F ield of Invention The present invention is directed to the use of a compound (BZD9L1 ) that inhibits sirtuin enzymes.

B ac kg round The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

S irtuins (S IRT) are NAD+ -dependent class ΙΠ histone deacetylases (HDACs) that share extensive homologies with the yeast silent information regulator 2 (S IR2). T he mammalian sirtuin family comprises seven proteins (S IRTT7) that possess NAD-dependent deacetylase, deacylase, and ADP-ribosyltransferase activities. S irtuins have different subcellular localization, clearly reZecting the distinctive features of each sirtuin member. S IRT1, S IRT6, and S IRT7 are mainly nuclear, S IRT2 is predominantly cytoplasmic, although nucleo- cytoplasmic shuttling has been reported, and S IRT3, S IRT4, and S IRT5 are largely localized to the mitochondria .

Mounting evidence has recently highlighted the importance of sirtuins in various biological processes and diseases. Their potential roles in cancer, metabolic and neurodegenerative diseases have stimulated investigation into their biological functions to seek potent and selective sirtuin inhibitors which could potentially lead to new therapeutic breakthrough. At present, a number of sirtuin inhibitors have been developed for different therapeutic purposes. S irtuin inhibitors such as S irtinol, Tenovin-6, S alermide and Cambinol, which targets S IRT1 and/or S IRT2, have been associated with antitumour activities in vitro and in vivo. However, sirtuin modulators usually vary in potency, depending on their chemical modifications, sirtuin expression and localization, cell-cycle phase and the complex roles of each sirtuin. Therefore the search for more potent and selective sirtuin modulators remains a significant area of pharmaceutical research. It is well established that cancer is an epigenetic as well as a genetic disease. Modifications in oncogenes or tumor suppressor genes are not only triggered by mutations, but they may also be due to post-translational modification that regulates the acetylation and deacetylation of histones and nonhistone proteins. S irtuins play a crucial role in the regulation of metabolism, differentiation, cell cycle progression, senescence and cell death. S IRT1 and S IRT2 have been demonstrated to play a role in carcinogenesis, both as positive drivers and also negative regulators of tumourigenesis, as their functions are frequently altered in cancer cells. Despite the controversy, we and others have found S IRT1 and S IRT2 to be implicated in tumorigenesis and drug resistance through deacetylation of numerous targets (Oon et al., unpublished). S IRT1 and S IRT2 are involved in tumourigenesis through deacetylation of numerous histone and non-histone proteins. Among the substrates for S IRTI are p53, nuclear factor-kappaB and Bax/Bcl-2, whereas S IRT2 is able to deacetylate p53, histone H4 and several cytoplasmic substrates, including -tubulin, which are implicated in growth regulation and tumourigenesis. Over-expression of S IRT1 and S IRT2 provides a tumour cell survival advantage and resistance to therapy by inhibiting apoptosis, resisting senescence and allowing unchecked cell division. S irtuin-2 (S IRT2), which acts as a G2 checkpoint mitotic regulator is crucial in cell cycle regulation, hence making it a potential target for cancer therapeutics. Down-regulation of S IRT2 has been shown to impede cell cycle progression and induce cell-cycle arrest, while overexpression has been reported to cause prolongation of the mitotic phase of the cell cycle leading to the inhibition of cell proliferation.

Anti-angiogenic agents have been described as being useful in research and therapy for angiogenesis-dependent diseases (1 ), such as inflammation (2), immunomodulation (3, 4), rheumatoid arthritis (5, 6), AIDS complications ^" Karposi S arcoma (7), retinal/blinding diseases (8), macular degeneration (9), skin diseases (10) (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy (1 1 ), Alzheimer (12), cerebral malaria (13), fibrosis (14), endometriosis(15), hemangioma (16), cancer, obesity and other metabolic diseases (17).

