WO2014083095A1

WO2014083095A1 - Combinations of metformin with other compounds for the treatment of cancer and for immunosuppression

Info

Publication number: WO2014083095A1
Application number: PCT/EP2013/074945
Authority: WO
Inventors: Marco Colombi; Don Benjamin; Michael Hall; Christoph Moroni
Original assignee: Universitaet Basel
Priority date: 2012-11-29
Filing date: 2013-11-28
Publication date: 2014-06-05

Abstract

The invention relates to combinations of metformin or related biguanides such as phenformin, buformin or proguanil, with particular metformin sensitizing compounds that have the capability to sensitize rapidly proliferating cells to metformin or related biguanides in such a way that combinations cause cell lethality at concentrations where neither metformin alone nor such metformin sensitizing compounds alone exert appreciable cell lethality. The invention further relates to the use of such combinations in the treatment of cancer and for achieving immunosuppression.

Description

Combinations of metformin with other compounds for the

treatment of cancer and for immunosuppression

Field of the Invention

The invention relates to combinations of metformin with particular compounds that have the capability to sensitize rapidly proliferating cells to metformin or related biguanides in such a way that combinations cause cell lethality at concentrations where neither metformin alone nor such metformin sensitizing compounds alone exert appreciable cell lethality. The invention further relates to the use of such combinations in the treatment of cancer and for achieving immunosuppression.

Background Art Anti-cancer therapy utilizes a combination of therapeutic interventions such as surgery, radiation therapy and chemotherapy. Surgery and radiation therapy are generally confined locally to the main site of tumor growth, while chemotherapy is applied to prevent tumor re-growth or against distant tumor foci. Chemotherapeutic agents are also used to reduce tumor growth to manage disease progression when radiotherapy or surgery is not an option.

Chemotherapeutic drugs are most effectively used in combination therapy. The rationale is to apply drugs that work via different mechanisms in order to decrease the probability of developing drug-resistant cancer cells. Combination therapy also allows, for certain drug combinations, an optimal combined dose to minimize side effects. This is crucial as standard chemotherapeutic agents target essential cellular processes such as DNA replication, cell division or induce DNA damage and thus have a general cytotoxic effect. Finally, combination treatment of two compounds may uncover unanticipated synergisms and trigger effects not induced by a single compound. In recent years, drugs are also used in a neoadjuvant setting, i.e. prior to surgery, to reduce the tumor mass or to improve long-term survival.

Synthetic lethality, originally described in yeast, arises when a combination of mutations in two genes (A and B) leads to cell death, whereas a mutation in only A or B is non-lethal (Tucker, C.L. & Fields, S., Nat Genet 2003, 35:204-205). As an example in the context of cancer chemotherapy, mutation of the haem oxygenase gene has been reported to be synthetically lethal with loss of the tumor suppressor fumarate hydratase (Frezza, C. et al., Nature 201 1 , 477:225-228). Extended to the protein level, two drugs targeting proteins A and B, respectively, may exert similar synthetic lethality by pharmacologically inhibiting the functions of both proteins simultaneously, whereas single inhibition is non-lethal. The concept of synthetic lethality also applies to a situation where a gene is mutated in cancer cells and genetic knockdown or pharmacological inhibition of a second gene or protein respectively, leads to cell death. This, for example, has been reported for Poly(ADP- ribose) polymerase (PARP) inhibitors and mutations of BRCA1 or BRCA2 in breast cancer. Mutations in BRCA1 or BRCA2 profoundly sensitize cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis (Farmer, H. et al., Nature 2005, 434:917-921 ). Similarly, RNAi screening has identified multiple synthetic lethal interactions with mutated Ras oncogenes found in many cancers (Luo, J. et al., Cell 2009, 137:835-848). Strategies used to identify synthetically lethal interactions are currently playing an increasing role in cancer drug development (Chan, D.A. & Giaccia, A.J., Nat Rev Drug Discov 201 1 , 10:351 -364). A synthetic lethal interaction has also been reported for the combination of metformin treatment of cells with glucose withdrawal (Menendez, J.A. et al., Cell Cycle 2012, 1 1 :2782-92). In summary, combinations of two mutations, or of a mutation plus drug treatment, or of simultaneous treatment with two suitable drugs have the potential to exert synthetic lethality in cancer cells, thus widely opening options for future cancer drug development. The attraction of synthetic lethality in cancer treatment lies in the fact that single knockout or inhibition has no or inconsequential effects, while the double inhibition is lethal. This is different from current cancer chemotherapy, where often two (or more) drugs are combined, for example oxaliplatin and irinotecan. The difference lies in the fact that in standard chemotherapy, each single drug is used at an effective dose and is thus toxic by itself, whereby combination leads not only to additive effectiveness but also to additive systemic toxicity. Thus, achieving synthetic lethality with drugs of little or no intrinsic toxicity at the given concentration holds great promise for cancer therapy.

