WO2013044596A1 - Kidney-type glutaminase inhibitor and preparation method and use thereof - Google Patents

Abstract

Disclosed in the present invention are a compound and a pharmaceutical composition used for inhibiting the activity of a kidney-type glutaminase, and the use of the compound in treatment, especially in treating or preventing diseases, in particular cancer, related to the increase in activity of glutaminase.

Description

 Renal glutaminase inhibitor, preparation method and use thereof

 The present invention relates to an inhibitor of kidney type glutaminase, a preparation method thereof, and use thereof for treating a disease associated with an increase in renal glutaminase activity. BACKGROUND OF THE INVENTION The rapid growth of tumor cells requires not only energy but also the production of new cells by nucleic acids, fatty acids and proteins. Glutamine, the most abundant amino acid in the human body, plays a crucial role in the growth and development of tumor cells (see, DeBerardinis, RJ et al. (2008) The biology of cancer: Metabolic reprogramming fuels cell growth And proliferation. Cell Metabolism 7: 11-20 and the literature Hsu, PP and Sabatini, DM (2008) Warburg and beyard. Cell 134: 703-707). Therefore, many tumor cells are described as cells that are "addicted to glutamine" (see Wise DR and Thompson CB (2010) Glutamine addiction: a new therapeutic target in cancer. Trends in Biochemical Sciences 35: 427- 433).

One of the important enzymes in the metabolism of glutamine is glutaminase, which is located in the lining of the mitochondria in cells (see Shapiro, RA et al. (1985) The orientation of phosphate-dependent glutaminase on the inner membrane Of rat renal mitochondria. Arch Biochem Biophys 243: 1-7), which catalyzes the reaction of glutamate to glutamate, which is converted to α-ketoglutaric acid by the action of glutamate dehydrogenase. Entering the Krebs cycle as a substrate to provide metabolic intermediates for macromolecular synthesis of tumor cells (see Lu WQ et al. (2010) Cancer metabolism: is glutamine sweeter than glucose. Cancer cell 18: 199-200) . Glutamine can be divided into two isoforms, the liver type glutaminase, which is expressed only in the peripheral cells of the liver after birth; the other is called the kidney type. Glutamine enzyme, which is abundantly expressed in various parts of the body such as kidney, brain, intestine, liver, and lymphocytes. It is important to be present in tumor cells (see Szeliga, M and Obara-Michlewska, M). (2009) Glutamine in neoplastic cells: focus on the expression and role of glutaminases. Neurochem Int 55: 71-75 ). These two sub Although they are highly similar in amino acid sequence, they are derived from different related genes (see

Elgadi, KM et al. (1999) Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alterative splicing. Physiol Genomics 27: 367-376 ), with different protein structures and kinetic characteristics to perform different functions, And the adjustment mechanisms involved are also different.

Malignant transformation of cells is accompanied by a significant increase in nucleic acid and protein synthesis. For the rapid growth of tumor cells, the high-speed synthesis of proteins requires constant supply of essential and non-essential amino acids. Glutamine is the most abundant amino acid in the human body, which guarantees this huge demand (see Medina, MA (2001). Glutamine metabolism: Nutritional and clinical significance, glutamine and cancer. J Nutr 131: 2539S-2542S ). Glutamine metabolism is carried out in the mitochondria of cells, so glutamine must be transported from the cell to the cytoplasm through the cell membrane and then transported from the cytoplasm through the mitochondrial membrane into the mitochondria (see Bode, B. (2001) recent molecular Advances in mammalian glutamine transport . J. Nutr. 131: 2475S-2485S )o Studies have shown that tumor cells transport glutamine through the cell membrane much faster than normal cells. Studies on Ehrlich ascites cancer cells have also demonstrated that a special glutamine transport system present on the mitochondrial membrane of this cancer cell can transport glutamine into the mitochondria faster than normal cells ( See Molina, M. et al. (1995) Glutamine transport by vesicles isolated from tumor cell mitochondrial inner membrane. Biochem J. 308: 629-633). Since the activity of glutaminase is dependent on the concentration of inorganic monuments (see Medina, M. et al. (1988b) Effects of palmitate, acetate and glucose in glutamine metabolism in Ehrlich ascites tumor cells. Biochimie 70: 833-834), However, the concentration of inorganic monument in the mitochondria of tumor cells is high, so its glutaminase activity is high. In fact, scientific studies have shown that the high activity of glutaminase is closely related to the rapid growth of tumor cells (see Souba, WW (1993) Glutamine and cancer. Ann. Surg. 218: 715-728). Detoxification of Ehrlich ascites cancer cells with antisense mRNA of glutaminase not only inhibited their growth but also changed their morphology. Cancer cells transfected with antisense mRNA are inoculated into mice, and such cancer cells completely lose the ability to produce tumors. Such mice are identical to healthy animals (see Lobo, C. et al. (2000) Inhibition Of glutaminase expression by antisense mRNA decreases growth and tumourigenicity of tumor cells. Biochem. J. 348: 257-161). These scientific findings fully demonstrate that glutaminase activity is closely related to tumorigenesis and development, and glutaminase has become a target gene of great interest in anti-tumor therapy. However, there have been no reports on drugs or therapies for effectively inhibiting tumors by effectively inhibiting glutaminase. Summary of the invention

 In a first aspect, the invention is a compound of the formula: or a pharmaceutically acceptable salt thereof:

1 is selected from the group consisting of H, halogen, (C _r C ₆ )alkyl; preferably H, F, Cl, Br, I; more preferably H;

R _{2 is} selected from H, OH, CKd-Ce) alkyl; preferably H, OH, OCH ₃ , OCH ₂ CH ₃ ; more preferably H;

R _{3 is} selected from the group consisting of H, halogen, (dC ₆ )alkyl, OH, 0-(dC ₆ )alkyl, 0-(dC ₆ )alkyl-COOH, 0-(C _r C ₆ )alkyl-aryl , OCO-(dC ₆ )alkyl, SH, S-(dC ₆ )alkyl, S-(C _r C ₆ )alkyl-aryl, N0 ₂ , NH ₂ , NH-(C _r C ₆ ) alkane , N((dC ₆ )alkyl) ₂ , N-id- ^)alkylaryl; preferably H, F, Cl, Br, I, OH, OCH ₃ , OCH ₂ CH ₃ , CH (CH _{3 2} , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; more preferably Br, CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ ;

