TECHNICAL FIELD

This invention relates, inter alia, to the use of certain compounds in the treatment of dengue fever or West Nile virus.

BACKGROUND

Dengue virus (DENV) is a mosquito-borne virus that causes significant disease worldwide. Endemic in more than 100 countries, DENV is estimated to cause 50 million infections each year. DENV infections can result in serious disease including dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and even death. Complicating matters further is the fact that DENV exists as four separate serotypes (DEN1V, DEN2V, DEN3V, and DEN4V) with infection by one serotype not providing protection from infections by the other serotypes.

According to the World Health Organization, DENV is considered to be the most important mosquito-borne viral disease in the world. Unfortunately, there are no vaccines approved to prevent DENV infection, and no approved antiviral drugs to treat the disease.

Every year, it is estimated that there are 50-100 million dengue virus infections with -1.5 million documented cases of dengue fever, and -500,000 cases of dengue hemorrhagic fever and shock syndrome. Reported cases increase annually. Approximately 40% of the world's population is at risk of dengue infection from living in regions endemic with the virus.

DENV is an enveloped, positive-strand RNA virus whose 1 1 kB genome is transcribed as a single polyprotein (See Tomlinson et al., 2009, Antiviral Res 82: 1 10-4) including the three structural (capsid, pre-m, and envelope) proteins at its 5' end followed by seven nonstructural proteins (Fields et al, 1996, Field's Virology, Third Edition, third ed. Lippincott Williams & Wilkins, Philadelphia). The N-terminal 180 residues of the NS3 protein encode the viral protease (Chambers et al, 1993, J Virol 67:6797-807) and -40 residues from the central hydrophilic domain of the NS2B protein (Yusof et al., 2000, J Biol Chem 275:9963- 9) encode the protease cofactor (Leung et al, 2001 , J Biol Chem 276:45762-71 ). Along with cellular proteases, the NS2B-NS3 protease complex (NS2B-NS3pro) is responsible for cleavage of the viral polyprotein (Cahour 992, J Virol 66: 1535-1542) and has been shown to be required for viral replication (Falgout et al., 1991 , J Virol. 65:2467-2475). As such, NS2B-NS3 protease provides a strategic target for inhibition in the development of dengue fever antivirals (Tomlinson et al., 2009, Infect Disord Drug Targets 9:327-43).

Flavivirus is a genus of the family Flaviviridae. This genus includes the West Nile virus and dengue virus as well as several other viruses that may cause encephalitis. Due to the similarities between dengue viruses and West Nile viruses, NS2B-NS3 protease also provides a strategic target for inhibition in the development of West Nile virus treatments.

West Nile virus (WN) belongs to the family Flaviviridae that comprises more than 60 viruses, many of which are important human pathogens. WN is a member of the Japanese encephalitis virus (JE) serocomplex of mosquito-borne flaviviruses that includes St. Louis encephalitis, JE, and Murray Valley encephalitis viruses (Calisher, CH. et al. 1989 J Gen Virol 70:27-43; Burke, D.S. & Monath, T . 2001 in: Fields Virology, eds. Knipe, D.M. & Howley, P.M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1 125). Like other members of the JE antigenic complex, WN is maintained in a natural cycle that involves mosquito vectors and birds, while humans and equines are usually incidental hosts. For many years WN has been recognized as one of the most widely distributed flaviviruses with a geographic range including Africa, Australia, Europe, the Middle East and West Asia (Burke, D.S. & Monath, T.P. 2001 in: Fields Virology, eds. Knipe, D.M. & Howley, P.M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1 125; Hayes, C.G. 1989 in: The Arboviruses: Epidemiology and Ecology, ed. Monath T.P. Boca Raton, FL CRC Press, Volume V, pp. 59-88).

There are no approved antiviral drugs for diseases caused by dengue or West Nile viruses. Currently, patients are treated with supportive care to relieve fever, pain, and dehydration. Therefore, there exists a need for additional vaccines or antiviral therapies to treat dengue or West Nile viruses.

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. SUMMARY OF INVENTION

We have now discovered, surprisingly, that certain thioguanine derivatives bearing certain substituents are useful as DENV2 NS2B-NS3 protease inhibitors.

A first aspect relates to a compound of formula (I):

R ¹ and R ⁴ each independently represent:

(a) H;

(b) Ci-12 alkyl, C ₂ .i ₂ alkenyl, C ₂ -i ₂ alkynyl, C ₃ -C ₅ cycloalkyl, C ₄ -C ₅ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci-4 alkyl, C _2-4 alkenyl, C ₂ _ ₄ alkynyl, C _3-5 cycloalkyl (which latter four groups are optionally substituted by one or more substituents selected from OH, =0, halo, C _1-4 alkyl and C _-4 alkoxy), phenyl, Het _a (which latter two groups are optionally substituted by one or more substituents selected from OH, halo, N0 ₂ , C(0)OR ^5a , Ci _-4 alkyl and C _1-4 alkoxy), OR ^5b , S(0) _n R ^5c , S(0) ₂ N(R ^5d )(R ^5e ), N(R ^5f )S(0) ₂ R ⁵⁹ , N(R ^5h )(R ⁵ⁱ ), and which C ₃ . ₁₅ cycloalkyl or Ci-15 cycloalkenyl groups may additionally be substituted by =0;

(d) phenyl, optionally substituted by one or more substituents selected from OH, halo, N0 ₂ , C(0)OR ^5a , d-4 alkyl and C _1-4 alkoxy;

(e) Hetb, optionally substituted by one or more substituents selected from OH, halo, N0 ₂ , C(0)0R ^5a , C _1-4 alkyl and C alkoxy; and

(f) S(0) _n R ^6a ,

R ² and R ³ each independently represent:

(a) H;

(b) S(0) _n R ^6b ; (c) C(0)R ^5k ; and

(d) C-i-12 alkyl, C ₂ .i ₂ alkenyl, C _2- 12 alkynyl, C ₃ -C ₁₅ cycloalkyl, C ₄ -C ₁₅ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci„4 alkyl, C _2-4 alkenyl, C ₂ . ₄ alkynyl, C ₃ . ₅ cycloalkyl (which latter four groups are optionally substituted by one or more substituents selected from OH, =0, halo, C _-4 alkyl and C _1-4 alkoxy), phenyl, Het _b (which later two groups are optionally substituted by one or more substituents selected from OH, halo, N0 ₂ , C(0)OR ^5a , Ci _-4 alkyl and C _1- alkoxy), OR ^5b , S(0) _n R ^5c , S(0) ₂ N(R ^5d )(R ^5e ), N(R ^5f )S(0) ₂ R ^¾ , N(R ^5h )(R ⁵ⁱ ), and which C ₃ -i ₅ cycloalkyl or Ci-15 cycloalkenyl groups may additionally be substituted by =0; or

R ² and R ³ , when taken together with the nitrogen atom to which they are attached, represent an -N=CH(R ⁷ ) group,

R ^6a and R ⁶ independently represent, at each occurrence, phenyl or Het _c , which two groups are optionally substituted by one or more substituents selected from OH, nitro, C(0)OR ^5j , CN, , halo, Ci-4 alkyl and C _1-4 alkoxy;

R ^5a to R ^5J and R ