarylalkyl, cycloalkyl or heterocycloalkyl, and Rn represents substituted or unsubstituted arylalkyl.
49. The method of claim 48, wherein R3 represents substituted or unsubstituted heteroarylalkyl.
50. The method of claim 26, wherein L represents CH2CH2, Y represents H, X represents S, Z represents R3(CO), Ri and R2 each represent H, R3 represents
C(Rg)(R9)(Rio), wherein Rg represents substituted or unsubstituted aryl, arylalkyl or heteroaryl, R9 represents H, R10 represents hydroxy, hydroxyalkyl or alkoxy, and Rn represents substituted or unsubstituted arylalkyl.
51. The method of any preceding claim, wherein the level of pyruvate carboxylase is determined by measuring an amount of pyruvate carboxylase protein expressed in a sample of multiple myeloma cells from the patient.
52. The method of claim 51 , wherein the amount of pyruvate carboxylase protein is measured as western blot band intensity.
53. The method of claim 51 , wherein the amount of pyruvate carboxylase protein is measured by immunohistochemistry.
54. The method of any one of claims 1-50, wherein the level of pyruvate carboxylase is determined by measuring the activity of pyruvate carboxylase.
55. The method of any of claims 1-50, wherein the level of pyruvate carboxylase is determined by whole transcriptome sequencing.
56. The method of any preceding claim, wherein the reference standard is a level of pyruvate carboxylase in a multiple myeloma cell line that is resistant to a compound of formula (II):
Figure imgf000071_0001
The method of claim 56, wherein the cell line is KMS-28PE or KMS-11 cells.
58. The method of claim 55, wherein the reference standard is expression of pyruvate carboxylase at a transcript count of about 220, wherein the transcript count is normalized against reference microarrays.
59. The method of any preceding claim, further comprising conjointly
administering one or more additional chemotherapeutic agents.
60. The method of claim 59, wherein conjointly administering one or more additional chemotherapeutic agents provide improved efficacy relative to each individual administration of the glutaminase inhibitor or the one or more additional chemotherapeutic agent.
61. The method of claim 60, wherein conjointly administering one or more additional chemotherapeutic agents provide a synergistic effect.
62. The method of claim 60, wherein conjointly administering one or more additional chemotherapeutic agents provide an additive effect.
63. The method of any of claims 59-62, wherein the glutaminase inhibitor and the one or more additional chemotherapeutic agents are administered simultaneously.
64. The method of any of claims 59-62, wherein the one or more additional chemotherapeutic agents are administered within about 5 minutes to within about 168 hours prior to or after administration of the glutaminase inhibitor.
65. The method of any of claims 59-64, wherein the one or more additional chemotherapeutic agents are selected from ABT-263, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, AZD5363, Bacillus Calmette-Guerin vaccine (beg), bicalutamide, bisphosphonate, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, cobimetinib, colchicine,
cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,
daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, eribulin, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil (e.g., 5-fluorouracil), fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ixabepilone, lenalidomaide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, miltefosine, mitomycin, mitotane, mitoxantrone, MK-2206, nilutamide, nocodazole, octreotide, oxaliplatin, olaparib, paclitaxel, pamidronate, pazopanib, pentostatin, perifosine, PF- 04691502, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, romidepsin, rucaparib, selumetinib, sorafenib, streptozocin, sunitinib, suramin, talazoparib, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trametinib, trastuzumab, tretinoin, veliparib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat (SAHA).
66. The method of claim 65, wherein the one or more additional chemotherapeutic agents are selected from bisphosphonate, bortezomib, carfilzomib, doxorubicin, lenalidomide, melphalan, pomalidomide, and vincristine.
67. The method of any of claims 59-64, wherein the one or more additionanl chemotherapeutic agents are immunooncology therapeutic agents selected from abagovomab, adecatumumab, afutuzumab, anatumomab mafenatox, apolizumab, blinatumomab, catumaxomab, durvalumab, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isatuximab, lambrolizumab, nivolumab, ocaratuzumab, olatatumab, pembrolizumab, pidilizumab, ticilimumab, samalizumab, and tremelimumab
68. The method of any preceding claim, further comprising conjointly
administering one or more immunomodulatory agents.
69. The method of claim 68, wherein conjointly administering one or more immunomodulatory agents provide improved efficacy relative to each individual administration of the glutaminase inhibitor or the one or more immunomodulatory agent.
70. The method of claim 69, wherein conjointly administering one or more immunomodulatory agents provide a synergistic effect.
71. The method of claim 69, wherein conjointly administering one or more immunomodulatory agents provide an additive effect.
72. The method of any of claims 68-71, wherein the glutaminase inhibitor and the one or more immunomodulatory agents are administered simultaneously.
73. The method of any of claims 68-71, wherein the one or more
immunomodulatory agents are administered within about 5 minutes to within about 168 hours prior to or after administration of the glutaminase inhibitor.
74. The method of any of claims 68-73, wherein the one or more
immunomodulatory agents are selected from granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod, IL-2, IL-7, IL-12, various chemokines, synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, glucans, and synthetic small molecules such as apremilast (CC-10004), CC-122, CC-11006, CC-10015, dexamethasone, prednisone, ImiDs, lenalidomide, pomalidomide, and thalidomide.
75. The method of claim 74, wherein the immunomodulatory agent is selected from dexamethasone, prednisone, and IMiDs.
76. The method of any preceding claim, further comprising conjointly
administering one or more anticancer agents selected from enzyme inhibitors (such as a kinase inhibitor), mitotic inhibitors, DNA-modifying agents, and cytidine analogs.
77. The method of claim 74, wherein the anti-cancer agent is selected from microtubule assembly inhibitors, AKT inhibitors, mTOR inhibitors, MEK inhibitors, RTK inhibitors, ATM inhibitors, ATR inhibitors, PI3K inhibitors, EGFR inhibitors, B-Raf inhibitors, C-kit inhibitors, PARP inhibitors, DNA cross-linking agents, DNA intercalating agents, and cytidine analogs.
78. The method of claim 75, wherein the anti-cancer agent is selected from vincristine, carboplatin, cisplatin, gemcitabine, MK2206, everolimus, trametinib, sunitinib, sorafenib, BEZ235, paclitaxel, docetaxel, erlotinib, selumetinib, sirolimus, trametinib, temsirolimus, pazopanib, olaparib, and GSK1120212.
PCT/US2015/041891 2014-07-24 2015-07-24 Treatment of multiple myeloma with heterocyclic inhibitors of glutaminase WO2016014890A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462028523P 2014-07-24 2014-07-24
US62/028,523 2014-07-24

