WO2014009891A1

WO2014009891A1 - Heterocyclic compounds for use in the treatment of cancers

Info

Publication number: WO2014009891A1
Application number: PCT/IB2013/055656
Authority: WO
Inventors: Nishigandha Ramchandra NAIK; Geetanjali Chandrashekhar Chimote
Original assignee: Piramal Enterprises Limited
Priority date: 2012-07-11
Filing date: 2013-07-10
Publication date: 2014-01-16

Abstract

The present invention relates to the method for the treatment of cancer in a subject comprising administering to the subject in need thereof, a therapeutically effective amount of a rho GTPase inhibitor selected from a compound of formula (I) or a compound of formula (II), wherein R₁, R₂, R₃, R₄, R₅ and R₆ are as defined in the specification, in all their stereoisomeric or tautomeric forms, and mixtures thereof in all ratios and their pharmaceutically acceptable salts thereof. The present invention also relates to pharmaceutical compositions comprising a rho GTPase inhibitor selected from one or more compounds of formula (I) or compounds of formula (II) for use in the treatment of cancer.

Description

HETEROCYCLIC COMPOUNDS FOR USE IN THE TREATMENT OF CANCERS

Field of the Invention

The present invention relates to a method for the treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I) (as described herein) or the compound of formula (II) (as described herein). The present invention also relates to pharmaceutical compositions comprising said compound of formula (I) or the compound of formula (II) for use in the treatment of cancers.

Background of the Invention

Cancer is a major health problem and a leading cause of death worldwide. The disease accounted for an estimated 12.4 million incident cases of cancer and 7.6 million deaths in 2008. Further, it is projected that there could be 27 million incident cases of cancer, 17 million deaths due to cancer and 75 million persons alive with cancer within five years of diagnosis (World Cancer Report, 2008).

About 85% of the cancers are of the epithelial origin and most of the times, metastasis of these cancers leads to death. Transcriptional up regulation of particular rho proteins have been reported in many cancers of epithelial cells including breast, lung, colon, colorectal and pancreatic cancer (British Journal of Cancer, 1998, 77(1 ), 147-152; Nature, 2000, 406, 532-535; The Journal of Cell Biology, 2000, 149(4), 775- 781 ).

Rho family of small molecular weight guanosine triphosphatases (hereinafter referred to as rho GTPases) are molecular switches that control signaling pathways regulating cytoskeleton organization, gene expression, cell cycle progression, cell motility and other cellular processes (Cell Communication and Signaling, 2010, 8, 23). Rho family GTPases are important signaling proteins that control diverse cellular functions related to cancer development, including actin cytoskeleton organization, transcription regulation, cell cycle progression, apoptosis, vesicle trafficking, and cell- to-cell and cell-to-extracellular matrix adhesions (Cell Communication and Signaling, 2010, 8 (23), 1 -14; Genes Dev., 1997, 1 1 , 2295-2322).

Rho GTPases work as sensitive molecular switches existing either in an inactive, guanasine diphosphate (GDP)-bound form or an active guanisine triphosphate (GTP)-bound form (Cell Communication and Signaling, 2010,8 (23), 1 - 14). This GDP-GTP cycle is tightly regulated by three classes of proteins: the guanine nucleotide exchange factors (GEFs) which activate GTPases by promoting the exchange of GDP for GTP, the GAPs (GTPase activating proteins) which enhance the intrinsic GTPase activity leading to the inactive rho GTPase form and the GDIs (guanine nucleotide-dissociation inhibitors) which sequester the GTPases in their GDP-bound state and regulate their intracellular localization (Biol. Cell, 2007, 99(2), 67- 86; TRENDS in Cell Biology, 2005, 15(7), 356-363). The exchange of GDP to GTP and thus the activation of rho GTPases is catalyzed by GEFs, which act downstream of numerous growth factor receptors, integrins, cytokine receptors, and cadherins. In turn, GTP bound active GTPases can interact with a plethora of different effectors which mediate the different cellular functions of this family of proteins. Rho GTPase effectors are a large group of proteins and include actin nucleation promoting molecules, adaptors, as well as kinases (Cell Communication and Signaling, 2010,8 (23), 1 -14).

Rho GTPases have been reported to contribute to most steps of cancer initiation and progression including the acquisition of unlimited proliferation potential, survival and evasion from apoptosis, tissue invasion and the establishment of metastasis (FEBS Letters, 2008, 582, 2093-2101 ).

The rho GTPase is a subfamily of the Ras super family proteins and the best characterized rho GTPases are rhoA, rad and cdc42. Rho GTPases contribute to the initiation and progression of cancer by subverting the normal regulation of specific intracellular signaling pathways (Current Opinion in Genetics and Development, 2005, 15, 62-68). In vitro, several Rho GTPases have oncogenic activity and/or can promote cancer cell invasion and this correlates with increased expression and activity in a variety of cancers (FEBS Letters, 2008, 582, 2093-2101 ).

Glioblastoma multiforme (GBM), or grade IV astrocytoma, represents the most common primary brain tumor in humans, comprising 12-15% of all intracranial neoplasms and 50-60% of astrocytic tumors. Current treatment options, which often involve a combination of chemotherapy, radiotherapy, and surgery, have proven to be of minor benefit and the clinical course from the time of diagnosis remains catastrophic, with a median survival range of 10-12 months (Anticancer Research, 2009, 29, 1 19-124; Anticancer Drugs, 2006, 17(9), 1003-1016). The current chemotherapy and clinical trials use cytotoxic drugs, anti-angiogenic compounds or target various cell-signaling pathways involved in cell proliferation. The drugs which are now in use for the treatment of glioblastoma include temozolomide, carmustine, cisplatin, oxiplatin and irrinotecan.

Studies have shown that rhoA appears to be up-regulated in human carcinomas, specifically in high-grade astrocytomas. Similarly, a large series of astrocytomas revealed a significant correlation between WHO grade of malignancy of astrocytoma and the expression of focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2), both of which are downstream targets of rhoA. Studies of Pyk2 in glioma cells revealed that silencing of this protein can suppress glioma motility and it appears to play an important role in glioblastoma cell migration (Anticancer Research, 2009, 29, 1 19-124; Neurosci. Lett., 2006, 407(2), 124-126; Acta Neuropathol., 2004, 108(3), 224-230).

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by unregulated growth of myeloid leukemia cells in the bone marrow and accumulation of these cells in the blood. CML represents approximately 15% of all adult leukemia (Ann. Intern. Med. ,1999, 131 , 207-219). A review article (Leukemia Research andTreatment, 2012, 1 -19) describes that the disease development is characterized by the presence of the Philadelphia chromosome which resulted from the reciprocal translocation between chromosomes 9 and 22. The pathogenesis of CML involves the constitutive activation of the Bcr-Abl tyrosine kinase, which governs malignant disease by activating multiple signal transduction pathways. The Bcr-Abl kinase inhibitor, imatinib, is the front-line treatment for CML. Mutation of Bcr-Abl is an important mechanism by which CML cells become resistant to imatinib. The T315I mutation is clinically significant since CML cells harboring this mutation are insensitive to imatinib and other Bcr-Abl targeted drugs (Leukemia, 2008, 22(6), 1 191-1 199).

Treatment options for CML patients with imatinib resistance include dasatinib, nilotinib or allogeneic stem cell transplantation. A notable common weakness of all second-generation kinase inhibitors, dasatinib, nilotinib and bosutinib is that these drugs target Bcr-Abl and none of these can inhibit cell expressing Bcr-Abl with T315I mutation.

Bcr-Abl protein which is characteristic for CML alters the activity of multiple proteins involved in rho signaling either by tyrosine phosphorylation or by intrinsic or induced GEF activity. Bcr-Abl contains also additional functional domains, in particular a DBL homology (DH) domain with guanine-exchange function which can activate small GTPases of Rho family. The DH domain of p210 Bcr-Abl acts as an activator of small GTPases of rho family. The best explored members of this family are rhoA, which induces stress fiber formation and is responsible for actomyosin contractility in non-muscle cells; rad , which is required for lamellipodia formation and dynamic assembly of focal adhesions; and cdc42, which regulates filopodia formation, directional migration and cell polarity. All of them can be regulated by DH domain contained in Bcr-Abl or by the domain with GAP activity which is present in the complementary fusion protein Abl-Bcr detectable in the majority of patients with CML (Cardiovascular and Haematological Disorders-Drug Targets, 2008, 8, 261 -267; BMC Cancer,2006, 6(262), 1 -1 1 ; Oncogene, 2008, 27(14), 2064-2071 ; Proc. Natl. Acad. Sci. USA, 1995, 92, 10282-10286).