R4 is selected from H, halo, (C _r C ₆₎ alkyl, 0- (C _r C _{6) alkyl, COOH, N0 2, NH 2} , NH- (dC 6) alkyl, N ((dC ₆₎ Alkyl) ₂ , preferably H, Cl, Br, I, COOH, N0 ₂ , N(CH ₃ ) ₂ ; more preferably H, Br, N0 ₂ ;

Alternatively, R ₃ and R ₄ may form an aryl or heteroaryl group together with the carbon atom or hetero atom to which they are attached, and the aryl or heteroaryl group may be an optionally substituted aryl or heteroaryl group. The aryl group is preferably a phenyl group, and the heteroaryl group is preferably a dioxole;

R _{5 is} selected from H, halogen, (dC ₆ )alkyl; preferably H, F, Cl, Br; more preferably H;

R4 and R ₅ may together with the carbon atom or hetero atom they are attached aryl or heteroaryl, said aryl or heteroaryl may be optionally substituted aryl or heteroaryl group, said aryl Base is preferably benzene Base

R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from H, halogen,

Base-aryl, OCO-id-^) pit foundation, SH, S-id-^) pit base, S-id-^) pit base-aryl group, N0 ₂ , NH ₂ , NH-(C _r C ₆ An alkyl group, N((dC ₆ )alkyl) ₂ , N-(C _r C ₆ )alkyl-aryl; preferably H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; more preferably H, CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ ;

Alternatively, R6 and R ₇ may together with the carbon atom or hetero atom to which they are attached form a C ₅ -C _10, saturated or unsaturated, substituted or unsubstituted cyclic group, said cyclic group The carbon in the carbon may be replaced by one or more hetero atoms selected from 0, S, N; preferably, the cyclic group is a cyclopentyl group, a cyclohexyl group, a phenyl group, an oxazole, a pyrazole, a thiazole, a different Oxazole, isothiazole, triazole;

Z is selected from C, 0, S, N; preferably C, N; more preferably <. According to a second aspect of the invention, there is provided a pharmaceutical composition comprising a compound of the invention and one or more pharmaceutically acceptable excipients. In a third aspect, the invention provides the use of a compound or composition of the invention for the manufacture of a medicament for inhibiting renal glutaminase. In a fourth aspect, the invention provides the use of a compound or composition of the invention described above for the manufacture of a medicament for the treatment or prevention of a condition associated with increased renal glutaminase activity. In a fifth aspect, the present invention also provides a method of treating or preventing a condition associated with an increase in renal glutaminase activity, the method comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of the present invention A compound or composition of the invention. DRAWINGS

Figure 1 shows the inhibition of cancer cell growth by the exemplary compounds of the invention. Figure 1A, non-small lung cancer cells CRL-5803 (ATCC) and breast cancer cells MDA-MB231 (ATCC) Western blot. Figure 1B shows growth inhibition of non-small lung cancer cells CRL-5803. Figure 1C shows growth inhibition of breast cancer cell MDA-MB231.

 Figure 2 shows the effect of an exemplary compound of the invention on the malignant transformation activity of cancer cells. Figure 2A shows the results of non-small lung cancer cells CRL-5803 and CRL-5800 (ATCC) in a saturation density assay. Figure 2B shows the results of breast cancer cells MDA-MB231 and SKBR3 (ATCC) in a saturation density experiment. Figure 2C shows the results of the Low serum assay. Figure 2D shows the results of the Soft agar assay.

 Figure 3 shows that the example compounds of the invention have no effect on the growth and morphology of normal cells. Figure 3A shows the effect on HMEC in human normal breast epithelial cells. Figure 3B shows the morphological effects on human normal mammary epithelial cells HMEC, two breast cancer cells MDA-MB231 and SKBR3.

 Figure 4 shows the amino acid sequence of mouse kidney glutaminase.

 Figure 5 shows that the example compounds of the invention inhibit renal glutaminase activity. Fig. 5A shows a recombinant protein expressing mouse kidney-type glutaminase in Escherichia coli, and its protease activity was measured after treatment with various concentrations of the compound. 100% means that 620 moles of glutamine can be hydrolyzed per minute per mole of glutaminase. Figure 5B, upper panel: MDA-MB231, SKBR3 cells were transfected with kidney glutaminase siRNA, or control siRNA, and then grown under low serum (1% FBS), respectively, on days 2, 4, and 6 Results of counting by different cells; Bottom panel: Western blot results showing the expression of glutaminase in MDA-MB231, SKBR3 cells after siRNA knockdown. Figure 5C shows the results of clustering of MDA-MB 231, SKBR3 cells transfected with kidney-type glutaminase siRNA, or control siRNA, and then grown in soft agar for ten days. Figure 5D, MDA-MB231. SKBR3 cells were grown in RPMI 1640 + 10% FBS medium, and cell counts were performed on days 2, 4, and 6 with or without glutamine. .

 Figure 6 shows the inhibitory effect of the exemplified compounds of the present invention on tumors in xenografted mice. Figure 6A shows the inhibition of MDA-MB231 cell growth. Figure 6B shows the inhibition of SKBR3 cell growth. Fig. 6C shows the inhibitory effect on P-493B lymphoma cells in mice.

Figure 7 shows the inhibitory effect of the exemplified compounds of the invention on glutaminase activity in malignant transformation of cells. The left panel shows the production of HMEC, MDA-MB231 and SKBR3 cells. Use: to determine the activity of mitochondria for glutaminase isolation from cells in different cases, 100% represents 750 moles of glutamine per minute per mole of glutaminase; The amount of glutaminase expressed and the amount of labeled protein in mitochondria of VDAC. detailed description

 According to a first aspect of the invention, in one embodiment, the invention provides a compound of the formula: or a pharmaceutically acceptable salt thereof:

 among them:

 Selected from H, F, Cl, Br, I;

R _{2 is} selected from the group consisting of H, OH, OCH ₃ , OCH ₂ CH ₃ ;

R _{3 is} selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ;

R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ , N(CH ₃ ) ₂ ;

Alternatively, R ₃ and R ₄ may, together with the carbon or hetero atom to which they are attached, form an optionally substituted aryl or heteroaryl;

R _{5 is} selected from the group consisting of H, Cl, Br;

R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ; Z is selected from C, 0, S, N, preferably C, N. In another embodiment of the present invention, there is provided a compound of the formula: or a pharmaceutically acceptable salt thereof:

 among them:

 Selected from H, Cl, Br;

R _{2 is} selected from the group consisting of H, OH, OCH ₃ , OCH ₂ CH ₃ ;

R _{3 is} selected from the group consisting of H, Cl, Br, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N ( CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ;

R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ , N(CH ₃ ) ₂ ;

Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl;

R _{5 is} selected from the group consisting of H, Cl, Br;

R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ; Z is selected from C, 0, S, N. In still another embodiment of the present invention, there is provided a compound of the formula: or a pharmaceutically acceptable salt thereof:

 among them:

 Ri is selected from H;

R _{2 is} selected from the group consisting of H, OCH ₃ and OCH ₂ CH ₃ ;

R _{3 is} selected from the group consisting of Br, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ;

R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ ;

Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl;

R _{5 is} selected from H;

R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ) ;

 Z is selected from C and N. In still another embodiment of the present invention, there is provided a compound of the formula: or a pharmaceutically acceptable salt thereof:

 among them:

 Ri is selected from H;

R _{2 is} selected from H;

R _{3 is} selected from the group consisting of Br, OCH ₃ , OCH ₂ CH ₃ . CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ ;

R4 is selected from the group consisting of H, F, Cl, Br, N0 ₂ ;

Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl;

R _{5 is} selected from H;

R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ) ;

z is selected from c. The term "d- ₆ alkyl" as used herein refers to a linear saturated hydrocarbon group or a branched saturated hydrocarbon group having 1 to 6 carbon atoms. Examples of the d- ₆ alkyl group include anthracenyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl groups. Preferably, the hydrocarbon group is linear.

As used herein, the term "aryl" refers to aromatic rings wherein at least one of a C ₆ _ ₁₂ monocyclic hydrocarbon or bicyclic hydrocarbon ring. Examples of such groups include phenyl (ph), naphthyl and tetrahydronaphthyl.

 The term "heteroaryl" as used herein, refers to a 5-6 membered aromatic monocyclic or fused 8-10 membered aromatic ring containing from 1 to 4 impurities selected from the group consisting of oxygen, nitrogen and sulfur. atom. Examples of such aromatic monocyclic rings include thiamidine, furyl, furazanyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazole Base, isoxazolyl, thiadiazolyl, pyranyl, pyrazolyl, pyrimidinyl, yl, pyridyl, pyridyl, triazinyl, tetrazinyl and the like. Examples of such aromatic bicyclic rings include quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, pteridinyl, porphyrinyl, phthalazinyl, naphthyridinyl, anthracene. , isoindolyl, azaindolyl, indolizinyl, carbazolyl, fluorenyl, pyrrolopyzyl, furanpyridyl, benzofuranyl, isobenzofuran Benzo, benzothiazepine, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzothiadiazolyl And imidazopyridyl.

O-id- ^)坑基-芳基、 OCO-id- ^)坑基、 SH、 S-(C_rC₆)烷基、 N0₂、 皿 2、 NH-(d-C₆)烷基、 N((C_rC₆)烷基 )2、 (C₃-C₈) 环烷基、 （C₃-C₇)杂环基。 The term "optionally substituted aryl or heteroaryl" as used herein refers to an aryl or heteroaryl group optionally substituted by: halo, OH, (dC ₆ )alkyl, CKd- ^) alkyl,

O-id-^) Pit-aryl, OCO-id-^) Pit base, SH, S-(C _r C ₆ )alkyl, N0 ₂ , Dish 2, NH-(dC ₆ )alkyl, N ((C _r C ₆ )alkyl)2, (C ₃ -C ₈ )cycloalkyl, (C ₃ -C ₇ )heterocyclyl.

The term "halogen" as used herein, unless otherwise specifically indicated, refers to fluoro, chloro, bromo or iodo. When the compound is geometrically heterogeneous, the compound of the formula ⁵ can be prepared as an isomeric mixture or a racemic compound, but the present invention relates to all such enantiomers or isomers, Either in optically pure form or as a mixture with other isomers. Separate enantiomers or isomers can be obtained by methods known in the art, such as optical resolution of a product or intermediate (e.g., chiral chromatographic separation (e.g., chiral HPLC)), or enantiomeric Structure synthesis method. Similarly, when a compound of the invention is present as an alternative tautomeric form (eg, a ketone/enol, amide) The present invention relates to isolated independent tautomers and to mixtures of tautomers in all ratios.

 The invention further relates to pharmaceutically acceptable salts of the above formulae, which are well known to those skilled in the art and include the basic salts of inorganic and organic acids, or the acid salts of inorganic or organic bases. The acid is, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, acetic acid R

 6. Trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, fumaric acid, succinic acid, maleic acid, benzoic acid, and the like. The base is, for example, an alkali metal or alkaline earth metal cation, an ammonium cation or the like. Each of the substituent groups, embodiments, or schemes of different preferred levels recited herein may be arbitrarily combined unless otherwise indicated. The compounds of the present invention can be obtained by the following general preparation methods or other known methods.

(Chemical of Heterocyclic Compounds (New York, NY, United States), 1996, vol. 32, No. 1, p. 30 - 34). The general routes used in the preparation of the compounds are merely illustrative of the methods by which the compounds of the invention can be synthesized, and for those skilled in the art having referenced the present disclosure, various modifications of the routes It can be made and is inspired.

Preparation of 5-(substituted phenyl)-2,2-dimercapto-2,3,5,6-tetrahydrobenzophenanthridine-4(1H)-one

F3⁄4, X, Ma

2)

Hi As indicated above, the compound (III) of the present invention can be reacted with 2-aminonaphthalene and substituted benzoquinone to form Schiff base (II), and then the resulting Schiff base and 5,5-dimercaptocyclohexyl-1 are produced. , 3-dione reaction to prepare. Specifically, the method includes:

 a. reacting 2-aminonaphthalene with a substituted benzofural in a benzene solvent to form a Schifffaction,

 b. reacting the Schiff base with 5,5-dimercaptocyclohexyl-1,3-dione in a mixed solvent of an alcohol and a benzene solvent to form a compound of the present invention,

 Wherein Rl, Κ·2, R3, R5, R6, R7, R Rl0, Rll, Rl2 and Rl3 are as defined above.

In the production method of the present invention, the alcohol solvent is, for example, an alcohol of d- ₅ , preferably ethanol. The benzene solvent is, for example, benzene, toluene, dinonylbenzene, triterpene benzene, chlorobenzene, bromobenzene or the like, and benzene is preferred. The Schiff base formed in the above step a can be purified or directly subjected to the next reaction without purification. Preferably, in the above steps a and/or b, the reaction is carried out at reflux temperature. According to a second aspect of the invention, there is provided a pharmaceutical composition comprising a compound of the invention described above together with one or more pharmaceutically acceptable excipients.