Metformin is a widely used biguanide drug for type 2 diabetes. It is related to buformin and phenformin, two biguanides not used anymore in diabetes due to toxicity. Metformin is safe and well-tolerated and has been used in long-term management of diabetes for over 50 years and is the most-prescribed anti-diabetic drug worldwide. The main clinical benefit of metformin in the treatment of type 2 diabetes is the suppression of hepatic gluco- neogenesis to reduce hyperglycemia and improved insulin sensitivity; these effects are believed to be exerted by metformin-dependent stimulation of AMP-activated protein kinase (AMPK) activity. Basic to this effect is the fact that metformin and other biguanides inhibit complex I of the respiratory chain (electron transfer chain) of mitochondria (El-Mir, M.J. et al., J Biol Chem 2000, 275:223-228). A meta-analysis of diabetic patients receiving metformin versus an unrelated anti-diabetic agent revealed that the metformin receiving cohort had lower incidence of cancer (Evans, J.M. et al., BMJ 2005, 330: 1304-5; Bowker S.L. et al., Diabetes Care 2006, 29:254-8). This has stimulated recent research into the use of metformin as an anti-cancer agent or prophylactic with numerous studies and trials in progress. Metformin has been reported to inhibit proliferation of selected cancer cell lines in vitro, and was also shown to be active in xenotransplantation experiments where such lines were grown in immuno-compromised mice (Dowling, R.J.O. et al., J Mol Endocrinol 2012, 48(3):R31 -43). Generally, the effective inhibitory dose of metformin in vitro is rather high (>10 mM), which limits its clinical usefulness. There is a need for agents which sensitize cells to metformin and provoke cell killing at lower concentrations. One such metformin sensitizing agent is syrosingopine, a drug which is synthetically lethal with non-lethal concentrations of metformin (WO 2012/095379).

Summary of the Invention The invention relates to combinations of metformin or related biguanides with particular compounds which have the capability to sensitize rapidly proliferating cells to metformin or related biguanides in such a way that combinations cause cell lethality, whereas neither metformin or related biguanides alone nor such metformin sensitizing compounds alone exert cell lethality.

In particular, the invention relates to a pharmaceutical composition comprising a metformin sensitizing compound and metformin or related biguanide.

Such metformin sensitizing compounds are, for example, compounds of formula (A)

wherein a dotted line indicates an optional bond; N adjacent to the dotted line carries hydrogen if the combination of a regular and a dotted line represents a single bond; Ri is hydrogen, methyl or halogen; and R₂ is absent or a substituent selected from methyl, trifluoromethyl and halogen located in any of the four available positions of the phenyl ring; and tautomers thereof. Particular examples of compounds of formula (A) are (£)-2-(2-(5-bromo-2-hydroxy- benzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one, (£)-2-(2-(2-hydroxybenzylidene)- hydrazinyl)-6-methylpyrimidin-4(3/-/)-one, (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)- pyrimidin-4(3/-/)-one, and (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydro- pyrimidin-4(1 H)-one.

Other metformin sensitizing compounds are, for example, 5,6-diphenyl-7-(tetrahydro-2- furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine, 5-amino-3-{2-[5-(1 ,3-benzothiazol-2- yl)-2-furyl]-1 -cyanovinyl}-1 H-pyrazole-4-carbonitrile, ethyl 1 -[(5-chloro-8-hydroxyquinolin- 7-yl)methyl]piperidine-3-carboxylate, ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3- (phenylmethylidene)cyclopent-1 -en-1 -yl]prop-2-enoate, 2-imino-4-(1 ,1 ,2,2-tetrafluoro- ethoxy)-2,3-dihydrobenzo[1 ,2-d:3,4-d']bis(thiazole)-7-amine, 5-methyl-N-{4-[4-(N-methyl- methanesulfonamido)phenyl]-1 ,3-thiazol-2-yl}-1 ,2-oxazole-3-carboxamide, 2-(2-cyano- ethyl)-3-methyl-1 -((3-morpholinopropyl)amino)benzo[4,5]imidazo[1 ,2-a]pyridine-4-carbo- nitrile, 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benz- amide, (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2-en- amide, 2-(4-chlorophenyl)-N'-[(2,4-dimethoxybenzoyl)oxy]ethanimidamide, and related compounds.

The invention further relates to the use of the mentioned combinations in the treatment of cancer, in particular in the treatment of carcinomas, sarcomas, tumors of the

haematopoietic system, tumors of the nervous system and melanoma, as well as for achieving immunosuppresion in diseases or situations where the immune system is hyperactivated such as in autoimmune diseases, allergies or following organ

transplantations.

Brief Description of the Figures

Figure 1 : Titration of (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methyl- pyrimidin-4(3/-/)-one (5) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 2: Titration of 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]- pyrimidin-4-amine (6) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 3: Titration of 5-amino-3-{2-[5-(1 ,3-benzothiazol-2-yl)-2-furyl]-1 -cyanovinyl}-1 H- pyrazole-4-carbonitrile (7) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 4: Titration of ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3- carboxylate (8) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ. Figure 5: Titration of ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenylmethylidene)- cyclopent-1 -en-1 -yl]prop-2-enoate (9) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 6: Titration of 2-imino-4-(1 ,1 ,2,2-tetrafluoroethoxy)-2,3-dihydrobenzo[1 ,2-d:3,4-d']- bis(thiazole)-7-amine (10) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 7: Titration of 5-methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]-1 ,3-thiazol- 2-yl}-1 ,2-oxazole-3-carboxamide (11 ) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 8: Titration of 2-(2-cyanoethyl)-3-methyl-1 -((3-morpholinopropyl)amino)benzo[4,5]- imidazo[1 ,2-a]pyridine-4-carbonitrile (12) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 9: Titration of 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)- phenyl]benzamide (13) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ. Figure 10: Titration of (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)- phenyl]prop-2-enamide (14) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 1 1 : Titration of 2-(4-chlorophenyl)-N'-[(2,4-dimethoxybenzoyl)oxy]ethanimidamide (15) on human HL60 leukemia cells (panel A) and normal human fibroblast cells (panel B) in the presence (dashed line) and absence (diamonds, solid line) of 4 mM metformin. Viable cells were quantified on day 3 (fibroblasts: day 5) by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in μΜ.