RhoA is frequently over expressed in cancer and the tumors derived from tissues of colon, breast and lung cancer showed over-expression of rhoA as compared with the normal tissues (Int. J. Cancer, 1999, 81 , 682-687). Depletion of rad strongly inhibits lamellipodia formation, cell migration and invasion in carcinoma cells, especially in glioblastoma and breast carcinoma cells (Oncogene, 2005, 24, 7821 - 7829). Another rho family gene, Cdc42 is also important for cell motility and able to induce a mesenchymal-amoeboid transition in melanoma cells (Current Biology, 2008, 18, 1456-1465). Rho GTPases and the signal pathways regulated by them have thus been proposed as potential anticancer therapeutic targets(FEBS Letters, 2008, 582, 2093-2101 ).

PCT Application Publication WO2007016539A2 describes methods for treating indication mediated by rho family proteins, particularly to treat diseases associated with rac disregulation. The compounds of this patent publication are employed to prepare compositions useful in treatment of diseases associated with abnormal cell proliferation and particularly suited for the treatment of cancers, such as solid tumors or lymphoid tumors.

PCT Application Publication WO2008024922A2 describes 8-hydroxyquinoline compounds, compositions comprising 8-hydroxyquinoline compounds and methods for treating metalloproteinase-related disorder, especially malignant neoplasm and angiogenesis.

PCT Application Publication WO200907457A2 describes an in vitro method of identification of inhibitory action of protoberberine, benzo[c]phenanthridine alkaloids or 3-arylisoquinolines derivatives on the activity of rho family GTPases, in particular on the activity of the members of the rac subfamily of rho GTPases. There still exists a continuing need for compositions and methods for treating cancers, especially glioblastoma, Chronic myelogenous leukemia (CML) or CML resistant to treatment with imatinib, for which many treatment options are not available. Furthermore, there is a compelling need for compositions and methods that will effectively target the signaling pathways related to rho GTPases and which can be useful for the treatment and therapeutic options for cancers mediated by the rho GTPase signaling pathways, particularly those mediated by rhoA, rhoB and rad . Examples of cancers that are mediated by rho GTPase signaling pathway are glioblastoma, breast cancer, chronic myelogenous leukemia (CML), CML resistant to treatment with imatinib, lung cancer, non-small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, colon cancer, renal cancer, ovarian cancer, testicular cancer and melanoma.

Summary of the Invention

The present invention relates to a method for the treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I) (as described herein) or the compound of formula (II) (as described herein), in all its stereoisomeric and tautomeric forms, and mixtures thereof in all ratios or a pharmaceutically acceptable salt thereof.

In one aspect, the present invention relates to a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II), in all its stereoisomeric and tautomeric forms, and mixtures thereof in all ratios or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier, for use in the treatment of cancer.

In yet another aspect, the present invention also relates to use of a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer. In a still further aspect, the present invention relates to a pharmaceutical composition comprising a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II), or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient or carrier, for use in the treatment of cancer in combination with an anticancer agent.

These and other objectives and advantages of the present invention will be apparent to those skilled in the art from the following description.

Detailed Description of the Invention

Accordingly, the present invention relates to a method for the treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I);

formula(l)

or the compound of formul

5

formula(ll)

wherein in the compounds of formula (I) and the compounds of formula II:

Ri and R₂ are independently selected from (C₆-Ci₀)aryl and heteroaryl;

R₃ and R₄ are independently selected from hydrogen, (d-C₆)alkyl and (C₆-Ci₀)aryl;R₅ and R₆ are independently selected from (CrC₆)alkyl, (C₆-Ci₀)aryl and heteroaryl; n is an integer from 0 to 3;

wherein,

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano; (C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independentlyselected from (Ci-C₆)alkyl, halogen, halo(Ci-C₆)alkyl, -0-(C C₆)alkyl, halo(Ci-C₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(Ci-C₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

Definitions

Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein and the appended claims. These definitions should not be interpreted in the literal sense as they are not general definitions and are relevant only for this application.

It will be understood that "substituted" or "substituted with" means that one or more hydrogens of the specified moiety are replaced with a suitable substituent and includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and results in a stable compound.

Within the context of the present application, the term "(Ci-C₆)alkyl" or "alkyl", as used herein, alone or as part of a substituent refers to an aliphatic group, including straight or branched chain alkyl group. A straight chain or branched chain alkyl has six or fewer carbon atoms in its backbone, for instance, C C₆ for straight- chain and C₃- C₆ for branched chain. Suitable alkyl groups containing from one to six carbon atoms are, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, 1 - methylbutyl, secondary butyl, tertiary pentyl, neopentyl, 2,2-dimethylbutyl, 2- methylpentyl, 3-methylpentyl or 3-methylpentyl.

Furthermore, unless stated otherwise, the alkyl groups may be substituted or unsubstituted. The alkyl group may be optionally substituted with one or more substituents, such as (d-C₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(C C₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, carboxy, cyano or nitro. Examples of substituted alkyl include but not limited to hydroxymethyl, 2-chloropropyl, aminoethyl or benzyl.

Within the context of the present application and as used herein, the term "halo(Ci-C₆)alkyl" or "haloalkyl" refers to radicals wherein one or more of the hydrogen atoms of the alkyl group are substituted with one or more halogens. Examples of halo(Ci-C₆)alkyl include but are not limited to chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl or trifluoromethyl.

Within the context of the present application and as used herein, the term "-0- (CrC₆)alkyl," or "alkoxy" refers to an (d-C₆)alkyl having an oxygen radical attached thereto, that is bonded via the oxygen atom. Representative alkoxy groups include but not limited to methoxy, ethoxy, propoxy, isopropoxy, isobutoxy and tert-butoxy. Alkoxy is unsubstituted or optionally substituted with one or more groups such as (CrC₆) alkyl, hydroxy, halogen, amino, carboxy, cyano or nitro.

Within the context of the present application and as used herein, the term "halo(CrC₆)alkoxy" o r"haloalkoxy" refers to radicals wherein one or more of the hydrogen atoms of the alkoxy group are substituted with one or more halogens. Representative examples of halo(CrC₆)alkoxy groups include but not limited to, difluoromethoxy (OCHF₂), trifluoromethoxy (OCF₃) or trifluoroethoxy (OCH₂CF₃).

Within the context of the present application and as used herein, the term (C₆- Cio) aryl" or "aryl" refers to a monocyclic or bicyclic hydrocarbon ring system having up to ten ring carbon atoms, wherein at least one carbocyclic ring is having a π electron system. Examples of (C₆-Ci₀) aryl ring systems include, but not limited to, phenyl or naphthyl. Unless indicated otherwise, aryl group may be unsubstituted or optionally substituted with one or more identical or different substituents such as (CrC₆)alkyl, halogen, halo(Ci-C₆)alkyl, hydroxy, thiol, -0-(Ci-C₆)alkyl, halo(CrC₆)alkoxy, (C₆- Cio)aryl, heterocyclyl, heteroaryl, amino, cyano, nitro, C(0)(Ci-C₆)alkyl, carboxy, C(0)NH₂ or C(0)0(CrC₆)alkyl. Aryl groups can be substituted in any desired position.

Within the context of the present application and as used herein, the term "heterocyclyl" refers to a saturated, unsaturated and aromatic monocyclic or polycyclic ring system containing three to nine ring carbon atoms and one to four identical or different hetero atoms selected from: a nitrogen (N), a sulphur (S) or an oxygen (O) atom. Heterocyclyl includes saturated heterocyclic ring systems, which do not contain any double bond and unsaturated heterocyclic ring systems, which contain at least one double bond, but do not form an aromatic ring system. Heterocyclyl monocyclic or bicyclic ring systems having at least one aromatic ring containing three to tenring carbon atoms and one to four identical or different hetero atom/s selected from nitrogen (N), sulphur (S) or oxygen (O) atom are herein referred to by the customary term "heteroaryl". It is further indicated that all the definitions and explanations provided for heterocyclyl apply to the term "heteroaryl". Suitable saturated and unsaturated non-aromatic heterocyclyl groups include, but are not limited to, azetidine, oxetane, thietane, tetrahydrofuran, dihydropyran, tetrahydropyran, piperidine, piperazine, morpholine, thiomorpholine, isoxazolidine or pyrazolidine. Suitable heteroaryl groups include, but are not limited to, thiene, furan, pyridine, oxazole, thiazole, pyrazine, pyrimidine, pyrrole, pyrazole, isooxazole, triazole, tetrazole, pyridazine, isothiazole, benzothiazole, benzooxazole, benzimidazole, quinoline, isoquinoline, 3, 5-dimethylisooxazole, chroman, or thiochroman.

The heterocyclyl and heteroaryl may be unsubstituted or optionally substituted with one or more identical or different substituents selected from (d-C₆)alkyl, halogen, halo(Ci-C₆)alkyl, -0-(Ci-C₆)alkyl, halo(CrC₆)alkoxy, hydroxy, thiol, (C₆-

Cio)aryl, heterocyclyl, amino, cyano, nitro, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ or

C(0)0(CrC₆)alkyl. It is further indicated that the substituents on the heterocyclyl and heteroaryl may be present either on the ring carbon or the ring nitrogen atoms when the said heterocyclyl or heteroaryl contains nitrogen atom.