 The pharmaceutical compositions of the present invention comprise a compound of the invention and a pharmaceutically acceptable carrier, adjuvant or vehicle. The pharmaceutically acceptable carriers, adjuvants and vehicles which can be used in the pharmaceutical compositions of the invention are those conventionally used in the field of pharmaceutical formulations, including, but not limited to, sugars, sugar alcohols, starches, ion exchangers, aluminas. , aluminum stearate, lecithin, serum protein (eg human serum albumin), buffer substances (eg phosphate), glycerol, sorbic acid, sorbic acid clock, partial glyceride mixture of saturated plant fatty acids, water, salt or Electrolyte (eg, protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based material, polyethyl b Glycols, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polypropylene oxide-block polymers, polyethylene glycol and lanolin.

In a specific embodiment, the pharmaceutical compositions of the present invention further comprise one or more additional active pharmaceutical ingredients. The compounds of the invention may be administered in combination with one or more additional active pharmaceutical ingredients. The composition may be in the form of a single composition comprising a compound of the invention and one or more additional active pharmaceutical ingredients. Alternatively, the composition may be in the form of a combination of two or more separate compositions, wherein the compound of the invention is contained in one composition, one or more additional active pharmaceutical ingredients are included in one or more In a separate composition. In the pharmaceutical composition, the additional active pharmaceutical ingredient may, for example, be another anti-tumor drug. The anti-tumor drug Can be selected from: asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, L-asparaginase, cyclophosphamide, cytarabine, dacarbazine, radiation Enterin D, daunorubicin, doxorubicin, epirubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexammine, hydroxyurea, ifosfamide, irinotecan , decanoyltetrahydrofolate, lomustine, nitrogen mustard, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, guanidine Benzamidine, raloxifene, streptozotocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, vindesine, aminoglutethimide, L-asparaginase, Azathioprine, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2,2,-difluorodeoxycytidine, docetaxel, erythrohydroxy adenine, estriol, 5 - Fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluorotestosterone, flutamide, hydroxyprogesterone caproate, deoxyxanthine , interferon, hydroxyprogesterone acetate, sedative acetate, melphalan, mitoxantrone, paclitaxel, pentastatin, N-dioxan-L-aspartate (PALA) , pucamycin, semustine, teniposide, testosterone propionate, thiotepa, tridecyl melamine, uracil, and vinorelbine, oxaliplatin, gemcitabine, capecitabine, ang Bomycin and its natural and synthetic derivatives, tocilizumab, trabedectin, temozolomide, trastuzumab, cetuximab, bevacizumab, cetuzumab, ZD-1839 (Iressa OSI-774 (Tarceva) . CI-1033. GW2016. GP-724,714, HKI-272, EKB-569. STI-571 (Gleevec). PTK-787, SU-11248, ZD-474. AG- 13736. KRN-951. CP-547, 632, CP-673, 451, CHIR-258, MLN-518, AZD-2171. PD-325901. ARRY 142886, cycloheptyl benzoate (SAHA), LAQ- 824, LBH-589, MS-275, FR-901228, Potembumab and CCI-779. In a third aspect, the invention also provides the use of a compound or composition of the invention for the manufacture of a medicament for inhibiting renal glutaminase. In a fourth aspect, the invention provides the use of a compound or composition of the invention described above for the manufacture of a medicament for the treatment or prevention of a condition associated with increased renal glutaminase activity. The conditions associated with increased renal glutaminase activity of the present invention are known to those skilled in the art, such as tumors, particularly lung tumors, breast tumors, lymphomas, malignant transformations, and the like. In a fifth aspect, the present invention provides a therapeutic or prophylactic and renal glutaminase A method of increasing the activity associated with a condition, the method comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of a compound or composition of the invention described above. Example

 The invention is further illustrated by way of example with reference to the embodiments. The compounds or reagents used in the examples are commercially available or can be prepared by conventional methods known to those skilled in the art; the experimental apparatus used is commercially available; unless otherwise stated, the following biological examples The cell lines used were all from the American Type Culture Collection (ATCC). It is to be understood that the examples are not intended to limit the scope of the invention. Preparation examples:

 Example 1:

Preparation of _N _(4-bromo-3-nitrophenylhydrazinyl)-2-naphthylamine (Ila )

 2-naphthylamine (2.00 g, 13.97 mmol) was added to the reaction flask, then dry benzene (30 ml) was added, stirred to dissolve, and finally 4-bromo-3-nitrobenzaldehyde ( 3.20 g, 13.97 mmol) was added. ). After the addition, the mixture was heated to reflux with an oil bath, and the water produced was separated by a water separator. After refluxing for 1 hour, TLC showed that the starting material had been reacted and the reaction was completed. The reaction mixture was concentrated to dryness to dryness crystals crystals crystals crystals

Preparation of 5-(4-bromo-3-nitrophenyl)-2,2-dimercapto-2,3,5,6-tetrahydrobenzophenanthryl-4(1H)-one (Compound 001)

The obtained black solid was added to a solution of (EtOH / benzene = 1:1) (50 ml), then bis-fluorenone (1.96 g, 13.97 mmol) was added, and the reaction system was heated to reflux. After 2 hours, TLC showed disappearance of starting material. The reaction is over. The reaction solution was cooled to 10 ° C, a solid was precipitated, suction filtered, and a solid was collected. The solid was washed with decyl t-butyl ether to give 3 _{g of a} brown solid. It was then recrystallized from (nitrobenzene: benzene = 1:1) 100 ml to give 1.3 g of a white solid.

 ESI-MS (Μ+Η+): 92·1, 478.1;

^ NMR (300MHz, DMSO-< ₆ ) δ 9.85 (s, 1H, NH), 7.93 (d, 1H, ArH, J = 8.4 Hz), 7.88 (d, 1H, ArH, J = 1.8 Hz), 7.83 (d, 1H, ArH, J = 9.0 Hz), 7.82 (s, 1H, ArH), 7.67 (d, 1H, ArH, J = 8.1 Hz), 7.42 (t, 1H, ArH, J = 7.6 Hz), 7.37 (dd, 1H, ArH, J = 8.7 Hz, 2.0 Hz), 7.33 (s, 1H, ArH), 7.32 (d, 1H, ArH, J = 9.0 Hz), 5.89 (s, IH, CH), 2.53 (d, IH, CH, = -16.2 Hz), 2.41 (d, IH, CH, J = -16.5), 2.23 (d , IH, CH, J = -16.2 Hz), 2.04 (d, IH, CH, J = -16.2Hz), 1.02 (s, 3H, CH ₃ ), 0.83 (s, 3H, CH ₃ ).