S ummary of Invention

The present invention is directed to the usefulness of compounds of formula l as anti-angiogenic agent and in particular inhibition of sirtuin enzymes using said compounds. Thus, in a first aspect of the current invention, there is provided a use of a compound of formula

I:

wherein:

R ¹ represents hydrogen, halogen, hydroxyl, carboxyl, C i- ₅ alkyl, imidazolyl, piperazinyl, morpholinyl, benzyl, R ⁴ OH or R ⁴ C OOH, where R ⁴ is (C l-h)™ and m is an integer of from 1 to 4;

R ² represents hydrogen, phenyl or 3-(2-oxopyrrolidin-1 -yl)propyl;

R ³ represents OR ⁵ or NR ⁶ R ⁷ ;

R ⁵ represents C i-6 alkyl; and

R ⁶ and R ⁷ are independently H, H2 (provided at least one is H), C 1-6 alkyl or, together with the N form a heterocyclic group having from 1 to 5 carbon atoms, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof,

in the manufacture of a medicament for the treatment of a condition or disorder ameliorated by the inhibition of angiogenesis.

P referably, the compound comprises a hydrogen at R ² and/or a ethyl group at R ⁵ . The invention also includes salts, solvates, prodrugs and metabolites of the compounds ethyl 2- phenyl-1 H-benzo[d]imidazole-5-carboxylate, ethyl 2-(4-bromophenyl)-1 H-benzo[d]imidazole-5- carboxylate and ethyl 2-(4-(piperidin-1 -yl)phenyl-1 H-benzo[d]imidazole-5-carboxylate (BZD9L1 ) as disclosed herein.

B ZD9L 1 The condition or disorder ameliorated by the inhibition angiogenesis may be selected from one or more of the group consisting of leukemia, AIDS complications (e.g. Karposi S arcoma), hemangioma, inflammation (e.g. including rheumatoid arthritis), immunomodulation, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases. For example, the condition or disorder ameliorated by the inhibition angiogenesis is selected from one or more of the group consisting of retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases.

Other aspects of the invention relate to:

(a) a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, for use in the treatment of a condition or disorder ameliorated by the inhibition of angiogenesis;

(b) a method of treatment of a condition or disorder ameliorated by the inhibition of angiogenesis, which method comprises the administration of an effective amount of a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, to a patient in need of such treatment; and

(c) a pharmaceutical formulation comprising a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. In said aspects of the invention, the compounds of formula I and conditions may be the same as the first aspect of the invention.

Drawing & B rief desc ription of d rawing The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawings of embodiments of the present invention, in which: F igure 1 provides data showing dose dependent effect of BZD9L1 on sprouting angiogenesis as depicted in the rat aortic ring assay (400x magnifications).

F igure 2 depicts data showing that the S IRT1 inhibitor EX527 increases C D34 vessel markers versus control groups.

F igure 3 depicts the effect of S irtuin inhibitors (IC50) on 3D spheroid sprouting.

F igure 4 is the synthetic scheme for making the compound (BZD9L1 ). Other compounds described herein may be made by analogy.

F igure 5 is the in vitro data showing BZD9L1 significantly reduced cell viability of EAHY 926 endothelial cells 72 hour post- treatment. F igure 6 is the data showing dose dependent effect of BZD9L1 on apoptosis and necrosis cell death 72 hour post- treatment.

F igure 7 is the data showing BZD9L1 (50 ι M) arrested EAHY 926 endothelial cells at G2/M phase 72 hour post-treatment.

F igure 8 A) depicts the effect of BZD9L1 on vessel outgrowth as depicted in the mouse choroid microvascular assay. B) is the data showing the effect of BZD9L1 on vessel outgrowth as depicted in the mouse choroid microvascular assay. F igure 9 A) depicts the effect of BZD9L1 on the ability to regress vessels. B) is the data showing BZD9L1 in increasing doses could possibility exhibit vessel regression abilities compared to control group.