The term "heteroatom" as used herein, includes nitrogen (N), oxygen (O) and sulfur (S). Any heteroatom with unsatisfied valency is assumed to have a hydrogen atom or an (Ci-C₆)alkyl group to satisfy its valency.

The term "halogen" or "halo" as used herein, unless otherwise indicated refer to bromine, chlorine, fluorine or iodine atom.

The term "amino" refers to the group "NH₂" which may be optionally substituted by one or more substituents. Examples of substituents include, but not limited to, (C

C₄)alkyl or (C₆-C₁₀) aryl.

The term "carboxy" refers to a group of formula -COOH; also referred to as a carboxylic acid group.

The term "rho GTPase inhibitor" refers to a substance that inhibits the activity of rho GTPases i.e. partially or totally blocks stimulation, inactivates, desensitizes or down regulates rho GTPases, particularly, rho GTPases such as rhoA, rad and cdc42. Within the context of the present invention, the rho GTPase inhibitor refers to the compound of formula (I) or compound of formula (II) as described herein.

The term "compounds of the present invention", or "the compounds of formula

(I) or formula (II)" or "the compounds of formula (I) or formula (II) of the present invention" used interchangeably, includes the compounds of formula (I) or the compounds of formula (II) and their stereoisomeric and tautomeric forms and mixtures thereof in all ratios, and pharmaceutically acceptable salts thereof. Within the context of the present invention, the compounds of formula (I) (as described herein) are pyrazolo [3, 4-d] pyrimidine-4-one derivatives; and the compounds of formula (II) (as described herein) are quinolinol derivatives. Accordingly, the compounds of formula (I) and the compounds of formula (II) are heterocyclic compounds.

Within the context of the present invention and as used herein, the term

"stereoisomer" is a general term used for all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or E/Z) isomers, and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers).

Within the context of the present invention and as used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ from each other only in the position of one (or more) mobile atoms and in electron distribution, for example, keto-enol tautomers.

Within the context of the present invention and as used herein, the term "pharmaceutically acceptable salts" includes salts of the active compounds which are prepared with acids or bases, depending on the particular substituents found on the compounds described herein.

Within the context of the present invention and as used herein, the term

"cancer" refers to cancer mediated by rho GTPase signaling pathway. The cancer is regulated by the intracellular signaling pathways related to one or more rho family of small GTPase proteins, wherein the rho GTPase is selected from rhoA, rad or cdc42.

As used herein, the term "rho family of GTPase proteins" or "rho GTPases" refers to a subfamily of ras super family of proteins and are small membrane bound, ras-related GTP-binding proteins that function by binding and hydrolyzing GTP. Rho GTPases function as molecular switches, cycling between an inactive GDP-bound conformation and an active GTP-bound conformation and include but not limited to, rhoA sub-family (rhoA, rhoB, rhoC), rac related sub-family (rad , rac2, rac3 or rhoG), the cdc42 related sub-family (cdc42, TC10, TCL), the Rnd subfamily (Rnd1 , Rnd2 and RhoE/Rnd3), the RhoBTB subfamily and the like (Journal of Cell Science, 2004, 1 17, 1301 -1312).

The term "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. The term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig as well as human.

Within the context of the present invention and as used herein, the term "sample" refers to a standard rho GTPase protein inhibitor or a test compound. For instance, the standard rad GTPase inhibitor is NSC-23766(N6-[2-[4-(diethylamino)-1 - methylbutylamino]-6-methylpyrimidin-4-yl]-2-methylquinoline-4,6-diamine).

As used herein, the term "test compound" refers to a substance which is being tested for rho GTPase protein inhibitory activity. Within the context of the present invention, the term "test compound" refers to the compound of formula (I) or the compound of formula (II).

Embodiments

In one aspect, the present invention relates to a method for the treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I)

₅

formula(ll)

wherein in the compounds of formula (I) and the compounds of formula II:

Ri and R₂ are independently selected from (C₆-Ci₀)aryl and heteroaryl;

R₃ and R₄ are independently selected from hydrogen, (d-C₆)alkyl and (C₆-Ci₀)aryl; R₅ and R₆ are independently selected from (CrC₆)alkyl, (C₆-Ci₀)aryl and heteroaryl; n is an integer from 0 to 3;

wherein,

(Ci -C₆) alkyl is unsubstituted or substituted with one or more groups independently selected from (d-C₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano;

(C-6-C10) aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(C C₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(CrC₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

In one embodiment, in the method of treatment according to present invention, the rho GTPase inhibitor is selected from the compound of formula (I)

formula(l)

wherein,

R-i and R₂ are independently selected from (C₆-C₁₀)aryl and heteroaryl;

R₃ and R₄ are independently selected from hydrogen, (CrC₆)alkyl and (C₆-Ci₀)aryl; n is an integer from 0 to 3;

wherein,

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano;

(C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(d-C₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof. In another embodiment, in the method of treatment according to the present invention, the rho GTPase inhibitor is selected from the compound of formula (I), wherein

Pt₂ is heteroaryl;

R₃ and R₄ are hydrogen;

n is 1 ;

wherein,

(C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (d-C₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(C C₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(CrC₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

In one embodiment, in the method of treatment according to the present invention the rho GTPase inhibitor is selected from the compound of formula (II);

formula(ll)

wherein,

Rs and R₆ are independently selected from (CrC₆)alkyl, (C₆-Ci₀) aryl and heteroaryl; wherein,

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy,hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano;

(C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(C C₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(CrC₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

In another embodiment, in the method of treatment according to the present invention, the rho GTPase inhibitor is selected from the compounds of formula (II), wherein R₅ is (Ci-C₆)alkyl;

wherein,

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (d-C₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀) aryl, heterocyclyl, amino and cyano;

(C₆-Cio)aryl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, -0-(CrC₆)alkyl, halo(CrC₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)(CrC₆)alkyl, carboxy, C(0)NH₂ and C(0)0(d-C₆)alkyl;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

In a specific embodiment, in the method of treatment according to the present invention, the rho GTPase inhibitor is selected from:

6-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-5-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4(5H)- one; or

N-((8-hydroxyquinolin-7-yl) (p-tolyl)methyl)acetamide;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

In one particular embodiment, the present invention relates to rho GTPase inhibitors selected from the compounds of formula (I) (as described herein) or the compounds of formula (II) (as described herein) and all their stereoisomeric and tautomeric forms, and mixtures thereof in all ratios or pharmaceutically acceptable salts thereof, for use in the treatment of a cancer.

In one particular embodiment, the present invention relates to a rho GTPase inhibitor selected from the compounds of formula (I) (as described herein) and all its stereoisomeric and tautomeric forms, and mixtures thereof in all ratios, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer.

In one particular embodiment, the present invention relates to a rho GTPase inhibitor selected from the compounds of formula (II) (as described herein) and all its stereoisomeric and tautomeric forms, and mixtures thereof in all ratios, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer.

In a specific embodiment, the rho GTPase inhibitor for use in the treatment of cancer is selected from:

6-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-5-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4(5H)- one; or N-((8-hydroxyquinolin-7-yl)(p-tolyl)methyl)acetamide;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

When the compounds of formula (I) or the compounds of formula (II) (as described herein) contain an acidic group, they can form an addition salt with a suitable base. For example, pharmaceutically acceptable base addition salts of the compounds of the formula (I) or the compounds of the formula (II) may include their alkali metal salts such as sodium, potassium, calcium, magnesium, ammonium or an organic base addition salt. Examples of pharmaceutically acceptable organic base addition salts of the compounds of the present invention include those derived from organic bases like lysine, arginine, guanidine, diethanolamine or other organic bases known to the person skilled in the art.

When the compounds of formula (I) or the compounds of the formula (II) (as described herein) contain one or more basic groups, they can form an addition salt with an inorganic or an organic acid. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like boric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogen phosphoric acid, dihydrogenphosphoric acid, sulfuric acid, or other inorganic acids known to the person skilled in the art. Furthermore, examples of pharmaceutically acceptable acid addition salts include the salts derived from organic acids like acetic acid, propionic acid, isobutyric acid, oxalic acid, malic acid, tartaric acid, citric acid, ascorbic, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, glucuronic acid, galacturonic acid, naphthoic acid, camphoric acid or other organic acids known to the person skilled in the art. Certain specific compounds of the present invention may contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

In one embodiment, the present invention relates to a rho GTPase inhibitor, selected from the compound of formula (I) (as described herein) or the compound of formula (II) (as described herein) for use in the treatment of cancer, wherein cancer includes, but not limited to; glioblastoma, breast cancer, chronic myelogenous leukemia (CML), CML resistant to treatment with imatinib, renal cancer, pancreatic cancer, colon cancer, lung cancer, non-small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, skin cancer, ovarian cancer, bladder cancer, testicular cancer and melanoma.