Publications (1)

Publication Number Publication Date
WO2016014890A1 true WO2016014890A1 (en) 2016-01-28

Family

ID=55163809

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/041891 WO2016014890A1 (en) 2014-07-24 2015-07-24 Treatment of multiple myeloma with heterocyclic inhibitors of glutaminase

Country Status (1)

Country Link
WO (1) WO2016014890A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016160980A1 (en) * 2015-03-30 2016-10-06 Calithera Biosciences, Inc. Methods of administering glutaminase inhibitors
WO2017062354A1 (en) * 2015-10-05 2017-04-13 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors and immuno-oncology agents
US9682952B2 (en) 2012-09-04 2017-06-20 Celgene Corporation Isotopologues of 3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl) piperidine-2-6-dione and methods of preparation thereof
US9687485B2 (en) 2014-06-13 2017-06-27 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors
US9732064B2 (en) 2006-09-26 2017-08-15 Celgene Corporation 5-substituted quinazolinone derivatives and compositions comprising and methods of using the same
US9751853B2 (en) 2011-03-11 2017-09-05 Celgene Corporation Solid forms of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, and their pharmaceutical compositions and uses
US10125130B2 (en) 2014-04-30 2018-11-13 Pfizer Inc. Cycloalkyl-linked diheterocycle derivatives
US10278968B2 (en) 2016-08-25 2019-05-07 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors
US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10316030B2 (en) 2014-08-07 2019-06-11 Calithera Biosciences, Inc. Crystal forms of glutaminase inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10441587B2 (en) 2015-04-06 2019-10-15 Calithera Biosciences, Inc. Treatment of lung cancer with inhibitors of glutaminase
IT201800009974A1 (en) * 2018-10-31 2020-05-01 Universita' Degli Studi Di Milano - Bicocca COMBINED PHARMACOLOGICAL TREATMENT FOR CANCER