Preparation of _N _(4-tert-butylbenzylidene)-2-naphthylamine (lib )

 2-Naphthylamine (3.0 g, 21.0 mmol) was added to a 100 ml three-necked flask, then dry benzene (50 ml) was added, stirred to dissolve, and finally 4-tert-butylbenzaldehyde (3.4 g, 21.0 mmol) was added. After the addition, the mixture was heated to reflux with an oil bath, and the water produced was separated by a water separator. After refluxing for 1 hour, TLC showed that the starting material had been reacted and the reaction was completed. The reaction solution was concentrated to dryness m vacuo.

Preparation of 5-(4-tert-butylphenyl)-2,2-dimercapto-2,3,5,6-tetrahydrobenzophenanthridine-4(1H)-one (Compound 002)

The obtained black solid was added to a solution of (EtOH / benzene = 1:1) (70 ml), then bis-fluorenone (2.9 g, 21.0 mmol) was added, and the reaction system was warmed to reflux. After 2 hours, TLC showed the starting material. Disappeared and the reaction is over. The reaction solution was cooled to 10 ° C, a solid was precipitated, suction filtered, and a solid was collected. The solid was washed with decyl t-butyl ether to give 2.7 _{g of a} brown solid. It was then recrystallized from (nitrobenzene: benzene = 1:1) 100 ml to give 1.5 g of white solid.

 ESI-MS (Μ+Η+): 410·2;

^ NMR (300MHz, DMSO-< ₆ ) δ 9.65 (s, 1H, NH), 7.97 (d, 1H, ArH, J

= 8.1 Hz), 7.76 (t, 2H, ArH, J = 9.0 Hz), 7.41 (t, 1H, ArH, J = 7.0 Hz), 7.30-7.26 (m, 2H, ArH), 7.13 (s, 4H, ArH), 5.74 (s, 1H, CH), 2.51 (d, 1H, CH, J = -16.8 Hz), 2.41 (d, IH, CH, J = -16.8 Hz), 2.19 (d, IH, CH, J = -15.9 Hz), 2.03 (d, 1H, CH, J = -15.9 Hz), 1.13 (s, 9H, 3CH ₃ ), 1.01 (s, 3H, CH ₃ ), 0.87 (s, 3H, CH ₃ ). Example 3:

 Preparation of N-(4-decylthiobenzylidene)-2-naphthylamine (lie )

 2-Naphthylamine (3.0 g, 21.0 mmol) was added to a 100 ml three-necked flask, then dry toluene (50 ml) was added, stirred to dissolve, and finally 4-(indolyl)benzaldehyde (3.2 g, 21.0 mmol) was added. ). After the addition, the mixture was heated to reflux with an oil bath, and the water produced was separated by a water separator. After refluxing for 1 hour, TLC showed that the starting material had been reacted and the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure to give a white solid.

Preparation of 5-(4-indolethiophenyl)-2,2-dimercapto-2,3,5,6-tetrahydrobenzophenanthryl-4(1H)-one (compound 003)

The obtained black solid was added to a solution of (EtOH / benzene = 1:1) (70 ml), then bis-fluorenone (2.95 g, 21.0 mmol) was added, and the reaction system was warmed to reflux. After 2 hours, TLC showed the starting material. Disappeared and the reaction is over. The reaction solution was cooled to 10 ° C, a solid was precipitated, suction filtered, and a solid was collected. The solid was washed with MTBE to give 3 _g brown brown solid. Then, it was recrystallized from (nitrobenzene: benzene = 1:1) (100 ml) to give a white solid.

 ESI-MS (Μ+Η+): 400·1;

^NMR (300MHz, DMSO-< ₆ ) δ 9.70 (s, 1H, NH), 7.91 (d, 1H, ArH, J

= 8.4 Hz), 7.77 (t, 2H, ArH, J = 7.0 Hz), 7.40 (s, 1H, ArH, J = 6.0 Hz), 7.30-7.17 (m, 2H, ArH), 7.15 (d, 2H, ArH, J= 8.4 Hz), 7.00 (d, 2H, ArH, J = 8.9 Hz), 5.74 (s, 1H, CH), 2.56-2.28 (m, 2H, CH ₂ ), 2.32 (s, 3H, SCH ₃ ), 2.20 (d, 1H, CH, = -16.2Hz), 2.01 (d, 1H, CH, = -15.9Hz), 1.02 (s, 3H, CH ₃ ), 0.84 (s, 3H, CH ₃ ) Biological Activity Example

The following examples are the biological activity tests of the compounds prepared in the above examples. Example 1 Inhibition of renal glutaminase activity

 1.1 The effect of renal glutaminase on the growth of cancer cells

 Kidney-type glutaminase siRNA (Stealth Select RNAi Duplexes from Invitrogen, catalog numbers: GLSMSS204740 and GLSMSS204742), or non-specific oligonucleotides used as control siRNA (Invitrogen catalog number: 12935-112), Breast cancer cells MDA-MB231 and SKBR3 were transfected with Lipofectamine 2000, then grown under low serum (1% FBS) conditions, and different cells were counted on days 2, 4, and 6 (Fig. 5B upper panel). At the same time, the expression of glutaminase in MDA-MB231, SKBR3 cells after siRNA knockdown was shown by Western blotting (Fig. 5B lower panel). The results showed that the growth of MDA-MB231 and SKBR3 breast cancer cells under low serum conditions was significantly inhibited when two different siRNAs directed against glutaminase were used to reduce glutaminase expression. The siRNA had no effect on the growth of these two breast cancer cells.

 MDA-MB231, SKBR3 cells were transfected with kidney-type glutaminase siRNA or control siRNA, and then grown in soft agar, and the formed clusters were counted ten days later. The results showed that treatment of siRNA against glutaminase inhibited the formation of clusters of these two breast cancer cells in adherent-independent growth (Fig. 5C).

 MDA-MB231. SKBR3 cells were grown in RPMI 1640 + 10% FBS medium supplemented with glutamine or without glutamine, and cell counts were performed on days 2, 4, and 6 respectively. The results showed that when glutamine was removed from the cell culture medium of MDA-MB 231 and SKBR3, their growth under low serum conditions was greatly inhibited (Fig. 5D), further demonstrating the growth of tumor cells against glutamine. Amide dependence. It can be seen that glutamine plays an important role in the metabolism of tumor cells.