In one embodiment, the present invention provides use of a rho GTPase inhibitor, the compounds of formula (I) (as described herein), for the manufacture of a medicament in the treatment of cancer.

In another embodiment, the present invention provides use of a rho GTPase inhibitor, the compound of formula (II) (as described herein), for the manufacture of a medicament in the treatment of cancer.

In a specific embodiment, the present invention provides use of a rho GTPase inhibitor selected from the compound of formula (I) (as described herein) or the compound of formula (II) (as described herein) for the manufacture of a medicament in the treatment of cancer, wherein cancer includes, but not limited to glioblastoma, breast cancer, chronic myelogenous leukemia (CML), CML resistant to treatment with imatinib, renal cancer, pancreatic cancer, colon cancer, lung cancer, non-small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, skin cancer, ovarian cancer, bladder cancer, testicular cancer and melanoma.

In a further embodiment, the present invention provides use of a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II), for the manufacture of a medicament in the treatment of cancer, wherein cancer is selected from glioblastoma, breast cancer, chronic myelogenous leukemia (CML) or CML resistant to treatment with imatinib.

In accordance with one embodiment, the present invention is directed to a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, comprising the steps of: a) contacting a sample with a solid support having a rho GTPase protein link; b) detecting the interaction of the sample, with rho GTPase protein, using a primary antibody and an enzyme linked secondary antibody.

In one embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the sample comprises a standard rho GTPase inhibitor or a test compound.

In one embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the test compound may be selected from the compounds of formula (I) or the compounds of formula (II).

In one embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the rho GTPase protein is selected from rhoA, rad or cdc42.

In one embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the solid support is a microtitre plate or an immunomodule.

In one embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the detection of interaction of sample with a rho GTPase protein is done using absorbance.

In another embodiment, the present invention provides a method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, wherein the primary antibody is selected from anti-rhoA, anti-rad or anti- cdc42 specific monoclonal antibody and the secondary antibody is Goat anti-mouse IgG-HRP (Immunoglobulin G-Horseradish peroxidase) conjugate.

According to one embodiment, the present invention relates to a pharmaceutical composition, comprising a rho GTPase inhibitor selected from one or more compounds of formula (I) or the compounds of formula (II) or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient, for use in the treatment of a cancer.

According to another embodiment, the present invention relates to a pharmaceutical composition comprising one or more compounds of formula (I) or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient, for use in the treatment of a cancer.

According to a yet another embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds of formula (I I) or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient, for use in the treatment of a cancer.

According to a further embodiment, the present invention provides a pharmaceutical composition comprising a rho GTPase inhibitor selected from the compound of formula (I) or the compound of formula (II), or pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable excipient or carrier; for use in the treatment of cancer in combination with an anticancer agent.

According to a still further embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds of formula (I) or pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable excipient or carrier; for use in the treatment of cancer in combination with an anticancer agent.

According to a still further embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds of formula (I I) or pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable excipient or carrier; for use in the treatment of cancer in combination with an anticancer agent.

An anticancer agent in combination with one or more compounds of formula (I) or one or more compounds of formula (II) for use in the treatment of cancer can be selected from, but not limited to, one or more of the compounds selected from: (i) kinase inhibitors such as gefitinib, imatinib, erlotinib, lapatinib, bevacizumab (avastin), sorafenib, Bcr-Abl kinase inhibitors or LY- 317615 (3-(1 -methyl-1 H-indol-3-yl)-4-[1 -[1 - (pyridin-2-ylmethyl)piperidin-4-yl]-1 H-indol-3-yl]-1 H-pyrrole-2,5- dionemonohydrochloride; (Cancer Res., 2005, 65(16), 7462-7469)); (ii) alkylating agents such as mitomycin C, busulfan, oxaliplatin, cisplatin, carboplatin, procarbazine or dacarbazine; (iii) antimetabolites such as methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, fluorouracil, vinblastine, vincristine, gemcitabine or paclitaxel; (iv) antibiotics such as anthracyclines, dactinomycin or bleomycin (v) hormonal agents such as tamoxifen, flutamide, GnRH (Gonadotropin-Releasing Hormone) agonists or aromatase inhibitors or (v) cancer vaccines such as avicine, oregovomab or theratope.

The term "pharmaceutically acceptable" as used herein in the present invention means that the carrier, diluents, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

The term "pharmaceutically acceptable carrier" as used herein means a nontoxic, inert, solid, semi-solid, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; malt; gelatin; talc; as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents; preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The term "therapeutically effective amount" as used herein in the present invention means an amount of the rho GTPase inhibitor which may be the compound of formula (I) or the compound of formula (II) or pharmaceutical composition containing the compounds e.g. compounds of formula (I) or compounds of formula (II) sufficient to significantly induce a positive modification in the condition to be regulated or treated, but low enough to avoid undue or severe side effects, within the scope of sound medical judgement. The therapeutically effective amount of the said compound or the said composition will vary with the type of cancer being treated, the age and physical condition of the subject, the severity of the cancer being treated or prevented, the duration of the treatment, the nature of concurrent therapy, the specific compound or composition employed, the particular pharmaceutically acceptable carrier utilized and other factors.

The term "treatment", "treat" and "therapy" as used herein and the like refer to alleviate, slow the progression, prophylaxis, attenuation or cure of cancer (e.g. cancer mediated by rho GTPase signaling pathway such as glioblastoma, breast cancer, chronic myelogenous leukemia (CML), CML resistant to treatment with imatinib, renal cancer, pancreatic cancer, colon cancer, lung cancer, non-small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, skin cancer, ovarian cancer, bladder cancer, testicular cancer and melanoma). Accordingly, the compounds of formula (I) or formula (II) (as described herein) may be used at very early stages of cancer, or before early onset, or after significant progression, including metastasis. Further, the term "treatment" designates in particular a reduction of the burden in a patient, such as a reduction in cell proliferation rate, a destruction of diseased proliferative cells, a reduction of tumor mass or tumor size, a delaying of tumor progression, as well as a complete tumor suppression.

The pharmaceutical compositions according to the present invention are prepared in a manner known and familiar to one skilled in the art. Pharmaceutically acceptable inert inorganic and/or organic carriers and/or additives can be used in addition to the compound(s) of formula (I) or formula (II) and/or its pharmaceutically acceptable salts. For the production of pills, tablets, coated tablets and hard gelatin capsules it is possible to use, for example, lactose, corn starch or derivatives thereof, gum arabic, magnesia or glucose, etc. Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, natural or hardened oils, etc. Suitable carriers for the production of solutions, for example injection solutions, or of emulsions or syrups are, for example, water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the various solvents which have been mentioned.

Further, the pharmaceutical compositions for use according to the present invention also contain additives such as, for example, fillers, antioxidants, emulsifiers, preservatives, flavours, solubilisers or colourants. Furthermore, in addition to at least one compound of formula (I) or its pharmaceutically acceptable salts, the pharmaceutical composition of the present invention can also contain one or more other therapeutically or prophylactically active ingredients. Analogously, in addition to at least one compound of formula (II) or its pharmaceutically acceptable salts, the pharmaceutical composition of the present invention can also contain one or more other therapeutically or prophylactically active ingredients.

The pharmaceutical compositions normally contain about 1 % to 99%, for example, about 10% to 80% by weight of the compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts.

The amount of the compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts in the pharmaceutical compositions can, for example, be from about 1 mg to 500 mg. The desirable dosage of the compounds of formula (I) or formula (II) (as described herein), can be selected over a wide range. The daily dosage of the compounds to be administered is selected to achieve the desired therapeutic effect in subjects being treated for cancers. A dosage of about 0.05mg/kg/day to 100 mg/kg/day of the compounds of formula (I) or formula (II) or their pharmaceutically acceptable salts thereof may be administered per day. If required, higher or lower daily dosages can also be administered. Actual dosage levels of the active ingredients in the pharmaceutical composition of this invention can be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level can be readily determined by a skilled medical practitioner in the consideration of the relevant circumstances, including the condition (cancer) to be treated, the chosen route of administration depending on a number of factors, such as age, weight and physical health and response of the individual patient, pharmacokinetics, severity of the disease and the like, factors known in the medical art.

The pharmaceutical compositions for the use according to the present invention can be administered orally, for example in the form of pills, tablets, coated tablets, capsules, granules or elixirs. Administration, however, can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injectable sterile solutions or suspensions, or topically, for example in the form of solutions or transdermal patches, or in other ways, for example in the form of aerosols or nasal sprays.