Desc ription It has been surprisingly found that the compounds of formula I (e.g. BZD9L1 ) are useful in the treatment of conditions or disorders that are ameliorated by the inhibition of angiogenesis. In addition, the compounds of formula I may also be surprisingly useful in the treatment of cancer. Thus, according to a first aspect of the invention, there is provided a use of a compound of formula I:

wherein:

R ¹ represents hydrogen, halogen, hydroxyl, carboxyl, C i- ₅ alkyl, imidazolyl, piperazinyl, morpholinyl, benzyl, R ⁴ OH or R ⁴ C OOH, where R ⁴ is (C l-h)™ and m is an integer of from 1 to 4;

R ² represents hydrogen, phenyl or 3-(2-oxopyrrolidin-1 -yl)propyl;

R ³ represents OR ⁵ or NR ⁶ R ⁷ ;

R ⁵ represents C i-6 alkyl (e.g. ethyl); and

R ⁶ and R ⁷ are independently H, H2 (provided at least one is H), C 1-6 alkyl or, together with the N form a heterocyclic group having from 1 to 5 carbon atoms, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof,

in the manufacture of a medicament for the treatment of a condition or disorder ameliorated by the inhibition of angiogenesis.

R ² in certain embodiments may be hydrogen. References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds. P harmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. S uch salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). S alts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

E xamples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

E xamples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+)-camphoric, camphor-sulphonic, (+)-(1 S )-camphor- 10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2- disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L- glutamic), -oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (e)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (e)-DL- mandelic, meta phosphoric, methanesulphonic, 1 -hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino- salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, meta phosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. P referred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). E xamples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. S olvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TG E ), differential scanning calorimetry (DS C) and X-ray crystallography.

The solvates can be stoichiometric or non-stoic hiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., S olid-S tate C hemistry of Drugs, S econd E dition, published by S S CI, Inc of West Lafayette, IN, USA, 1999, IS BN 0-967-06710-3.

^' P harmaceutically functional derivatives , of compounds of formula l as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I. This term may also include metabolites of the compounds of formula I which retain pharmacological activity.

The term prodrug, of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

P rodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. P rodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

E xamples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. ^' Design of P rodrugs, p. 1-92, E lsevier, New York-Oxford (1985). Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the compounds of formula I_.

Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ichira I pool ^" method), by reaction of the appropriate starting material with a ichira I auxiliary ^" which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

Unless otherwise stated, the term "alkyl" refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term "alkyl" refers to an acyclic group, it is preferably CMO alkyl and, more preferably, Ci-e alkyl or, yet more particularly, Ci- ₅ alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term "alkyl" is a cyclic group (which may be where the group "cycloalkyl" is specified), it is preferably C3-12 cycloalkyl and, more preferably, C ₅ -io(e.g. C5-7) cycloalkyl.

When used herein, alkylene refers to CMO (e.g. C1-6) alkylene and, preferably C 1-3 a Ikylene, such as pentylene, butylene (branched or unbranched), preferably, propylene (n-propylene or isopropylene), ethylene or, more preferably, methylene (i.e. -CH2-). The term "halogen", when used herein, includes fluorine, chlorine, bromine and iodine.

The term "aryl" when used herein includes C 6-i ₄ (such as C 6-13 (e.g. C 6-10)) aryl groups. S uch groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-1 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Most preferred aryl groups include phenyl.

Compounds of formula I that may be mentioned include those of the examples described hereinafter. Particular compounds compounds of formula I that may be mentioned include:

(i) (ethyl 2-(4-(piperidin-1 -yl) phenyl)-1 H-benzo [d]imidazole-5-carboxylate);

(ii) ethyl 2-phenyl-1 H-benzo[d]imidazole-5-carboxylate;

(iii) ethyl 2-(4-bromophenyl)-1 H-benzo[d]imidazole-5-carboxylate;

or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof.

A particular compound of formula I that may be mentioned herein is the compound of formula la.

or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof.