1.2 Inhibition of recombinant renal glutaminase activity

 The recombinant protein of mouse kidney-type glutaminase (molecular weight 65864D, shown in Figure 4) was expressed in E. coli, and its enzyme activity was measured after treatment with various concentrations of compound 002. Specific steps are as follows:

The gene encoding mouse glutaminase (residues 128-603) was cloned into the pET 28a vector (Novagen catalog number: 69864-3) with histidine attached to the N-terminus. The glutaminase protein was further purified by anion exchange column chromatography. Recombinant 1 μΜ glutaminase in 57 μΜ Tris-acetic acid (pH 8.6) and 0.25 μΜ ΕϋΤΑ buffer with different concentrations of compound 002— Warm the bath, the final volume is 80μ1, and rotate for 30 minutes. Compound 002 was diluted in DMSO so that the volume added remained constant (5 μl) in the different reactions. Then add glutamine to a final volume of 115 μΐ, a final concentration of 17 μΜ, and react at 37 ° C for 1 hour, then add 10 μΐ 3 M hydrogen chloride to stop the reaction. Remove 10 μΐ from the first reaction and add to the second In the reaction, the second reaction contained 114 μΜ Tris-HCK PH 9.4 ), 0.35 μΜ ΑΌΡ, 1.7 μΜ NAD and 6.3 U/ml glutamate dehydrogenase in a final volume of 228 μl. The absorption was measured at room temperature for 45 minutes and then at 340 nm to calculate the activity of glutaminase.

 The results showed that Compound 002 had a significant inhibitory effect on glutaminase activity, and the inhibitory effect was directly proportional to its concentration (Fig. 5A).

1.3 Inhibition of glutaminase activity in malignant transformation of cells

 Mitochondria were isolated from the same number of normal mammary epithelial cells HMEC (Gibco catalog number: A10565) and breast cancer cells MDA-MB231 and SKBR3. Two breast cancer cells were treated with compound 002 or not treated, and were isolated from cells in different cases. The mitochondria are assayed for the activity of glutaminase. The results showed that mitochondria isolated from MDA-MB231 and SKBR3 cells showed significantly higher activity than normal human mammary epithelial cells HMEC. When cells were treated with compound 002, glutaminase activity was strongly inhibited (Fig. 7, left panel). The expression of glutaminase in different cases and the expression level of the marker protein in VDAC mitochondria were determined by Western blotting. The results are shown in Figure 7 (right panel). Although the expression of glutaminase in SKBR3 cells was slightly lower than that of HMEC, the activity of glutaminase was much higher than that of HMEC, indicating that the activity of glutaminase was not determined by its expression.

 The above steps of mitochondrial separation are as follows:

Isolation of mitochondria from cells using the purchased QIAGEN "mitochondrial separation kit" (Catalog No. 37612): Transfer a suspension containing approximately 2 x 10 ⁷ cells into a 50 ml tube and centrifuge at 500 x g for 10 min at 4 ^G C. The supernatant was removed and the pellet was washed with 1 ml of 0.9% sodium chloride. Resuspend the cells with 2 ml of Lysis buffer pre-cooled on ice, warm the bath for 10 minutes on a shaker at 4 °C, centrifuge at 1000 °C for 10 minutes at 10 °C, carefully pour off the supernatant, use Resuspend the cell pellet in 1.5 ml of lysis buffer, pump the solution into the syringe with a 1 ml syringe with a needle, and then push it out once, ten times, using this method to completely lyse the cells. Centrifuge at 1000 x g for 10 minutes at 4 ° C, carefully transfer the supernatant to a clean Centrifuge at 6000 x for 10 minutes at 4 °C. The pellet of mitochondria was washed with 1 ml of Mitochondrial storage buffer and centrifuged at 6000 x for 20 minutes at 4 °C. The mitochondrial pellet is resuspended in mitochondrial storage buffer and used to determine glutaminase activity. Example 2 Effect on epidermal growth factor receptor (EGFR):

 The EGFR-mediated signaling pathway plays an important role in regulating cell growth, cell cycle progression, cell differentiation, and biological functions of apoptosis. Overactivation of EGFR can lead to a variety of human diseases, particularly cancer (see, Pavelic, K. et al. (1993) Evidence for a role of EGF receptor in the progression of human lung carcinoma. Anticancer Research 13, 1133-1137 and The literature Slamon, D. J" et al. (1989) Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 244: 707-712). The study found that the expression of EGFR can be used as a diagnosis of breast cancer and lung cancer. An indicator of patient survival (see Moasser, MM (2007) The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 26, 6469-6487).

 The present study found that the compounds of the present invention also produce an inhibitory effect on EGFR by inhibiting renal glutaminase activity:

 Non-small lung cancer cells CRL-5803 and breast cancer cells MDA-MB231 were cultured in RPMI 1640 supplemented with 10% FBS, while cells were treated with different compounds 001, 002 or 003 (each compound in culture) The final concentration was 10 μΜ or the cells were treated with DMSO, and the cells were lysed two days later, and Western blotting was performed using antibodies against EGFR and actin. The results are shown in Fig. 1A. As can be seen from the figure, the above compounds can significantly reduce the expression level of EGFR, thereby inhibiting the signal transduction pathway of epidermal growth factor activation. Example 3 Inhibition of cell growth

Non-small lung cancer cells CRL-5803 and breast cancer cells MDA-MB231 were cultured in RPMI 1640 medium, 10% FBS was added, and cells were treated with compound 001, 002 or 003 (the end of each compound in the culture solution) Cells were treated at a concentration of 10 μΜ or with DMSO and cell counts were performed up to six days. As a result, as shown in Figs. 1B and 1C, the compounds inhibited the growth of CRL-5803 and MDA-MB231 cells, but 001 and 003 for cancer. The inhibition of cell growth was significantly weaker than 002. Effect of Example 4 on the malignant transformation activity of cancer cells

 4.1 Non-small lung cancer cells CRL-5803 and CRL-5800; breast cancer cells MDA-MB 231 and SKBR3 were cultured in RPMI 1640 medium supplemented with 10% FBS, and treated with compound 002 (the end of the compound in the culture solution) The concentration was ΙΟμΜ) or not treated, and cell counts were performed on days 2, 4, and 6, respectively, and the results are shown in Fig. 2A and Fig. 2Β.

 4.2 Two breast cancer cells, MDA-MB231 and SKBR3, were cultured in RPMI 1640 medium supplemented with 1% FBS. The cells were treated with Compound 002 (the final concentration of the compound in the culture solution was ΙΟμΜ) or left untreated, and the cells were counted on days 2, 4, and 6, respectively, and the results are shown in Fig. 2C.