It is understood that modifications that do not substantially affect the operability of the various embodiments of this invention are included within scope of the invention disclosed herein. Accordingly, the following examples are intended to illustrate but not to limit scope of the present invention.

Experimental

The abbreviations and terms that are used herein:

CaCI₂ Calcium chloride NaHCOs Sodium bicarbonate

C0₂ Carbon dioxide Na₂C0₃ Sodium carbonate

KCI Potassium chloride NaCI Sodium chloride

KH₂P0₄ Potassium dihydrogen phosphate MgCI₂ Magnesium chloride

Na₂HP0₄ Disodium hydrogen phosphate MgS0₄ Magnesium sulphate

NaH₂P0₄ Sodium dihydrogen phosphate NaN₃ Sodium azide

s Second ng Nanogram

min Minute μ9 Microgram

h Hour(s) mg Milligram

RT Room Temperature (25°C) 9 Gram

mM Millimolar μΙ Microlitre

M Molar ml Milliliter

rpm Revolutions per minute nm Nanometre

Tris-HCI Tris hydrochloride mm Millimetre

PBS Phosphate Buffered Saline DMSO Dimethyl sulfoxide

FBS Fetal Bovine Serum NBT Nitrozoliumtetrablue HEPES N-[2-Hydroxy ethyl]piperazine (-N'-[2-ethane sulphonic acid])

PBST Phosphate Buffered Saline with Tween-20

HRP Horse Radish Peroxidase

BCIP 5-bromo-4-chloro-3'-indolyphosphate

DMEM Dulbecco's Modified Eagle Medium

F10 Ham's F-10 Nutrient Mixture (F-10)

MEGS Mammary epithelial growth supplements

3D MCS Three Dimensional Multicellular spheroids

ELISA Enzyme Linked Immunosorbent assay

PAGE Polyacrylamide Gel Electrophoresis

NCCS National Centre for Cell Science, Pune, India

RPMI Roswell Park Memorial Institute

ACTREC Advanced Centre for Treatment, Research and Education in Cancer, Tata

Memorial Centre, Navi Mumbai, India

OECD Organization for Economic Co-operation and Development

Example 1

6-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-5-phenyl-1 H-pyrazolo[3,4- d]pyrimidin-4(5H)-one

The title compound was procured from Chembridge library (Catalogue number: 7740160). Example 2

N-((8-hydroxyquinolin-7-yl)(p-tolyl)methyl)acetamide

The title compound was procured from Chembridge library (Catalogue number: 5848587).

Pharmacology

The pharmacological activity of the compounds of formula (I) and the compounds formula (II) (as described herein) for use in the treatment of cancers, particularly the cancers mediated by rho GTPase signaling pathway can be determined by a number of pharmacological assays well known in the art. The exemplified pharmacological assays, which are herein described below, has been carried out with the compounds of formula (I) and the compounds formula (II), referred to in the above Examples 1 and 2. Example 3

Primary screening method for rho GTPase interacting compounds (ELISA based assay)

The ELISA based assay was employed for screening and identifying compounds for rho GTPase protein inhibitory activity. Particularly, the test compounds (compound of Example 1 and compound of Example 2) were identified for their ability to inhibit rho GTPase rad . The ELISA based assay method involved fixing active rho GTPase in a 96 well plate and detecting them using a primary antibody and an enzyme linked secondary antibody. The rho GTPase inhibitory activity of the compounds was detected by the alteration in the absorbance intensity which is indicative of the effect of the compounds on the conformation of rho GTPase which is crucial for their activity. The assay was validated using a known rho GTPase inhibitor compound, NSC-23766 (source: Calbiochem) for rad protein. A) Materials and reagents

a) Constitutively active rad protein (commercially available as Histidine tagged) and stock preparation

The original stock (1 mg/ml) of constitutively active rad protein was prepared by dissolving constitutively active rad protein (10 μg) in sterile distilled water (10 μΙ). The original stock (1 μΙ) was snap freezed in liquid nitrogen and stored at -80°C. The original stock (2 μΙ) was mixed with carbonate-bicarbonate buffer (998 μΙ) to obtain a constitutively active rad protein stock (2 μg/ml). This stock (1000 μΙ) was mixed with carbonate-bicarbonate buffer (3000 μΙ) to obtain a working stock (0.5 ng/μΙ). 16 μΙ aliquots of working stock (0.5 ng/μΙ or 0.5 μg/ml) were snap freezed in liquid nitrogen and stored at -80°C. For experimentation, the working stock of constitutively active rad protein (16 μΙ) was mixed with the carbonate-bicarbonate buffer (24 μΙ).

b) Carbonate-bicarbonate buffer (0.1 M)

To Na₂C0₃ solution (0.2 M, 32 ml) was added NaHC0₃ solution (0.2 M, 68 ml) and the volume was adjusted to 200 ml using water to obtain a carbonate-bicarbonate buffer (0.1 M, pH 9.6) which was stored at 4°C.

c) Sodium phosphate buffer (PBS) (0.1 M)

To Na₂HP0₄ solution (0.2 M, 95 ml) was added NaH₂P0₄ solution (0.2 M, 450 ml) and the volume was adjusted to 1000 ml using water. Sodium chloride (9 g) was added to obtain the buffer. The buffer (pH 7.4) was stored at 4°C.

d) PBST wash buffer

PBS (1000 ml) was mixed with Tween-20 (500 μΙ) to obtain PBST wash buffer. e) Blocking buffer

1 g non-fat milk powder was mixed with PBS (100 ml) to obtain the blocking buffer.

f) Antigen presenting buffer

Trichloroacetic acid (TCA) solution was prepared by dissolving TCA (1000 g) in water (1000 ml). TCA solution (1 ml) was diluted with water (to 100 ml) to obtain antigen presenting buffer. The buffer was stored at RT.

g) Antibody dilution buffer

PBS was used as antibody dilution buffer,

h) Primary and secondary antibodies

The primary antibody used in the ELISA based assay was anti-rad specific monoclonal antibody (Catalogue number ARC03, Cytoskeleton Inc., USA). The primary antibody was reconstituted using antibody dilution buffer (antibody is supplied as lyophilized powder and therefore needed to be reconstituted) and stored at 4°C. The secondary antibody employed in the ELISA based assay was Goat anti-mouse IgG-HRP conjugate (Catalogue number sc-2302, Santa Cruz Biotechnology, Inc., USA) (200 μg in 0.5 ml). The secondary antibody was stored at 4°C.

i) HRP (Horse Radish Peroxidase) developing solution

To a solution A prepared by dissolving a Sigmafast OPD (O-phenylenediamine dihydrochloride) tablet set (Catalog number P9187, Sigma-Aldrich) in water (10 ml) was added solution B obtained by dissolving urea hydrogen peroxide tablet package in water (10 ml). Aliquots of 1 ml of the buffer were prepared and stored at -70°C.

j) HRP stop solution

HRP stop solution was obtained by adding concentrated sulphuric acid (5.4 ml) to cold water (70 ml) and diluted with water (to 100 ml). The solution was stored at 4°C.

B) Sample preparation

The rho GTPase inhibitor stock (10 mg/ml) was prepared by dissolving the known rad inhibitor, NSC-23766, in DMSO. A working stock A (100 ng/ml) of the inhibitor was prepared by diluting the stock 100 times in water.

The original stock of test compounds (Example 1 or Example 2; 10 mM) was prepared in DMSO. The working stock B (100 μΜ) of the test compound was prepared by diluting the original stock with water (in the ratio of 1 :100).

C) Assay protocol

The assay conditions for detecting rad interacting compounds using ELISA based assay, particularly, respective volumes of test compounds, known rad inhibitor, blank and the rad control used in the assay have been indicated below in Table 1 .

Table 1 : Condil ions for ELISA based assay rad protein Working stock A Working stock Carbonate- bicarbonate stock (μΙ) (Ul) B(ul) buffer (μΙ)

0 0 50

40 0 - 10

40 10 -

40 10 - - Final concentrations in the well: rad protein control is 8 ng; rad inhibitor, NSC- 23766 is 1000 ng; and test compound is 10 μΜ

Samples as given in Table 1 , was added directly to the 96-well maxisorp plates used in the assay. The carbonate-bicarbonate buffer devoid of the active rad protein was used as a blank. The plate was incubated on an orbital shaker at 300 rpm and at 4°C overnight (16 to 18 h). The wells were washed twice with PBST (200 μΙ) on an orbital shaker at 1350 rpm for 10 s. The wells were blocked by adding 1 % non fat milk (150 μΙ) and incubated at 37°C for 90 min. The wells were washed twice with PBST (200 μΙ) on an orbital shaker at 1350 rpm for 10 s. The wells were further treated with the antigen presenting buffer (200 μΙ) and incubated at RT for 2 min. The wells were then washed thrice with PBST on an orbital shaker at 1350 rpm for 10 s.