Figure 12: Titration of metformin on human HL60 leukemia cells in absence (solid lines) and presence (broken lines) of a metformin sensitizing compound. The metformin sensitizing compounds and the concentrations used were:

panel A: (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)- one (5) at 1 μΜ;

panel B: 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine (6) at 5 M;

panel C: ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3-carboxylate (8) at 3 μΜ;

panel D: ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenylmethylidene)cyclopent-1 - en-1 -yl]prop-2-enoate (9) at 3 μΜ;

panel E: 5-methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]-1 ,3-thiazol-2-yl}-1 ,2- oxazole-3-carboxamide (11 ) at 5 μΜ;

panel F: 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]- benzamide (13) at 10 μΜ;

panel G: (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2-en- amide (14) at 5 μΜ.

Viable cells were quantified on day 2 by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates

concentration of metformin in mM. Figure 13: Titration of phenformin on human HL60 leukemia cells in absence (solid lines) and presence (broken lines) of a metformin sensitizing compound. The sensitizing compounds used were:

panel A; 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine (6) at 5 μΜ;

panel B: (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2- enamide (14) at 5 μΜ.

concentration of phenformin in μΜ. Figure 14: Titration of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin- 4(3H)-one (16) on human hepatocellular carcinoma cell lines HepG2 (left panel) and Huh7 (right panel) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 5 by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates

concentration in nM.

Figure 15: Titration of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)-one (17) on human hepatocellular carcinoma cell lines HepG2 (left panel) and Huh7 (right panel) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 5 by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates

concentration in nM. Figure 16: Titration of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydro- pyrimidin-4(1 /-/)-one (18) on human hepatocellular carcinoma cell lines HepG2 (left panel) and Huh7 (right panel) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 5 by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in nM.

Figure 17: Titration of (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methyl- pyrimidin-4(3/-/)-one (5) on human hepatocellular carcinoma cell lines HepG2 (left panel) and Huh7 (right panel) in the presence (dashed line) and absence (solid line) of 4 mM metformin. Viable cells were quantified on day 5 by alamarBlue assay. Relative growth (in percent) normalized to untreated cells (100%) is shown on the Y-axis. X-axis indicates concentration in nM.

Detailed Description of the Invention

Metformin sensitizing compounds as understood in the present invention comprise compounds which kill cancer cells if and only if combined with metformin or a related biguanide. This is a case of so-called "synthetic lethality", wherein metformin or the related biguanide is, at the concentration used, not cytotoxic for a cancer cell, and likewise the metformin sensitizing compound is not cytotoxic, but when combined together, the combination is lethal for the cancer cell. In the classic way of looking at a combination therapy, such a combination exerting synthetic lethality may be termed synergistically active.

Metformin or related biguanides according to the invention comprise the following compounds:

Metformin, 3-(diaminomethylidene)-1 ,1 -dimethylguanidine hydrochloride of formula (1 ):

A biguanide related to metformin as understood in the present invention is, for example, phenformin, buformin, or proguanil (an anti-malaria prodrug), preferably phenformin.

Phenformin, 1 -(diaminomethylidene)-2-(2-phenylethyl)guanidine of formula (2):

Buformin, 2-butyl-1 -(diaminomethylidene)guanidine of formula (3):

Proguanil, 1 -(4-chlorphenyl)-5-isopropylbiguanide of formula (4):

etformin sensitizing compounds are, for example, compounds of formula (A)

wherein

a dotted line indicates an optional bond;

N adjacent to the dotted line carries hydrogen if the combination of a regular and a dotted line represents a single bond;

Ri is hydrogen, methyl or halogen; and

R₂ is hydrogen or a substituent selected from methyl, trifluoromethyl and halogen located in any of the four available positions of the phenyl ring;

and tautomers thereof.

Halogen is fluorine, chlorine or bromine. Halogen R-i is preferably fluorine or chlorine.

Tautomers are, in particular, keto/enol and imine/enamine tautomers. For example, if both dotted lines represent a bond, the resulting compound can also be regarded as a 1 ,3- pyrimidine-4-ol. If only the dotted line connecting N is a bond and the other dotted line is no bond, then the C=N double bond may be shifted to any of the other nitrogen atoms of the -N=C(NH-)₂, i.e. guanidino, partial structure. Particular preferred examples of compounds of formula (A) are compounds 5, 16, 17, and 18:

(£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one of formula (5):

and tautomers thereof, E)-2-(2-(2-hydroxybenzylidene)hydrazi of formula (16):

(16)

and tautomers thereof, (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)-one of formula (17):

and tautomers thereof, and (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydropyrimidin-4(1 H)-one of formula (18):

(18)

and tautomers thereof.

Other metformin sensitizing compounds are, for example,

5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine of formula

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl rings,

5-amino-3-{2-[5-(1 ,3-benzothiazol-2-yl)-2-furyl]-1 -cyanovinyl}-1 /-/-pyrazole-4-carbonitrile of formula (7):

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the benzothiazole and/or furane ring, ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3-carboxylate of formula (8):

(8) and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the quinoline ring, and/or wherein the ethoxy group is replaced by methoxy or isopropoxy, ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenylmethylidene)cyclopent-1 -en-1 -yl]-

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl ring, and/or wherein the ethoxy group is replaced by methoxy or isopropoxy,

4-imino-7-(1 ,1 ,2,2-tetrafluoroethoxy)-3,10-dithia-5,12-diazatricyclo[7.3.0.0{2,6}]dodeca- 1 (9), 2(6), 7,1 1 -tetraen-1 1 -amine (also named 2-imino-4-(1 ,1 ,2,2-tetrafluoroethoxy)-2,3- dihydrobenzo[1 ,2-d:3,4-d]bis(thiazole)-7-amine) of formula (10):

5-methyl-N-{4-[4-(N-methylmeth^

carboxamide of formula (11 ):