 4.3 Two breast cancer cells, MDA-MB231 and SKBR3, were grown on soft agar for 14 days, treated with compound 002 or not, and then counted for all communities larger than 50 mm in diameter, plotted against the percentage of total community. As shown in Figure 2D (top). Both breast cancer cells form large colonies in soft agar, and stop growing when they are treated with compound 002, just like a single cell (Figure 2D below).

 In the present study, the compounds of the present invention inhibited the highly aggressive lung cancer cell CRL-5803 and the breast cancer cell MDA-MB-231, and the mild lung cancer cell CRL-5800 breast cancer cell SKBR3 cells under high density conditions. Growth (Fig. 2A, 2B); inhibited the growth of breast cancer cells under low serum conditions (Fig. 2C); more importantly, inhibited anchorage-independent growth of these two cells to form clusters (Fig. 2D), Adherent-independent growth is an important feature of malignant transformation of cells. Example 5 Effect on normal cells

 5.1 Culture human normal mammary epithelial cells HMEC in MEGM (Lonza) complete medium, treat cells with compound 002 or compound 003 (each compound in the final concentration of 10 μΜ in culture), or treat with DMSO, respectively 2, 4, 6 days for cell counting. The results showed that Compound 002 or Compound 003 had no significant effect on the growth of normal HMEC (Fig. 3Α).

5.2 Human normal mammary epithelial cells HMEC and two breast cancer cells MDA-MB231. SKBR3 treated with compound 002 (the end of the compound in the culture solution) The concentration was ΙΟμΜ) or not treated, photographed to compare their morphological changes. The results showed that the morphology of MDA-MB231 cells and SKBR3 cells treated with compound 002 changed greatly, most of the cells were dead, but HMEC morphology No significant change (Figure 3Β). Effect of Example 6 on Cell Glutamine Metabolism

 As shown in Figures 6A and 6Β, MDA-MB231 cells and SKBR3 cells were grown in RPMI 1640 + 1% FBS culture medium, and cells were treated with compound 002 (the final concentration of the compound in the culture solution was ΙΟμΜ), or in the treatment At the same time, a cell-permeable analog (α-KG) of α-ketoglutaric acid was added, and cell counts were performed on days 2, 4, and 6, respectively, and the results showed that whether it was MDA-MB231 or SKBR3 cells, compound 002 was used. At the time of treatment, they all stopped growth under low serum conditions, and when treated with Compound 002 while adding the analog a-KG of ketoglutarate, the growth of the cells was almost the same as that of the untreated normal cells.

 As described above, glutamine in the mitochondria is converted to glutamate under the catalysis of glutaminase, and glutamic acid is converted to a-ketoglutaric acid under the catalysis of glutamate dehydrogenase, as a substrate. The form enters the tricarboxylic acid cycle and provides a metabolic intermediate for the growth of cancer cells. In the present study, the inhibition of the growth of tumor cells by Compound 002 was counteracted by the analog when a ketoglutarate analog which can penetrate into the cells was added. This further demonstrates that the compound inhibits the production of a-ketoglutarate by inhibiting the activity of glutaminase of tumor cells, thereby preventing cell growth and metabolism. Example 7 Inhibition of tumors in mice

Glutaminase has been shown to be overexpressed in P-493B lymphoma cells (see, document Gao, P. et al. (2009) c-Myc suppression of miR-23a/b enhance mitochondrial glutaminase expression and glutamine metabolism. Nature 458 :762-765). P-493B (ATCC) lymphoma cells (2 x 10 ⁷ ) were injected subcutaneously into the flank of severely immunodeficient mice SCID (National Cancer Institute) (mouse weight 120 g-125 g), and tumors grew after 12 days. 170 mm ³ , followed by treatment of the tumor with Compound 002 by intraperitoneal injection of 200 μΐ per day, a total of 200 μg of Compound 002, for 12 days.

Control animals were injected with DMSO dissolved in PBS in the same manner. Among them, the volume of the tumor was calculated by the method of Le et α/. (2010). The results are shown (Fig. 6C), after 12 days of tumor The size has been reduced by half. It shows that when the compound is injected intraperitoneally into the liver of mice and is not degraded, it can reach the tumor and play a positive therapeutic role on the tumor. The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical solution of the present invention in any way. It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention without departing from the spirit and scope of the invention. Those skilled in the art can understand that various features of the technical solutions of the present invention described in the present application can be appropriately combined as needed.

Claims

Rights request A compound having the formula: or a pharmaceutically acceptable salt thereof:

 among them: Selected from H, halogen, (dC ₆ )alkyl; more preferably H; R _{2 is} selected from H, OH, CKd-Ce) alkyl; more preferably H; R _{3 is} selected from the group consisting of H, halogen, (dC ₆ )alkyl, OH, 0-(dC ₆ )alkyl, 0-(dC ₆ )alkyl-COOH, 0-(C _r C ₆ )alkyl-aryl , OCO-(dC ₆ )alkyl, SH, S-(dC ₆ )alkyl, S-(C _r C ₆ )alkyl-aryl, N0 ₂ , NH ₂ , NH-(C _r C ₆ ) alkane A group, N((dC ₆ )alkyl) ₂ , N-(C _r C ₆ )alkyl-aryl; more preferably Br, CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ . R4 is selected from the group consisting of H, halogen, (C _r C ₆ )alkyl, 0-(C _r C ₆ )alkyl, COOH, NO ₂ , NH ₂ , NH-(C _r C ₆ )alkyl, N ((C _r C ₆ )alkyl) ₂ ; more preferably H, Br, N0 ₂ ; Alternatively, R ₃ and R ₄ may form an aryl or heteroaryl group together with the carbon atom or hetero atom to which they are attached, and the aryl or heteroaryl group may be optionally substituted, and the aryl group is preferably a phenyl group. The heteroaryl group is preferably a dioxole; R _{5 is} selected from H, halogen, (dC ₆ )alkyl; preferably H, F, Cl, Br; more preferably H; R4 and R ₅ may together with the carbon atom or hetero atom which they are attached aryl or heteroaryl, said aryl or heteroaryl may optionally be substituted, the aryl group is preferably phenyl; R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from H, halogen,