The wells were incubated with primary antibody (50 μΙ per well) (rad specific monoclonal antibody-antibody dilution buffer volume ratio: 1 :250) at RT on an orbital shaker at 300 rpm for 1 h. The wells were washed thrice with PBST (200 μΙ) on an orbital shaker at 1350 rpm for 10 s.

The wells were further incubated with secondary antibody (50 μΙ per well) (Goat anti-mouse IgG-HRP conjugate-antibody dilution buffer volume ratio 3:250) at RT on an orbital shaker at 300 rpm for 1 h. The wells were further washed thrice with PBST (200 μΙ) on an orbital shaker at 1350 rpm for 10 s.

The HRP developing solution (50 μΙ per well) was added to the wells which were incubated at 37°C for 10-15 min. The HRP stop solution (50 μΙ per well) was added to the wells and the absorbance was measured at 490 nm.

D) Results

20% change in the ELISA signal is considered to be a positive signal.

Accordingly, for the test compound Example 1 , a 24% increase in the rad ELISA signal intensity was observed as compared to that of the active rad protein control. For the test compound Example 2, a 22% increase in the rad ELISA signal intensity was observed as compared to the active rad protein control.

E) Conclusion

The results indicate that the test compounds interact with rad rho GTPase and are capable of treating cancer mediated through the rho GTPase, rad . Example 4

Cell proliferation assay

A) Cell Counting Kit-8 assay

Cell Counting Kit-8 (CCK-8) allows convenient assay by utilizing Dojiondo's highly water-soluble tetrazolium salt which produces a water-soluble formazan dye upon bioreduction in the presence of an electron carrier, 1 -Methoxy phenazium methyl sulphate. CCK-8 assay is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. The antiproliferative activity of the test compounds (compounds of Example 1 and Example 2) were measured according to the procedure described in Journal of Experimental and Clinical Cancer Research, 201 1 , 30(1 10), 1 -14, the disclosure of which is incorporated herein in its entirety.

B) Cell lines for the CCK-8 assay

Cell lines were procured from various sources (Table 2 and Table 3) and were maintained under optimum conditions of growth as suggested by the supplier. Fifteen cell lines were employed in the assay: glioblastoma (4), breast cancer (2), imatinib sensitive CML cell lines (3) and imatinib resistant CML cell lines (3), acute promyeloblastic cell line (1 ), normal breast epithelial cell line (1 ) and normal human fibroblast cell line (1 ). The cell lines were routinely passaged twice weekly. Each of these cell lines were grown at 37°C in a humidified atmosphere with 5% C0₂ in the medium mentioned herein below in Tables 2 and 3 respectively.

Table 2: Description of cancer cell lines

MCF7 Breast adeno NCCS DMEM + FBS (10%) +

carcinoma HEPES (10 mM)

2.

BT-549 Ductal carcinoma NCCS DMEM +FBS (10%) of the breast +HEPES (10 mM)

K562 Bcr-Abl Leukemia ACTREC RPMI + 10% FBS

(imatinib sensitive)

KCL22 Bcr-Abl Leukemia Dr. Nagai, RPMI + FBS (10%)

3. (imatinib sensitive) Japan

KU812 Bcr-Abl Leukemia Dr. Nagai, RPMI + 10% FBS

(sensitive to Japan

imatinib)

BAF₃ Bcr-Abl Leukemia Dr. Drucker, RPMI + 10% FBS

(imatinib resistant) USA

/bcr-abl

/T315I

4. KCL22 Bcr-Abl Leukemia Dr.T. Nagai, RPMI + FBS (10%) +

/SR cell line (imatinib Japan 10 μΜ imatinib

resistant)

KU812 Bcr-Abl Leukemia Dr.T. Nagai, RPMI + FBS (10%) +

/SR cell line (imatinib Japan 10 μΜ imatinib

resistant)

Bcr-abl negative RPMI + FBS (10%)

5. HL60 promyeloblast

Table 3: Description of normal cell lines

C) Sample preparation

The test compounds (Example 1 or Example 2; 40 mM stock) were prepared in

DMSO and subsequent dilutions to obtain 0.1 μΜ to 10 μΜ concentrations, were made using sterile deionized water. NSC-23766 was used as standard and DMSO was used as vehicle control.

D) Assay protocol

Cells were seeded at a density of 3X10³ to5X10³ (0.180 ml) per well in a 96 well tissue culture plate and allowed to incubate at 37^<Ό, 5% C0₂ incubator for 24 h. 20 μΙ of the test sample/ NSC-23766/ vehicle control were added to each well in triplicate. Plates were further incubated at 37^<Ό, 5% C0₂ incubator for 48 h. CCK-8 solution (10 μΙ) was added to each well and the plates were further incubated for 4 h. The absorbance was read at 450 nm using a microplate reader.

Anti-proliferative activity of the test compounds (Example 1 and Example 2) in various cancer cell lines and normal cell lines and the antiproliferative activity of the standard compound NSC-23766 expressed as IC₅o values (μΜ) or percent (%) inhibition were calculated in comparison with control values. The results are provided in Table 4 and Table 5 respectively.

Table 4: Antiproliferative activity in cancerous cell lines

ND: Not Determined

Table 5: Antiproliferative activity in normal cell lines

No. Description of the cell Cell line ICso (μΜ)/% inhibition at 10 μΜ

line Example 1 Example 2

1. Mammary epithelial MCF10A 24 % 5.99

non-tumorigenic

2. Human fibroblast WI-38 33 % 41 % E) Antiproliferative activity of test compounds (compounds of Example 1 and Example 2) in 3D MCS glioblastoma cells

The antiproliferative activity of the test compounds (Example 1 and Example 2) in 3D MCS glioblastoma cells (which is biochemically and physiologically representative of in vivo glioblastoma cells). The 3D MCS glioblastoma cells were cultured according to the procedure described in the reference Cancer Research, 1998, 58, 1305-1314.

The antiproliferative activity in the 3D MCS glioblastoma cell lines was measured according to the CCK-8 assay procedure and the conditions herein described above in Example 4D. The anti-proliferative activity of the test compounds (compounds of Example 1 and Example 2) in 3D MCS glioblastoma cell lines expressed as IC₅o values (μΜ) and percent (%) inhibition were calculated in comparison with control values. The results are provided in Table 6.

Table 6: Antiproliferative activity in 3D MCS glioblastoma cell lines

F) Conclusion

The compounds (as described herein) exhibited significant antiproliferative activity against glioblastoma cell lines, 3D MCS glioblastoma cell lines, breast cancer cell lines, imatinib sensitive CML cell lines and imatinib resistant CML cell lines.

Example 5

Cell migration assay

A) Wound scratch healing assay

This wound healing assay is a simple method which mimics cell migration during wound healing in vivo. This method is based on the observation that, upon creation of a new artificial gap, so called "scratch", on a confluent cell monolayer, the cells on the edge of the newly created gap will move toward the opening to close the "scratch" until new cell-cell contacts are established again. The basic steps involve creation of a "scratch" on monolayer cells, capture of images at the beginning and regular intervals during cell migration to close the scratch, and comparison of the images to determine the rate of cell migration (Nature Protocols, 2007, 2(2), 329-333).

The wound scratch healing assay was utilised to study the effect of the test compounds (compounds of Example 1 and Example 2) in glioblastoma cell lines and designed according to the procedure described in the reference, BMC Cancer, 201 1 , 1 1 , 388, the disclosure of which is incorporated herein in its entirety.

B) Assay protocol

The glioblastoma cell lines for the assay were maintained under same conditions as herein indicated above in Table 2.

The test compounds (compounds of Example 1 and Example 2; 40 mM stock) were prepared in DMSO and subsequent dilutions were made using sterile deionized water.

Cells were seeded at a density of 1 X10⁴ per well in a 96 well tissue culture plate and allowed to incubate at 37°C, 5% C0₂ incubator for 72 h, after that a smooth scratch was created in the monolayer formed across the well using a sterile tip. The test compounds (compounds of Example 1 and Example 2) at their respective IC₅₀ concentrations in monolayer glioblastoma cell lines (LN-229: compound of Example 1 (0.76 μΜ) and compound of Example 2 (2.02 μΜ); LN-18: compound of Example 1 (0.80 μΜ) and compound of Example 2 (4.12 μΜ); U87MG: compound of Example 1 (1 .88 μΜ) and compound of Example 2 (7.28 μΜ)) and the corresponding DMSO dilution (control) were added to each well and allowed to incubate at 37°C, 5% C0₂ incubator for 24 h. Images at the beginning and at the end of the 24 h incubation period were captured which signified the migration of the cells towards the scratch and the width of the gap created was measured. The inhibition of migration of the cells by the test compounds were measured by quantifying the width or the gap closed by migration of cells towards the scratch because of the test compounds and the width or gap closed with respect to the control. The results are presented in Table 7. Table 7: Inhibition of cell migration in glioblastoma cell lines

C) Conclusion

The compounds (as described herein) exhibited significant inhibition of migration of glioblastoma cell lines which is indicative of these compounds capable of inhibiting metastasis of cancer.