The compound for use mentioned in the above-mentioned aspect of the invention may be utilised in a method of medical treatment. Thus, according to further aspects of the invention, there is provided: (i) a compound of formula I or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, for use in the treatment of a condition or disorder ameliorated by the inhibition of angiogenesis; and

(ii) a method of treatment of a disorder or condition ameliorated by the inhibition of angiogenesis, which method comprises the administration of an effective amount of a compound of formula I as defined hereinbefore, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, to a patient in need of such treatment.

The term ^' disorder or condition ameliorated by the inhibition of angiogenesis . will be understood by those skilled in the art to include: cancer, leukemia, AIDS complications (e.g. Karposi S arcoma) or more particularly hemangioma, or, yet more particularly, inflammation (e.g. including rheumatoid arthritis), or yet further more particularly, immunomodulation, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases.

Thus, further aspects of the invention relate to the following.

(a) A compound of formula I, as hereinbefore defined, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, for use in the treatment of a condition or disorder selected from hemangioma, or, more particularly, inflammation (e.g. including rheumatoid arthritis), or yet more particularly, immunomodulation, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases,

(b) Use of a compound of formula I, as hereinbefore defined, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof, for the preparation of a medicament for the treatment of a condition or disorder selected from hemangioma, or, more particularly, inflammation (e.g. including rheumatoid arthritis), immunomodulation, retinal/blinding diseases (e.g. macular degeneration), or yet more particularly, skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases. A method of treatment of a disorder or condition selected from hemangioma, or, more particularly, inflammation (e.g. including rheumatoid arthritis), or yet more particularly, immunomodulation, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases, which method comprises the administration of an effective amount of a compound of formula I, as hereinbefore defined, or a pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative thereof. Particular disorders that may be mentioned in relation to the aspects of the invention described hereinbefore include AIDS complications (e.g. Karposi S arcoma), hemangioma, inflammation (e.g. including rheumatoid arthritis), or more particularly, immunomodulation, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, Alzheimer ^' s disease, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases (e.g. rheumatoid arthritis, or more particularly, retinal/blinding diseases (e.g. macular degeneration), skin diseases (e.g. psoriasis, keloids, rosacea, dermatitis), retinopathy, cerebral malaria, fibrosis, ischemic chronic wounds, endometriosis, obesity and other metabolic diseases). Yet further particular disorders or conditions that may be mentioned in relation to the aspects of the invention described hereinbefore include rheumatoid arthritis, or more particularly, macular degeneration, psoriasis, ischemic chronic wounds and retinopathy.

For the avoidance of doubt, in the context of the present invention, the term ^' treatment, includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.

The terms patient, and patients, include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. T he term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

The term effective amount, refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

F urther embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.

The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term "isotopically labelled" includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.

The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include ² H, ³ H,

"C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ 0, ¹⁸ 0, ³⁵ S , ¹⁸ F, ³⁷ CI, ⁷⁷ Br, ⁸² Br and ¹²⁵ l).

When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non- radioactive isotope.

Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, trans mucosa lly (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. S uch pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. S uitable pharmaceutical formulations may be found in, for example, Remington The S cience and P ractice of Pharmacy, 19th ed., Mack Printing Company, E aston, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. S uitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, S cience (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the pa rticular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule may contain from 0.001 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer. A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may conta in from 0.001 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.001 mg to about 1000 mg per day of a compound of formula I.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic tha n, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise. E xamples