Base-aryl, OCO-id-^) pit foundation, SH, S-id-^) pit base, S-id-^) pit base-aryl group, N0 ₂ , NH ₂ , NH-(C _r C ₆ Alkyl, N((dC ₆ )alkyl) ₂ , N-(C _r C ₆ )alkyl-aryl; preferably 11^, < 1, 81 1, 011, CH ₃ , OCH3, OCH2CH3, CH2CH3 , CH2CH2CH3, CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; more preferably H, CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ ; Alternatively, R6 and R ₇ may together with the carbon atom or hetero atom to which they are attached form a C ₅ -C _10, saturated or unsaturated, substituted or unsubstituted cyclic group, said cyclic group The carbon in the carbon may be replaced by one or more hetero atoms selected from 0, S, N; preferably, the cyclic group is a cyclopentyl group, a cyclohexyl group, a phenyl group, an oxazole, a pyrazole, a thiazole, a different Oxazole, isothiazole, triazole; Z is selected from C, 0, S, N; preferably C, N; more preferably <. 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein and R ₅ are each independently selected from H, F, Cl, Br. Compound 1 or 2, a pharmaceutically acceptable salt thereof according to claim 1, wherein R ₂ is selected from _{H, OH, OCH 3, OCH} 2 CH 3. 4. A compound according to any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R ₃ is selected from H, F, Cl, Br, I, OH, OCH 3, OCH 2 CH 3, CH (CH 3) 2, C (CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ . 5. A compound according to any of the preceding claims, or a pharmaceutically acceptable salt thereof, selected from the group consisting of H, Cl, Br, I, COOH, NO ₂ , N(CH ₃ ) ₂ . 6. A compound according to any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R ₃ and carbon atom or heteroatom which they are attached form a dioxole or phenyl group. 7. A compound according to any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R ₅ and with the carbon atom or hetero atom to which they are attached form a phenyl together.  8. A compound according to any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Z is < or >.  9. A compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein:  Selected from H, F, Cl, Br, I; R _{2 is} selected from the group consisting of H, OH, OCH ₃ , OCH ₂ CH ₃ ; R _{3 is} selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ , N(CH ₃ ) ₂ ; Alternatively, R ₃ and R ₄ may, together with the carbon or hetero atom to which they are attached, form an optionally substituted aryl or heteroaryl; R _{5 is} selected from the group consisting of H, Cl, Br;  Z is selected from C, 0, S, N.  The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Selected from H, Cl, Br; R _{2 is} selected from the group consisting of H, OH, OCH ₃ , OCH ₂ CH ₃ ; R _{3 is} selected from the group consisting of H, Cl, Br, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N ( CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ , N(CH ₃ ) ₂ ; Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl; R _{5 is} selected from the group consisting of H, Cl, Br; R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ) ;  Z is selected from C, 0, S, N.  11. The compound of claim 1 wherein  Ri is selected from H; R _{2 is} selected from the group consisting of H, OCH ₃ and OCH ₂ CH ₃ ; R _{3 is} selected from the group consisting of Br, OH, OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH ₃ ) ₂ ; R4 is selected from the group consisting of H, Cl, Br, I, N0 ₂ ; Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl; R _{5 is} selected from H; R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ) ;  Z is selected from C and N.  The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein  Ri is selected from H; R _{2 is} selected from H; R _{3 is} selected from the group consisting of Br, OCH ₃ , OCH ₂ CH ₃ . CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , SCH ₃ ; R4 is selected from the group consisting of H, F, Cl, Br, N0 ₂ ; Alternatively, R ₃ and R 4 may, together with the carbon or hetero atom to which they are attached, form an optionally substituted phenyl or dioxolyl; R6, R ₇ , R ₉ , Rio. R _u , R ₁₂ and R ₁₃ are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , OCH ₂ COOH, OCH ₂ Ph, OCOCH ₃ , SCH ₃ , N(CH ₃ ) ₂ , N(CH ₂ CH _{3 2} ) ;  R5 is selected from H;  z is selected from c.  13. A pharmaceutical composition comprising a compound according to any of the preceding claims and one or more pharmaceutically acceptable excipients, preferably further comprising one or more additional active pharmaceutical ingredients, such as antineoplastic agents.  14. The composition of claim 13, wherein the additional active pharmaceutical ingredient is an antitumor drug, preferably, the antitumor drug is selected from the group consisting of asparaginase, bleomycin, carboplatin, carmustine, Chlorambucil, cisplatin, L-asparaginase, cyclophosphamide, cytarabine, dacarbazine, actinomycin 0, daunorubicin, doxorubicin, epirubicin (adriamycine), Epirubicin, etoposide, 5-fluorouracil, hexammine melamine, hydroxyurea, hetero-epoxide amide, irinotecan, decanoyltetrahydrofolate, lomustine, nitrogen mustard, 6-mercaptopurine, beauty Sodium, methotrexate, mitomycin <, mitoxantrone, prednisolone, prednisone, guanidine, raloxifene, streptozotocin, tamoxifen, thioguanine , topotecan, vinblastine, vincristine, vindesine, aminoglutethimide, L-asparaginase, azathioprine, 5-azarconia, cladribine, busulfan, diethylstilbestrol, 2,,2,-fluorodeoxycytidine, docetaxel, erythrohydroxy adenine, estriol, 5- Fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluorotestosterone, flutamide, hydroxyprogesterone caproate, deoxyxanthine, interferon, guanidine acetate Ketone, medroxyprogesterone acetate, melphalan, mitoxantrone, paclitaxel, pentastatin, N-dioxosyl-acetyl-L-aspartate (PALA), pucamycin, semustine , teniposide, testosterone propionate, thiotepa, tridecyl melamine, uracil, and vinorelbine, oxaliplatin, gemcitabine, capecitabine, epothilone, and their natural and synthetic derivatives , tosimotozumab, trabedectin. Temozolomide, trastuzumab, cetuximab, bevacizumab, bottuzumab, ZD-1839 (Iressa). OSI-774 (Tarceva). CI -1033. GW2016. GP-724,714, HKI-272, EKB-569. STI-571 (Gleevec). PTK-787, SU-11248, ZD-474. AG-13736. KRN-951. CP-547, 632, CP-673, 451, CHIR-258, MLN-518, AZD-2171. PD-325901. ARRY 142886, cycloheptyl benzoate (SAHA), LAQ-824. LBH-589, MS-275, FR-901228, Potembumab and CCI-779. 15. A compound according to any of the preceding claims 1-12 or a group according to claims 13-14 Use of a compound for the preparation of a medicament for inhibiting glutaminase activity.  16. Use of a compound according to any one of claims 1-12 or a composition according to claims 13-14 for the manufacture of a medicament for the treatment or prevention of a condition associated with an increase in renal glutaminase activity.  17. Use according to claim 15 or 16, wherein the condition is a tumor, preferably a lung tumor, a breast tumor, a lymphoma or a malignant transformation of cells.