Example 6

Matrix metalloproteinases (MMP) secretion assay

The effect of the test compounds (compounds of Example 1 and Example 2) on

MMP secretion which contributes to metastasis of cancer was estimated by gelatinase zymography according to the procedure described in the reference Methods Mol. Med., 2001 , 57, 163-174 which is incorporated herein in its entirety. A) Assay protocol

The assay procedure was carried out in the glioblastoma cell line LN-229. The cell line was maintained according to the conditions prescribed in Table 2, herein indicated above.

Cells were seeded at a density of 3X10³ per well in a 96 well tissue culture plate and allowed to incubate at 37°C, 5% C0₂ incubator for 48 h. Cells were incubated with serum deprived medium containing the test compounds at their respective IC₅o concentrations in monolayer glioblastoma cell line LN-229 (compound of Example 1 (0.76 μΜ) and compound of Example 2 (2.02 μΜ)) and the corresponding DMSO dilution (control). After further incubation for 16 h, the conditioned medium was analysed by zymography. For zymography, conditioned medium were mixed with 4X sample buffer (Tris-HCI (1 M, 2.5 ml, pH-6.8), SDS (0.8 g), glycerol (4 ml), Bromophenol Blue (40 mg) in milli-Q water (10 ml)) in the volume ratio of 3:1 and loaded on PAGE containing gelatin. The gels were washed in a buffer containing mild detergent followed by incubation at 37°C overnight in calcium containing renaturation buffer (Tris-HCI (50 mM), CaCI₂ (10 mM), NaCI (150 mM), NaN₃ (0.05%) in milli-Q water (500 ml)) without detergent. The gel was then stained with Commassie brilliant blue in isopropanol (40%) and destained. Densitometry analysis was carried out to estimate the effect of the test compounds on MMP secretion in LN-229 cells. B) Results

The test compound of Example 1 exhibited 30% inhibition of MMP-9 secretion in glioblastoma cells (LN-229) and the test compound of Example 2 exhibited 50% inhibition of MMP-9 secretion in glioblastoma cells (LN-229). C) Conclusion

The compounds (as described herein) showed significant inhibition of MMP secretion in glioblastoma cancer cells which is indicative of its effectiveness in inhibition of cell invasion and metastasis of cancers. Example 7

In vivo studies

Animals were housed and cared in accordance with the guidelines in force prescribed by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), India. Procedures using laboratory animals were approved by the IAEC (Institutional Animal Ethics Committee) of Piramal Enterprises Limited (formerly known as Piramal Healthcare Limited), Goregaon, Mumbai, India.

In vivo efficacy studies of the present invention in glioblastoma xenograft model

In vivo efficacy of the compounds of formula (I) or formula (II) of the present invention was carried in glioblastoma (cell line: LN-229) xenograft tumor model, according to the procedure described in the reference Cancer Research, 2005, 65, 957-966.

A) Cell Lines

The cell lines used in this study, human glioblastoma cell lines (LN-229) were procured from NCCS and maintained as herein above indicated in Table 2. B) Sample preparation

Aliquots of the test compounds dissolved in DMSO were maintained at -20 °C in a freezer. Test samples (3 mg/ml) for animal injections were thawed fresh every day. C) Efficacy study in SCID mice

Severely Combined Immune Deficient (NOD-SCID strain), female mice (procured from ACTREC, Navi Mumbai, India) 6-8 weeks old, weighing 20 to 22 g were used.

LN-229 cells were grown in DMEM medium supplemented with FBS (10%) and HEPES (10 mM) in 5% C0₂ incubator at 37^<C. Cells were pelleted by centrifugation at 1000 rpm for 10 min. Cells were suspended in saline to get a count of 1 X10⁸ cells per ml. The cell suspension (0.1 ml) was injected by subcutaneous (s.c.) route in donor mice. When tumours of about 1 cm diameter were formed, the mice were sacrificed and tumours were removed. The tumour mass was cut into small pieces of about 2 mm X 2 mm size. These tumour pieces were implanted subcutaneously into experimental mice. Mice were observed for palpable tumour mass. Once the tumour size reached 4-5 mm in diameter, the animals were randomised and divided into respective treatment groups. The dosage of the test compounds (compounds of Example 1 and Example 2) and the control as indicated in Table 8, was administered for a period of 5 days.

Table 8: Treatment groups in the xenograft models

ql dx 5: single administration for 5 days, n: number of animals

Tumor growth in test compound treated animals was calculated as

T/C (Treated/Control) X100 %

and tumor growth inhibition (%) was calculated as [100 - T/C %].

Results are presented in Table 9. Table 9: In vivo tumor growth inhibition (TGI in xenograft tumor model)

D) Conclusion

The compounds (as described herein) showed significant tumor growth inhibition in LN-229 glioblastoma xenograft model. Data clearly demonstrates the in vivo efficacy of the test compounds in inhibiting glioblastoma LN-229 cells in xenograft tumor model.

Example 8

Fish Embryo Acute Toxicity Assay in Zebrafish model

The test compounds (compounds of Example 1 and Example 2) were tested for acute toxicity studies in compliance with Fish Embryo Toxicity (FET) test as laid down by the OECD guideline for testing of chemicals. A) Maintenance of Zebrafish embryos

A breeding stock of wild type zebrafish was used for egg production. The breeding stock of zebrafish was maintained in a temperature controlled room at 28.5 °C with a 14/10 h day/night light regime (i.e. the fish are exposed to light for 14 h and are maintained in the dark for 10 h). The zebrafish were randomly selected for mating and housed in breeding tanks. The male and female zebrafish in the breeding tanks were maintained in a ratio of 2:1 . After spawning, the embryos were collected from the breeding trap and washed thoroughly to eliminate any debris lying at the bottom of the tank. The zebrafish embryos obtained after spawning were staged and dispensed in distilled water and maintained in an incubator at 28.5±0.5 ^qC in dark.

B) Assay methodology

The fish embryo acute toxicity assay for the test compounds were carried out in accordance with the OECD guideline for testing chemicals, 1992, 1 -18, and the test procedure as substantially described in the reference ALTEX, 2002, 19(1 ), 38-48, the disclosure of which is incorporated herein in its entirety. The acute toxicity test was performed in a 96 well microtitre plate and was initiated immediately after fertilization. The zebrafish embryos obtained after spawning were transferred to 96 well plates with one embryo per well in the microtitre plate. The extended embryos test, wherein the semi-static technique which comprised of renewing the stock solution of the test compounds (Example 1 and Example 2) every 24 hours in the 96 well microtitre plate (containing the embryos) was employed. Varying concentrations (0.1 μΜ to 150 μΜ) of the test compounds were delivered to the 96 well microtitre plate. The stock solutions of the test compounds (Example 1 and Example 2) were prepared in DMSO, such that the final concentration of DMSO was 0.1 %. 12 embryos were exposed to five concentrations of the each of the test compounds (Example 1 at 0.1 , 1 , 5, 10, 25 and 50 μΜ and Example 2 at 0.1 , 0.5, 1 , 5 and 10 μΜ) at 6 hours post fertilization(hpf). A dilution-water control and a DMSO control were used for each plate. A negative control containing 0.1 % DMSO was also used for each plate. The plates were incubated at 28°C in an incubator. The lethal end point, i.e. the median lethal concentration (LC₅₀) which corresponds to the concentration of the test compound which resulted in 50% mortality of the embryos was measured using an Zeiss Axio Observer A.1 , inverted microscope at the end of 48 h of embryonic development. The apical end points considered in the assay included coagulations of eggs, irregular formation of somites, absence of tail detachment and lack of the heartbeat. The observance of any of these apical end points was considered to be indicative of the terminal toxic effect of the test compounds. The no effect observed concentration (NOEC) suggestive of the concentration at which the test compounds had no effect on the larval morphology, growth and development of the embryos was also measured.

C) Results

The median lethal concentration (LC₅₀) values determined in the assay for the test compound of Example 1 was found to be >150 μΜ and the LC₅o value for the test compound of Example 2 was found to be 12.06 μΜ. The NOEC concentration for the test compound of Example 1 was found to be 25 μΜ and for the test compound of Example 2 was found to be 10 μΜ.

D) Conclusion

The results of the acute toxicity test indicate that the test compounds are nontoxic at concentrations (LC₅o values) which are several folds above the antiproliferative or cytotoxic inhibitory activity (IC₅o) concentration of the test compounds. The NOEC values indicate that the test compounds are safe at concentrations which are several folds higher than the antiproliferative or cytotoxic inhibitory activity.