(11 )

and compounds carrying additional fluorine, chlorine, methyl and/or tnfluoromethyl groups attached to the phenyl ring,

12-(2-cyanoethyl)-1 1 -methyl-13-{[3-(morpholin-4-yl)propyl]amino}-1 ,8-diaza- tricyclo[7.4.0.0{2,7}]trideca-2(7),3,5,8, 10,12-hexaene-10-carbonitrile (also named 2-(2-cyanoethyl)-3-methyl-1 -((3-morpholinopropyl)amino)benzo[4,5]imidazo[1 ,2-a]- pyridine-4-carbonitrile) of formula (12):

(12)

and compounds carrying additional fluorine, chlorine, methyl and/or tnfluoromethyl groups attached to the phenyl ring, 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide of formula (13):

(2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazoM -yl)phenyl]prop-2-enamide of formula (14):

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl ring, and/or wherein the methoxy groups are replaced by ethoxy or isopropoxy, ethanimidamide of formula (15):

(15)

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl rings, and/or wherein the methoxy groups are replaced by ethoxy or isopropoxy.

Preferred are compounds 5 to 15 and compounds 16 to 18, and tautomers thereof.

Particularly preferred are compounds 5, 16, 17, and 18, and tautomers thereof. Whenever a compound is listed by name and/or formula the description of such a compound likewise encompasses salts thereof, in particular acid addition salts of basic compounds with amino functions. Such acid addition salts are formed with

pharmaceutically acceptable inorganic or organic acids.

Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane- 1 ,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1 ,5-naphthalene- disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl- sulfamic acid, or other organic protonic acids, such as ascorbic acid. Pharmaceutical compositions according to the invention are, for example, compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. The compositions comprise a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin, alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the combination of a metformin sensitizing compound and metformin or a related biguanide depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration. The pharmaceutical compositions comprise from approximately 1 % to approximately 95% (for compounds of formula 6 to 15) and from approximately 0.001 % to approximately 95% (for compounds of formula 5, 16 to 18) of the combination of a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin. Single-dose

administration forms comprising in the preferred embodiment from approximately 20% to approximately 90% of a combination of a metformin sensitizing compound and metformin, and forms that are not of single-dose type comprising in the preferred embodiment from approximately 5% to approximately 20% combination of a metformin sensitizing compound and metformin or related biguanide. Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories, or capsules. Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, drops, sprays, and dispersions. Examples are capsules containing from about 0.05 g to about 3.0 g combination of a metformin sensitizing compound and metformin or related biguanide, preferably from about 0.25 g to about 1.5 g.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.

Preference is given to the use of solutions of the combination of a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the combination of a metformin sensitizing compound and metformin or a related biguanide, alone or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example

preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80^® (polyoxyethylene(20)sorbitan mono-oleate).

Suspensions in oil comprise as the oil component the vegetable, synthetic, or semisynthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di- or trivalent, alcohol, especially glycol and glycerol. As mixtures of fatty acid esters, vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful. The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

Suitable carriers for preferred solid oral dosage forms are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above- mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof. Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of the combination of a metformin sensitizing compound and metformin or a related biguanide.

Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the combination of a metformin sensitizing compound and metformin or a related biguanide in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the combination of a metformin sensitizing compound and metformin or a related biguanide is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added. Pharmaceutical compositions suitable for rectal administration are, for example, suppositories that consist of a combination of a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin, and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin

hydrocarbons, polyethylene glycols or higher alkanols.

For parenteral administration, aqueous solutions of a combination of a metformin sensitizing compound and metformin or a related biguanide, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable. The combination of a metformin sensitizing compound and metformin or a related biguanide, optionally together with excipients, can also be in the form of a lyophilizate and can be made into a solution before parenteral administration by the addition of suitable solvents. Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.

Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbiocides, such as sorbic acid or benzoic acid. On the basis of the studies described in more detail below, a pharmaceutical composition comprising a metformin sensitizing compound and metformin or a related biguanide according to the invention shows therapeutic efficacy against different types of cancer including carcinomas, sarcomas, tumors of the haematopoietic system, tumors of the nervous system and melanomas, in particular gliomas, leukemias, lymphomas, epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ductal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and glomus tumors, naevi and melanomas, soft tissue tumors including sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors, osseous and chondromatous neoplasms, giant cell tumors, miscellaneous bone tumors, gliomas, glioblastomas, oligodendrogliomas, neuroepitheliomatous neoplasms, meningiomas, nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, Hodgkin^'s and non-Hodgkin^"s lymphomas, other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias including acute and chronic leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes. Organs with malignant growth to be targeted include but are not limited to lung, breast, stomach, gut, liver, pancreas, bone, brain, organs of the lymphoid system, bone marrow, ovary, uterus, testis including tumors of ectodermal, mesodermal and endodermal origins. On the basis of the studies described in more detail below, the combination of a metformin sensitizing compound and metformin or a related biguanide is useful for achieving immunosuppression in diseases where the immune system is hyperactivated. Such diseases are, in particular, autoimmune diseases and allergic states, which are sensitive to blockade of T cell proliferation and include, but are not limited to, connective tissue diseases such as lupus erythematodes, sclerodermia, polymyositis/ dermatomyositis, mixed connective tissue disease, rheumatoid arthritis, Sjogren-syndrome, panarteritis nodosa, Wegeners granulomatosis; systemic autoimmune diseases such as rheumatoid arthritis, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, Guillain-Barre syndrome, multiple sclerosis; localized autoimmune diseases such as type 1 diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, celiac disease, Crohn's disease, ulcerative colitis, Addison's disease, primary biliary cirrhosis, autoimmune hepatitis, giant cell arteritis, psoriasis and chronic infections. The combination of a metformin sensitizing compound and metformin or a related biguanide is also useful for the treatment of immunological organ rejection following organ transplantation.