P reparation of ethyl 2-phenyl-1 H-benzo[d]imidazole-5-carboxylate

4-fluoro-3-nitrobenzoic acid (0.05g, 0.27 mmol) was esterified in the presence of catalytic sulfuric acid in ethanol (10mL) by refluxing at 65 eC for 6 hours. Ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 hour, treated with tin (Π) chloride (190mg, 1 mmol) and stirred for a further 0.5 hour. The resulting mixture was then treated with benzaldehyde (0.3 mmol) and sodium bisulfite (57 mg, 0.3 mmol) and left to stir for another 3 hour. The solution was cooled to room temperature and subsequently evaporated under reduced pressure. It was resuspended in ethyl acetate (10mL), washed with 10% sodium carbonate (20mL) and water (20mL x2), dried over sodium sulfate and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70- 230 mesh; C HC I ₃ -MeOH 9:1 ) to obtain the final products (70-90%). P reparation of ethyl 2-(4-bromophenyl)-1 H-benzo[d]imidazole-5-carboxylate

This compound is prepared using the method as described above for ethyl 2-phenyl-1 H- benzo[d]imidazole-5-carboxylate except bromobenzaldehyde is used instead of benzaldehyde. 4-fluoro-3-nitrobenzoic acid (0.05g, 0.27 mmol) was esterified in the presence of catalytic sulfuric acid in ethanol (10mL) by refluxing at 65 eC for 6 hours. Ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 hour, treated with tin (II) chloride (190mg, 1 mmol) and stirred for a further 0.5 hour. The resulting mixture was then treated with bromobenzaldehyde (0.3 mmol) and sodium bisulfite (57 mg, 0.3 mmol) and left to stir for another 3 hour. T he solution was cooled to room temperature and subsequently evaporated under reduced pressure. It was resuspended in ethyl acetate (10mL), washed with 10% sodium carbonate (20mL) and water (20mL x2), dried over sodium sulfate and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70-230 mesh; C HC I ₃ -MeOH 9:1 ) to obtain the final products (70- 90%). Preparation of ethyl 2-(4-(piperidin-1 -yl)phenyl)-1 H-benzo[d] imidazole-5-carboxylate (BZD9L 1 )

The synthetic scheme is depicted in F igure 4.

4-fluoro-3-nitrobenzoic acid (0.05 g, 0.27 mmol) was esterified in the presence of catalytic H2S O4 in E tOH (10 mL) by refluxing at 65 °C for 6 h, with the reaction monitored by thin layer chromatography (TLC). U pon completion of the reaction, ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 h, treated with S nC (190 mg, 1 mmol) and stirred for a further 0.5 h. The resulting mixture was then treated with 4-(1 - piperidinyl)benzaldehyde (0.3 mmol) and NaHS 0 ₃ (57 mg, 0.3 mmol) and left to stir for another 3 h. The solution was cooled to room temperature and subsequently evaporated under reduced pressure. The residue was resuspended in E tOAc (10 mL), washed with 10% a2C 0 ₃ (20 mL) and water (20 mL x 2), dried over a2S 0 ₄ and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70-230 mesh; C HC I ₃ -MeOH 9:1 ) to obtain the final product BZD9L1 (75% from starting material).

As can be appreciated by the skilled artisan, the above synthetic scheme is not intended to comprise a comprehensive list of all means by which the compound described and claimed in this application may be synthesized. F urther methods will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps described above may be performed in an alternate sequence or order to give the desired compounds.

E xample 1

BZD9L1 as sirtuin inhibitor

S IRT1 and S IRT2 activities were tested using the F luordeLys kit (B iomol International) according to standard manufacturer's protocol. F luorescence was measured at360nm(excitation)and460nm (emission) on a fluorometric reader(Infinite 200TE CAN) and the inhibition was expressed in arbitrary unit for that of S IRT1 and S IRT2 activity under each experimental condition. Resveratrol (S igma) was used as a positive control in addition to Nicotinamide (provided in the kit) as negative control. (Oon et al., 2015). Ref: Oon, C.E., Strell, C, Yeong, K.Y., Ostman, A., Prakash, J . SIRT1 inhibition in pancreatic cancer models; contrasting effects in vitro and in vivo. European Journal of Pharmacology (2015), vol (15)757:59-67 Results

Table 1 lists the half maximal inhibitory concentration (IC ₅ o) of BZD9L1 in inhibiting SIRT1 and SIRT2 activities compared to commercial sirtuin inhibitors. As shown in Table 1, the IC ₅ o of BZD9L1 for SIRT1 is better than most commercial sirtuin inhibitors (AGK2, Cambinol, Salermide, Sirtinol and Tenovin-6). This is also true for the inhibitory effect of BZD9L1 on SIRT2, which is better than all of the commercial sirtuin inhibitors it was compared with, except AGK2. As inhibitors of sirtuin enzymes have been reported to have anti-cancer properties, BZD9L1 is a prospective anti-cancer agent.