Example 9

Cardiotoxicity Assay in Zebrafish model

Cardiotoxicity assay of the test compounds were carried out in accordance with the test procedure substantially as described in the reference Circulation, 2003, 107, 1355-1358, the disclosure of which is incorporated herein in its entirety. A) Maintenance of Zebrafish embryos

The maintenance of Zebrafish embryos was carried out in exactly using the same conditions as described in Example 8.

B) Assay protocol

The cardiotoxicity assay was performed in a 12 well microplate. The stock solutions of the test compounds (Example 1 and Example 2) were prepared in DMSO. 20 embryos were suspended per well in an embryo medium (NaCI (13.6 mM), KCI (0.25 mM), Na₂HP0₄ (0.25 mM), KH₂P0₄ (0.44 mM), CaCI₂ (1 .29 mM), MgS0₄ (2 mM), NaHC0₃ (4 mM) in distilled water) supplemented with N-phenyl thiourea (0.0003%) at 24 hpf. Various concentrations of the test compounds (Example 1 : 50, 75, 100 and 150 μΜ; and Example 2: 1 and 10 μΜ) were added to the wells from the DMSO stocks (wherein the final concentration of DMSO is 2%) at 48 hpf for 3h. The distilled water and DMSO vehicle control were used as standards in the plate. The heart rate measurement of the embryos was carried out with a Carl Zeiss microscope and analysis was performed using Carl Zeiss Axiovision software.

C) Results

The concentration at which, the test compound Example 1 , exhibited cardio toxicity was found to be 150 μΜ and the test compound Example 2, exhibited cardiotoxicity was found to be 10 μΜ.

D) Conclusion

The results indicate that the test compounds are not associated with bradycardia (pathological slowing down of cardiac rhythm) or tachycardia (pathological fastening of cardiac rhythm) in concentrations which are several folds above the concentrations (IC₅o) at which the compounds exhibit antiproliferative or cytotoxic inhibitory activity. Example 10

Angiogenesis Assay

The angiogenesis assay for the test compounds (Example 1 and Example 2) were carried out in invivo zebrafish embryo model system in accordance with the procedure substantially as described in the reference Angiogenesis, 1999, 3, 353-359.

A) Maintenance of Zebrafish embryos

Zebrafish embryo generation was carried out in accordance with the procedure described in Example 8. After spawning, the Zebrafish embryos obtained were maintained in 12 well plates with 20 embryos per well in embryo medium supplemented with Phenyl-1 -thiourea (PTU) (0.003%).

B) Assay methodology

The stock solutions of the test compounds (Example 1 and Example 2) were prepared in DMSO (wherein the final concentration of DMSO was 0.1 %). Various concentrations of the test compounds were added to the embryos maintained in the individual wells of the 12 well microplate 24 hpf. The microplate was then incubated at 28°C until 72 hpf. The distilled water and DMSO were used as controls in the wells. The endothelial cells of the embryos were stained for endogenous alkaline phosphatase activity. The embryos were fixed in paraformaldehyde (4%) in PBST buffer and further treated with methanol (50%) and methanol (100%) for 5 min. The embryos were transferred to glass petriplates containing pre-cooled acetone. The plates were stored in a refrigerator at -20°C for 30 min and the larvae were transferred to 6 well microplate. The larvae were rinsed twice with PBST for 5 min. For staining, the embryos were equilibrated in NTMT buffer (Tris-HCI (0.1 M) (pH 9.5); MgCI₂ (50 mM); NaCI (0.1 M); Tween 20 (0.1 %)) for 15 min. The zebrafish was stained with NBT/BCIP solution for 30 min. The embryos were then examined for staining of sub- intestinal baskets using a Zeiss AxioObserver A.1 microscope. The sub-intestinal baskets were scored on the basis of length (extent from line of neuromast, distance in μΜ), shape of basket, spiky projections and presence of interconnecting vessels. Deferoxamine, an iron-chelating agent utilized to create hypoxic environment was used as a pro angiogenic control.

C) Results

The test compounds (Example 1 and Example 2) did not exhibit anti-angiogenic effect.

D) Conclusion

The results indicate that the test compounds (Example 1 and Example 2) are desirable for use in the treatment of malignancies of the nervous system, particularly glioblastoma.

Claims

We Claim :

A rho GTPase inhibitor selected from the compound of formula (I);

Formula (I)

or the compound of formula (II);

Formula (I I)

wherein,

Ri and R₂ are independently selected from (C₆-Ci₀)aryl and heteroaryl;

R₃ and R₄ are independently selected from hydrogen, (d-C₆)alkyl and (C₆-Ci₀)aryl; n is an integer from 0 to 3;

R₅ and R₆ are independently selected from (CrC₆)alkyl, (C₆-Ci₀)aryl and heteroaryl; wherein,

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, alkoxy, halo(C C₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano;

(C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, alkoxy, halo(Ci- C₆)alkoxy, hydroxy, (C₆-Ci₀) aryl, heterocyclyl, amino, cyano, C(0)alkyl, carboxy, C(0)NH₂ and C(0)Oalkyl;

or a stereoisomer or a tautomer or a pharmaceutically acceptable salt thereof; for use in the treatment of a cancer.

2. A rho GTPase inhibitor for the use according to claim 1 , wherein the rho GTPase inhibitor is selected from:

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

3. A rho GTPase inhibitor for the use according to claim 1 or claim 2, wherein the cancer is selected from: glioblastoma, breast cancer, chronic myeloid leukemia (CML), chronic myeloid leukemia (CML) resistant to treatment with imatinib, lung cancer, non- small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, colon cancer, renal cancer, ovarian cancer, testicular cancer or melanoma.

4. A method for the treatment of cancer in a subject, comprising administering to the subject in need thereof, a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I);

Formula (I)

or the compound of formula (II);

Formula (I I)

wherein,

Ri and R₂ are independently selected from (C₆-Ci₀)aryl and heteroaryl;

(CrC₆)alkyl is unsubstituted or substituted with one or more groups independently selected from (CrC₆)alkyl, halogen, halo(CrC₆)alkyl, alkoxy, halo(C C₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino and cyano; (C₆-Cio)aryl or heteroaryl is unsubstituted or substituted with one or more groups independently selected from (Ci-C₆)alkyl, halogen, halo(d-C₆)alkyl, alkoxy, halo(C C₆)alkoxy, hydroxy, (C₆-Ci₀)aryl, heterocyclyl, amino, cyano, C(0)alkyl, carboxy, C(0)NH₂ and C(0)Oalkyl;

or a stereoisomer or a tautomer or a pharmaceutically acceptable salt thereof.

5. The method according to claim 4, wherein the rho GTPase inhibitor is selected from:

N-((8-hydroxyquinolin-7-yl)(p-tolyl)methyl)acetamide;

or a stereoisomer or a tautomer, or a pharmaceutically acceptable salt thereof.

6. The method according to claim4, or claim 5, wherein the cancer is selected from glioblastoma, breast cancer, chronic myeloid leukemia (CML), chronic myeloid leukemia (CML) resistant to treatment with imatinib, lung cancer, non-small cell lung carcinoma, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, colon cancer, renal cancer, ovarian cancer, testicular cancer or melanoma.

7. A pharmaceutical composition comprising a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, or the compound of formula (II) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier, for use in the treatment of cancer.

8. A pharmaceutical composition comprising a therapeutically effective amount of a rho GTPase inhibitor selected from the compound of formula (I) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, or the compound of formula (II) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, and at least one further anticancer agent, together with a pharmaceutically acceptable excipient or carrier, for use in the treatment of cancer.

9. Use of a rho GTPase inhibitor selected from the compound of formula (I) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, or the compound of formula (II) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament in the treatment of cancer.

10. A method for screening compounds which are useful for treating cancers mediated by one or more rho GTPases, comprising the steps of: a) contacting a sample with a solid support having a rho GTPase protein link; b) detecting the interaction of the sample, with rho GTPase protein, using a primary antibody and an enzyme linked secondary antibody.

1 1 . The method according to claim 10, wherein the sample comprises a standard rho GTPase inhibitor or a test compound.

12. The method according to claim 10 or claim 1 1 , wherein the test compound may be selected from the compound of formula (I) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, or a compound of formula (II) as defined in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof.

13. The method according to claim 10, wherein the rho GTPase protein is selected from rhoA, rad or cdc42.

14. The method according to claim 10, wherein the solid support is a microtitre plate or an immunomodule.

15. The method according to claim 10, wherein the detection of interaction of sample with a rho GTPase protein is done using absorbance.

16. The method according to claim 10, wherein the primary antibody is selected from anti-rhoA, anti-rad or anti-cdc42 specific monoclonal antibody and the secondary antibody is Goat anti-mouse IgG-HRP (Immunoglobulin G-Horseradish peroxidase) conjugate.