The combination of a metformin sensitizing compound and metformin or a related biguanide and pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide according to the invention may be applied in the form of fixed combinations. Such fixed combination may contain a metformin sensitizing compound and metformin or a related biguanide in a relative amount (weight per weight) of between 1 to 1 and 1 to 10Ό00, preferably between 1 to 5 and 1 to 5Ό00, such as a combination of between 1 to 1 Ό00 and 1 to 5Ό00 for combinations with metformin, whereby the maximum recommended daily dose of metformin based on the experience with diabetes type 2 is 2'550 mg, and a combination between 1 to 5 and 1 to 25 for combinations with phenformin for compounds of formula 6 to 15. For compounds of formula 5, 16, 17, and 18, such fixed combination may contain a metformin sensitizing compound and metformin or a related biguanide in a relative amount (weight per weight) of between 1 to 1 and 1 to 1 Ό00Ό00, preferably between 1 to 5 and 1 to 500Ό00, such as a combination of between 1 to 100Ό00 and 1 to 500Ό00 for combinations with metformin, whereby the maximum recommended daily dose of metformin based on the experience with diabetes type 2 is 2'550 mg, and a combination between 1 to 5Ό00 and 1 to 25Ό00 for combinations with phenformin Alternatively, the combination of a metformin sensitizing compound and metformin or a related biguanide may be applied in two different, separate pharmaceutical compositions, optionally being provided together in a kit. The administration of a metformin sensitizing compound and metformin or a related biguanide may also be staggered, or the compounds may be given independently of one another within a reasonable time window.

Pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide may be further combined with other chemotherapeutic agents. Therapeutic agents for possible combination are especially one or more cytostatic or cytotoxic compounds, for example a chemotherapeutic agent or several selected from the group comprising indarubicin, cytarabine, interferon, hydroxyurea, bisulfan, or an inhibitor of polyamine biosynthesis, an inhibitor of the mTOR pathway, an inhibitor of mTOR-complex 1 or mTOR complex 2, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor tyrosine kinase, a cytokine, a negative growth regulator, such as TGF-β or IFN-β, an aromatase inhibitor, a classical cytostatic, an inhibitor of the interaction of an SH2 domain with a phosphorylated protein, an inhibitor of Bcl-2 and modulators of the Bcl-2 family members such as Bax, Bid, Bad, Bim, Nip3 and BH3-only proteins. The combination of a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin, and pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide may be administered especially for cancer therapy in combination with radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies or neo-adjuvant therapy in combination with surgery. Other possible treatments are therapy to maintain the patient's status after tumor regression, or chemopreventive therapy, for example in patients at risk.

The present invention relates furthermore to a method for the treatment of diseases where the immune system is hyperactivated (as detailed above), and furthermore to treat immunological organ rejection following transplantation of organs such as kidney, liver, lung or bone marrow, which comprises administering a combination of a metformin sensitizing compound and metformin or a related biguanide in a quantity effective against said disease or organ rejection, respectively, to a warm-blooded animal requiring such treatment. The combination of a metformin sensitizing compound and metformin or a related biguanide can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases or organ rejection, respectively, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.05 g to approximately 3 g, preferably from approximately 0.25 g to approximately 1.5 g, of a combination of the present invention.

The invention relates to the use of a combination of a metformin sensitizing compound and metformin or a related biguanide and of pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide for the treatment of cancer, in particular for the treatment of the particular cancers mentioned above. More specifically, the invention relates to the use of a combination of a metformin sensitizing compound and metformin or a related biguanide and of pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide for the treatment of carcinomas, sarcomas, tumors of the haematopoietic system, tumors of the nervous system and melanomas. Furthermore, the invention relates to the use of a combination of a metformin sensitizing compound and metformin or a related biguanide and of pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide for achieving immunosuppression in diseases where the immune system is hyperactivated, such as autoimmunity, transplantation medicine and in other cases where immunosuppression is desirable, in particular in immunological diseases sensitive to blockade of T cell proliferation, systemic autoimmune diseases, and localized autoimmune diseases, as explained above. More specifically, the invention relates to the use of a combination of a metformin sensitizing compound and metformin or a related biguanide and of pharmaceutical compositions comprising a metformin sensitizing compound and metformin or a related biguanide for the treatment of autoimmune diseases, such as autoimmune diseases of the skin, nervous system, connective tissue, muscle, nervous system, blood forming system, bone and inner organs, in particular psoriasis, multiple sclerosis, and anemias.

The preferred relative amount of a metformin sensitizing compound and metformin or a related biguanide, e.g. phenformin, dose quantity and kind of pharmaceutical composition, which are to be used in each case, depend on the type of cancer or disease of the immune system, the severity and progress of the disease, and the particular condition of the patient to be treated, and has to be determined accordingly by the medical doctor responsible for the treatment.

The invention further relates to the use of a combination of a metformin sensitizing compound and metformin or a related biguanide for the preparation of a pharmaceutical composition for the treatment of cancer or disease of the immune system, as explained above.

Especially, the invention provides a method for treatment of cancer or disease of the immune system, which comprises administering a combination of a metformin sensitizing compound and metformin or a related biguanide or of a pharmaceutical composition comprising a metformin sensitizing compound and metformin or a related biguanide, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. The invention is based on screening results obtained with a compound library of some 15Ό00 compounds, screened for cytotoxicity dependent on the presence of 4 mM metformin. Based on such a screen it was possible to identify compounds which show synthetic lethality at given concentrations in the presence of metformin and related biguanides, with no toxicity in its absence. Such compounds have great value for cancer chemotherapy, as they are not toxic by themselves and do not trigger side effects, or only to a very limited extent. The compounds to be screened were added to the rapidly proliferating human leukemia HL60 cell line, and compounds selected when cell viability - measured by alamarBlue assay - differed by more than 50% in presence versus absence of 4 mM metformin. Candidate drugs scoring positive were retested, and compounds where synthetic lethality with metformin was confirmed were further analysed with titration experiments. The activity profiles of the resulting metformin sensitizing compound tested in presence and absence of metformin are shown in Figures 1 to 1 1 .