Table 1 E xample 2

The rat aortic ring assay was carried out according to Zhu et al., 2002. The thin prep rat aortic ring assay: a modified method for the characterization of angiogenesis in whole mounts Angiogenesis 2002; 5: 81 -6.

The rat aortic ring assay allows analysis of cellular proliferation, migration, tube formation and microvessel branching in vitro, while mimicking the real-life environment in which angiogenesis occurs. Rat aortic rings treated with BZD9L1 demonstrated inhibition of sprouting with increasing doses, suggesting that BZD9L1 is a potent a nti-angiogenic compound (F igure 1 ).

E xample 3

Animal work was carried out according to Oon et al., 2015. S ix- to seven-week-old male BALB/c S CID mice (Harlan S prague Dawley, Inc., Indiana) were injected subcutaneously with a 1 :1 ratio of a cell suspension containing 5x10 ⁶ PANC-1 cells and Matrigel (BD Biosciences).

E ach treatment group consisted of seven mice. The tumour growth was monitored two to three times per week my measuring the length (L), width (W) and height (H) of each tumour with a caliper. Tumour volumes were then calculated using the formula (V = 0.52xLxWxH). Treatment with PBS (control) or EX527 (10 mg/kg) were given intra peritonea lly every three days, starting from Day 7, when the tumours reached 50 mm ³ in size the mice were sacrificed.

Tumour sections were stained for C D34 vessel markers and the results are shown in F igure 2.

Ref: Oon, C.E ., S trell, C, Yeong, K.Y., Ostman, A., P rakash, J . S IRT 1 inhibition in pancreatic cancer models; contrasting effects in vitro and in vivo. E uropean J ournal of P harmacology (2015), vol (15)757:59-67

As shown in F igure 2, EX527 increased C D34 vessel number compared to the control P BS group in the pancreatic xenograft tumours (P<0.01 ). P rimary antibody omission was performed as negative control to demonstrate that the positive staining observed was not a result of high background staining. This shows that while EX527 is a S IRT 1 -selective inhibitor, it does not provide any a nti-angiogenic effects. The aortic ring assay (Zhu et al, 2002, Blacher et al., 2001 and the spheroid sprouting assay (Blacher et al., 2014) are two established assays that have been widely accepted as in vitro models to study angiogenesis. These two assays allow the key steps of angiogenesis to be studied as the cell aggregates/ tissues mimic the native tissue/ microenvironment more accurately compared to 2D culture.

Refs : Blacher S , Devy L, Burbridge MF, et al. Improved quantification of angiogenesis in the rat aortic ring assay. Angiogenesis 2001 ; 4: 133-42. Baker M, Robinson S D, Lechertier T, et al. Use of the mouse aortic ring assay to study angiogenesis. Nat P rotoc 201 1 ; 7: 89-104.

Blacher S , E rpicum C, Lenoir B, et al. Cell invasion in the spheroid sprouting assay: a spatial organisation a nalysis adaptable to cell behaviour. PLoS One;2014; 9: e97019.

E xample 4

EAHY926 endothelial spheroids were generated according to modified protocol from Blacher et al., 2014. F ull Matrigel (BD Biosciences) rather than collagen was used as the matrix for spheroid embedding.