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140142081A1 (en) * 2012-11-22 2014-05-22 Agios Pharmaceuticals, Inc Compounds and their methods of use
US20140142146A1 (en) * 2012-11-21 2014-05-22 Agios Pharmaceuticals, Inc Compounds and their methods of use
WO2014079150A1 (en) * 2012-11-22 2014-05-30 Agios Pharmaceuticals, Inc. Compounds and their methods of use
WO2014089048A1 (en) * 2012-12-03 2014-06-12 Calithera Biosciences, Inc. Treatment of cancer with heterocyclic inhibitors of glutaminase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140142146A1 (en) * 2012-11-21 2014-05-22 Agios Pharmaceuticals, Inc Compounds and their methods of use
US20140142081A1 (en) * 2012-11-22 2014-05-22 Agios Pharmaceuticals, Inc Compounds and their methods of use
WO2014079150A1 (en) * 2012-11-22 2014-05-30 Agios Pharmaceuticals, Inc. Compounds and their methods of use
WO2014089048A1 (en) * 2012-12-03 2014-06-12 Calithera Biosciences, Inc. Treatment of cancer with heterocyclic inhibitors of glutaminase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NAMBA, MASAYOSHI ET AL.: "Establishment of five human myeloma cell lines.", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY, vol. 25.8, 1989, pages 723 - 729 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9732064B2 (en) 2006-09-26 2017-08-15 Celgene Corporation 5-substituted quinazolinone derivatives and compositions comprising and methods of using the same
US9751853B2 (en) 2011-03-11 2017-09-05 Celgene Corporation Solid forms of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, and their pharmaceutical compositions and uses
US9969713B2 (en) 2011-03-11 2018-05-15 Celgene Corporation Solid forms of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, and their pharmaceutical compositions and uses
US9682952B2 (en) 2012-09-04 2017-06-20 Celgene Corporation Isotopologues of 3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl) piperidine-2-6-dione and methods of preparation thereof
US10125130B2 (en) 2014-04-30 2018-11-13 Pfizer Inc. Cycloalkyl-linked diheterocycle derivatives
US10676471B2 (en) 2014-04-30 2020-06-09 Pfizer Inc. Cycloalkyl-linked diheterocycle derivatives
US9687485B2 (en) 2014-06-13 2017-06-27 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors
US10316030B2 (en) 2014-08-07 2019-06-11 Calithera Biosciences, Inc. Crystal forms of glutaminase inhibitors
US10676472B2 (en) 2014-08-07 2020-06-09 Calithera Biosciences, Inc. Crystal forms of glutaminase inhibitors
EA037152B1 (en) * 2015-03-30 2021-02-11 Калитера Байосайенсиз, Инк. Method of treating cancer
CN107921031A (en) * 2015-03-30 2018-04-17 卡利泰拉生物科技公司 The method for giving glutamine enzyme inhibitor
WO2016160980A1 (en) * 2015-03-30 2016-10-06 Calithera Biosciences, Inc. Methods of administering glutaminase inhibitors
US10441587B2 (en) 2015-04-06 2019-10-15 Calithera Biosciences, Inc. Treatment of lung cancer with inhibitors of glutaminase
US10258619B2 (en) 2015-10-05 2019-04-16 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors and immuno-oncology agents
WO2017062354A1 (en) * 2015-10-05 2017-04-13 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors and immuno-oncology agents
US10940148B2 (en) 2015-10-05 2021-03-09 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors and immuno-oncology agents
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10385130B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US11535670B2 (en) 2016-05-11 2022-12-27 Huyabio International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10278968B2 (en) 2016-08-25 2019-05-07 Calithera Biosciences, Inc. Combination therapy with glutaminase inhibitors
IT201800009974A1 (en) * 2018-10-31 2020-05-01 Universita' Degli Studi Di Milano - Bicocca COMBINED PHARMACOLOGICAL TREATMENT FOR CANCER
WO2020089398A1 (en) * 2018-10-31 2020-05-07 Universita' Degli Studi Di Milano Bicocca Combinatorial drug treatment for cancer

Similar Documents

Publication Publication Date Title
AU2016246521B2 (en) Treatment of lung cancer with inhibitors of glutaminase
WO2016014890A1 (en) Treatment of multiple myeloma with heterocyclic inhibitors of glutaminase
AU2015274361B2 (en) Combination therapy with glutaminase inhibitors
US20200038398A1 (en) Combination therapy with glutaminase inhibitors
WO2015138902A1 (en) Combination therapy with glutaminase inhibitors
US10278968B2 (en) Combination therapy with glutaminase inhibitors
US10195197B2 (en) Combination therapy with glutaminase inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15824630

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15824630

Country of ref document: EP

Kind code of ref document: A1