Figure 1 shows the activity of (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6- methylpyrimidin-4(3/-/)-one (formula 5) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at submicromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was no appreciable toxicity at submicromolar concentrations in the presence of metformin. The slight toxicity seen in absence of metformin was abolished by the presence of metformin.

Figure 2 shows the activity of 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo- [2,3-d]pyrimidin-4-amine (formula 6) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was no toxicity with or without metformin.

Figure 3 shows the activity of 5-amino-3-{2-[5-(1 ,3-benzothiazol-2-yl)-2-furyl]-1 -cyano- vinyl}-1 H-pyrazole-4-carbonitrile (formula 7) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmedby microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>10 μΜ). Figure 4 shows the activity of ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3- carboxylate (formula 8) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>10 μΜ). Figure 5 shows the activity of ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenyl- methylidene)cyclopent-1 -en-1 -yl]prop-2-enoate (formula 9) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was no toxicity even at high concentrations. Figure 6 shows the activity of 4-imino-7-(1 ,1 ,2,2-tetrafluoroethoxy)-3,10-dithia-5,12-diaza- tricyclo[7.3.0.0{2,6}]dodeca-1 (9),2(6),7,1 1 -tetraen-1 1 -amine (also termed 2-imino-4- (1 ,1 ,2,2-tetrafluoroethoxy)-2,3-dihydrobenzo[1 ,2-d:3,4-d]bis(thiazole)-7-amine, formula 10) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was revealed by microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>10 μΜ). Figure 7 shows the activity of 5-methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]- 1 ,3-thiazol-2-yl}-1 ,2-oxazole-3-carboxamide (formula 11 ) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low micromolar concentrations in the presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>10 μΜ).

Figure 8 shows the activity of 12-(2-cyanoethyl)-1 1 -methyl-13-{[3-(morpholin-4-yl)- propyl]amino}-1 ,8-diazatricyclo[7.4.0.0{2,7}]trideca-2(7),3,5,8,10,12-hexaene-10- carbonitrile (also termed 2-(2-cyanoethyl)-3-methyl-1 -((3-morpholinopropyl)amino)- benzo[4,5]imidazo[1 ,2-a]pyridine-4-carbonitrile, formula 12) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at around a concentration of 10 μΜ in presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>20 μΜ).

Figure 9 shows the activity of 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3- thiazol-4-yl)phenyl]benzamide (formula 13) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at around a concentration of 10 μΜ in presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was no toxicity.

Figure 10 shows the activity of (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol- 1 -yl)phenyl]prop-2-enamide (formula 14) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at around a concentration of 10 μΜ in presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), there was toxicity only at high concentrations (>20 μΜ). Figure 1 1 shows the activity of 2-(4-chlorophenyl)-N'-[(2,4-dimethoxybenzoyl)oxy]ethan- imidamide (formula 15) tested in human leukemia HL60 cells (panel A) and normal human fibroblasts (panel B). The dashed line shows the activity in presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at around a concentration of 5 μΜ in presence of metformin, but not in its absence (panel A), resulting cell death was confirmed by microscopy. On fibroblasts (panel B), this compound showed similar toxicity in presence and absence of metformin.

To show that the metformin sensitizing compounds actually alter the dose response profile to biguanides, titrations were performed of metformin and phenformin in the presence and absence of a fixed concentration of a metformin sensitizing agent.

Figure 12 shows that addition of metformin up to 5 mM is essentially non-toxic, with even less than 20% toxicity at 10 mM (solid lines): However, when a metformin sensitizing compound is added to metformin, dramatic toxicity is induced (panels A-G, broken lines). Note that for these experiments, sub-toxic concentrations of the metformin sensitizing compounds were used as previously determined in the corresponding titration experiments (Fig. 1 -1 1 , see Brief description of Figures 12, 13). When for example 1 μΜ of compound (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin- 4(3/-/)-one 5 was added to metformin (Fig. 12, panel A), metformin was toxic with an estimated IC50 at about 0.5 mM. The estimated cytotoxic IC50 for metformin varied slightly between different compounds, but full cell killing was observed with all

combinations tested (Fig 12, A-G), as seen from the alamarBlue cell viability

measurements and also confirmed by microscopic examination and by apoptosis assay (data not shown). A similar effect is expected when combining a metformin sensitizing compound with another biguanide, for example phenformin. This compound is known to be more active than metformin, and when formerly used in diabetes type 2 therapy, was applied at lower concentrations than metformin. Figure 13 shows experiments where increasing concentrations of phenformin were combined either with 5,6-diphenyl-7-(tetrahydro-2- furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine 6 at 5 μΜ (panel A), or with (2£)-3-(3,5- dimethoxyphenyl)-N-[3-(1 /-/-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2-enamide 14 at 5 μΜ (panel B). As shown in Figure 13, phenformin is non-toxic up to a concentration of 50 μΜ whereas addition of the metformin sensitizing agents at non-toxic concentrations induces strong toxicity with approximate IC50 values for phenformin of about 20 μΜ.

Analogues of compound of formula 5 were tested for synthetic lethality with metformin in human hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7. HCC is a common form of cancer for which no effective therapy currently exists. These compounds show potent activity with metformin-dependent cell lethality in the nanomolar range.