Ref: Blacher S , E rpicum C, Lenoir B, et al. Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour. P LoS One;2014; 9: e97019. IC 50 of BZD9L1 successfully inhibited vessel sprouting compared to control untreated. AG K2 appeared to promote sprouting compared to control spheroids (F igure 3). The sprouting inhibitory ability of EX527 and Tenovin-6 is minimal compared to that of BZD9L1 (F igure 3).

E xample 5

BZD9L1 was examined for its effect on the growth of EAHY926 endothelial cells.

The extent of proliferation was determined by comparing EAHY926 endothelial cell counts for samples treated with different doses of BZD9L1 to that of untreated controls using CyQUANTs assay (LifeTechnologies). Cells were incubated with CyQUANT reagent according to the standard manufacturer's protocol. F luorescence intensities of samples were measured with a fluorescence microplate reader (Infinite 200TE CAN) using excitation at 485nm and fluorescence detection at 530nm. Results are depicted in F igure 5.

EAHY926 cells were treated with different doses of BDZ9L1 for 72hrs. Cell membranes were lysed with a hypotonic buffer (4mM S odium C itrate, 0.1 %T ritonX-100) containing 0.1 mM propidium iodide (PI), 2.5 ι g/ml Annexin V and 50 mg/ml R NaseA (LifeTechnologies) for 20 min at 4 °C under mild agitation. Aggregates were removed by filtration trough a 40 mm cell strainer. The total nuclei fluorescence F L2-A was measured under exclusion of debris/aggregates via the F L2-W versus F L2-A plot using the FACS Calibur (BDBiosciences). The data was analysed with the ModFIT Cell Cycle analysis software (Verity S oftwarehouse). Results are depicted in F igures 6 and 7. As shown in F igure 5, BDZ9L1 effectively impaired endothelial cell growth with increasing doses. The data depicted in F igures 6 and 7 demonstrate that the mechanism of action appears to be through the induction of apoptosis (F igure 6) and cell cycle arrest at G2/M phase compared to Control (F igure 7). Cell cycle arrest at G2/M phase has been associated with enhanced apoptosis in other studies and may improve the cytotoxicity of standard drugs.

E xample 6

Mouse choroid microvascular assay. C 57BL/6J mice were killed by cervical dislocation. The choroid explants from Post-natal day 3 C 57BL/6J mice were isolated through a very tedious process under a stereo microscope. Briefly, the cornea and lens were removed from the anterior of the eye followed by separation of the peripheral choroid-sclera complex from the retina. The choroid was then cut into 1 mm x 1 mm segments. The choroid explants were then incubated in reduced growth factor Matrigel (BD Biosciences). Vascular outgrowth including cells from the microenvironment from the choroidal tissues treated with DMS O vehicle control, 3 doses (low, medium and high) of BZD9L1 or negative control ALK1 inhibitor (Axon MedC hem) 3 days post-treatment were assessed through phase contrast microscope. The direct effect of BZD9L1 on the morphological changes of vessel sprouts was noted. Imaging and quantification of sprouting area was done using a phase contrast fluorescence microscope and TRI2 software. Results are depicted in F igure 8.

As shown in F igure 8, the mouse choroid microvascular assay depicts the effect of BZD9L1 on vessel outgrowth. BZD9L1 in increasing concentration effectively inhibited vessel formation and other neighbouring cells including pericytes, immune cells and fibroblast outgrowth from mouse choroid. At 50i M BZd9L1, tissue debris was observed as a result of toxicity from the high dose treatment. ALK1 was used as positive control. E xample 7

BZD9L1 was tested to investigate its ability to regress vessels which is often a useful approach in targeting vessels in tumours. C horoids were mounted and left to sprout in culture media for two days prior to treatment and then for another 24 hr with different doses BZD9L1, this time at a lower concentration of 5i M, 10i M and 20 ι M. ALK1 at 50nM was used as positive control. A trend was observed in which BZD9L1 in increasing doses could possibility exhibit vessel regression abilities compared to control group. Results are depicted in F igure 9A and 9B.

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