Figure 14 shows the activity of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyl- pyrimidin-4(3/-/)-one (formula 16) tested on human hepatocellular carcinoma cell lines HepG2 (panel A) and Huh7 (panel B). The dashed line shows the activity in the presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low nanomolar concentrations in the presence of metformin, but not in its absence. Resulting cell death was confirmed by microscopy.

Figure 15 shows the activity of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)- one (formula 17) tested on human hepatocellular carcinoma cell lines HepG2 (panel A) and Huh7 (panel B). The dashed line shows the activity in the presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low nanomolar concentrations in the presence of metformin, but not in its absence. Resulting cell death was confirmed by microscopy.

Figure 16 shows the activity of (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyl- tetrahydropyrimidin-4(1 /-/)-one (formula 18) tested on human hepatocellular carcinoma cell lines HepG2 (panel A) and Huh7 (panel B). The dashed line shows the activity in the presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low nanomolar concentrations in the presence of metformin, but not in its absence. Resulting cell death was confirmed by microscopy.

Figure 17 shows the activity of (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6- methylpyrimidin-4(3/-/)-one (formula 5) tested on human hepatocellular carcinoma cell lines HepG2 (panel A) and Huh7 (panel B). The dashed line shows the activity in the presence of 4 mM metformin, the solid line shows the activity in the absence of metformin. The compound was active at low nanomolar concentrations in the presence of metformin, but not in its absence. Resulting cell death was confirmed by microscopy.

In conclusion, the combination of a biguanide (metformin, phenformin and other biguanides) with a metformin sensitizing agent is a powerful novel method to induce cytotoxicity in cancer cells, by co-application of compounds for cancer therapy which are essentially non-toxic by themselves.

Examples

Cell culture

Human cancer cell lines Huh7, HepG2 (hepatocellular carcinoma) and HL60

(promyelocytic leukemia) were grown in Iscove's medium containing 10% FCS, 2 mM L-glutamine and 50 μΜ 2-mercaptoethanol. All cells were grown at 37°C in 5% C0₂.

Reagents

Metformin and phenformin (Sigma) were prepared as 1 M and 0.1 M stocks, respectively, in PBS and kept at 4°C. The metformin sensitizing compounds were from Enamine (formula 5, 8 to 14, 18) or Chembridge (formula 6, 7, 15, 16, 17 and 19), and were prepared as 10 mM stocks in DMSO.

Cell proliferation assay

Cells were seeded at appropriate density (5,000-15,000 cells per well depending on cell type, 150 μΙ medium per well) in flat-bottomed 96-well plates and compounds added at the desired concentrations. After 3 days, in some experiments after 2 or 5 days, proliferation was assayed by adding 0.1 volume of AlamarBlue (Invitrogen), and fluorescence was read at 535/595 nm excitation/emission after 4-6 hours of color development. Readings were normalized to non-treated control cells and growth expressed as percentage of control growth.

Apoptosis assay

Apoptosis was determined by Annexin V-FITC (Invitrogen) and propidium iodide counter- staining, and cells were analysed by FACS to distinguish between viable / early apoptotic / late apoptotic cell sub-populations. Cells were seeded at a density of 100,000 cells / 5 ml medium with addition of compounds as indicated. 500 μΙ of culture was harvested after 3 days and the cells were washed and stained with Pl/Annexin V before FACs analysis. Synthetic lethality screen using HL60 leukemia cells

A drug co-screen was performed using the compounds of commercial drug libraries from Enamine and from Chembridge on human HL60 leukemia cells in the presence and absence of metformin (4 mM) to identify compounds that would exert synthetic lethality with metformin as co-drug to kill these cells synergistically. The screen was done using robotic technology, and candidate hits were confirmed multiple times using 96 well microtiter plates and conditions described above.

Claims

1. A pharmaceutical composition comprising a metformin sensitizing compound and metformin or related biguanide.

2. The pharmaceutical composition of claim 1 comprising a metformin sensitizing compound and metformin, phenformin, buformin or proguanil.

3. The pharmaceutical composition of claim 1 comprising a metformin sensitizing compound and metformin or phenformin.

4. The pharmaceutical composition according to claims 1 to 3, wherein the metformin sensitizing compound is selected from the group consisting of - compounds of formula (A)

(A)

wherein

a dotted line indicates an optional bond;

Ri is hydrogen, methyl or halogen; and

and tautomers thereof; - 5,6-diphenyl-7-(tetrahydro-2-furanylm of formula (6):

- 5-amino-3-{2-[5-(1 ,3-benzothiazol-2-yl)-2-furyl]-1 -cyanovinyl}-1 /-/-pyrazole-4-carbonitrile of formula (7):

(₇)

(8) and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the quinoline ring, and/or wherein the ethoxy group is replaced by methoxy or isopropoxy, - ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenylmethylidene)cyclopent-1 -en-1 -yl]-

- 2-imino-4-(1 ,1 ,2,2-tetrafluoroethoxy)-2,3-dihydrobenzo[1 ,2-d:3,4-d]bis(thiazole)-7-amine) of formula (10):

- 5-methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]-1 ,3-thiazol-2-yl}-1 ,2-oxazole-3- carboxamide of formula (11 ):

(11 )

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl ring,

- 2-(2-cyanoethyl)-3-methyl-1 -((3-morpholinopropyl)amino)benzo[4,5]imidazo[1 ,2-a]- pyridine- -carbonitrile) of formula 12 :

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl ring, - 4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide of formula (13):

- (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazoM -yl)phenyl]prop-2-enamide of formula (14):

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl ring, and/or wherein the methoxy groups are replaced by ethoxy or isopropoxy, and

- -(4-chlorophenyl)-N'-[(2,4-dimethoxybenzoyl)oxy]ethanimidamide of formula (15):

and compounds carrying additional fluorine, chlorine, methyl and/or trifluoromethyl groups attached to the phenyl rings, and/or wherein the methoxy groups are replaced by ethoxy or isopropoxy,

5 The pharmaceutical composition according to claim 4, wherein the metformin sensitizing compound is a compound of formula (A), and tautomers thereof.

6. The pharmaceutical composition according to claim 5, wherein the metformin sensitizing compound is selected from the group consisting of

- (£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one of formula (5):

and tautomers thereof, -2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one of formula (16):

and tautomers thereof,

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)-one of formula (17):

and tautomers thereof, and

- (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydropyrimidin-4(1 H)-one of formula (18):

and tautomers thereof.

7. The pharmaceutical composition according to claims 1 to 4, wherein the metformin sensitizing compound is selected from the group consisting of

(£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one 5, 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine 6,

5-amino-3-{2-[5-(1 ,3-benzothiazol-2-yl)-2-furyl]-1 -cyanovinyl}-1 /-/-pyrazole-4-carbonitrile 7, ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3-carboxylate 8,

ethyl 3,3-dicyano-2-[(3Z)-2-(morpholin-4-yl)-3-(phenylmethylidene)cyclopent-1 -en-1 -yl]- prop-2-enoate 9,

2-imino-4-(1 ,1 ,2,2-tetrafluoroethoxy)-2,3-dihydrobenzo[1 ,2-d:3,4-d']bis(thiazole)-7-amine 10,

5-methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]-1 ,3-thiazol-2-yl}-1 ,2-oxazole-3- carboxamide 11 ,

2-(2-cyanoethyl)-3-methyl-1 -((3-morpholinopropyl)amino)benzo[4,5]imidazo[1 ,2-a]- pyridine-4-carbonitrile 12,

4- {[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide 13, (2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2-enamide 14, 2-(4-chlorophenyl)-N'-[(2,4-dimethoxybenzoyl)oxy]ethanimidamide 15, and

related compounds.

8. The pharmaceutical composition according to claim 4, wherein the metformin sensitizing compound is selected from the group consisting of

(£)-2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one 5, 5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine 6, ethyl 1 -[(5-chloro-8-hydroxyquinolin-7-yl)methyl]piperidine-3-carboxylate 8,

5- methyl-N-{4-[4-(N-methylmethanesulfonamido)phenyl]-1 ,3-thiazol-2-yl}-1 ,2-oxazole-3- carboxamide 11 ,

4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide 13, and

(2£)-3-(3,5-dimethoxyphenyl)-N-[3-(1 H-1 ,2,3,4-tetrazol-1 -yl)phenyl]prop-2-enamide 14.

9. The pharmaceutical composition according to claim 4, wherein the metformin sensitizing compound is

5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine 6, 5-methyl-N-{4-[4-(N-methylmetha

carboxamide 11 ,

4- {[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide 13, or

10. The pharmaceutical composition according to according to claims 1 to 4, wherein the metformin sensitizing compound is

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one 16,

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)-one 17, or

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydropyrimidin-4(1 H)-one 18.

1 1 . The pharmaceutical composition of any one of claims 1 to 8, wherein the relative amount (weight per weight) of the metformin sensitizing compound and metformin or related biguanide is between 1 to 1 and 1 to 10Ό00.

12. The pharmaceutical composition of claim 1 1 , wherein the relative amount (weight per weight) of the metformin sensitizing compound and metformin is between 1 to 1 Ό00 and 1 to 5Ό00.

13. A combination of a metformin sensitizing compound and metformin or related biguanide for use in the treatment of cancer or for achieving immunosuppression.

14. The combination according to claim 13, wherein the metformin sensitizing compound is

5,6-diphenyl-7-(tetrahydro-2-furanylmethyl)-7/-/-pyrrolo[2,3-d]pyrimidin-4-amine 6,

4-{[(cyanomethane)sulfonyl]methyl}-N-[3-(2-methyl-1 ,3-thiazol-4-yl)phenyl]benzamide 13, or

15. The combination according to claim 13, wherein the metformin sensitizing compound is

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methylpyrimidin-4(3/-/)-one 16,

(£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)pyrimidin-4(3/-/)-one 17, or (£)-2-(2-(2-hydroxybenzylidene)hydrazinyl)-6-methyltetrahydropyrimidi H)-one 18.

16. The combination of a metformin sensitizing compound and metformin or related biguanide for use in the treatment of carcinomas, sarcomas, tumors of the haematopoietic system, tumors of the nervous system and melanomas , according to claim 13.

17. The combination of a metformin sensitizing compound and metformin or related biguanide for use in immunosuppression in diseases where the immune system is hyperactivated, according to claim 13.

18. The combination of a metformin sensitizing compound and metformin or related biguanide according to any one of claims 13 to 17, wherein the metformin sensitizing compound and metformin or related biguanide are part of the same pharmaceutical composition.

19. The combination of a metformin sensitizing compound and metformin or related biguanide according to any one of claims 13 to 17, wherein the metformin sensitizing compound and metformin or related biguanide are in different pharmaceutical

compositions.

20. Use of a combination of a metformin sensitizing compound and metformin or related biguanide according to claim 13 for the preparation of a pharmaceutical composition for the treatment of cancer or for achieving immunosuppression.

21 . A method for treatment of cancer or for immunosuppression in diseases where the immune system is hyperactivated, which comprises administering a combination of a metformin sensitizing compound and metformin or related biguanide, or of a

pharmaceutical composition comprising a metformin sensitizing compound and metformin or related biguanide, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.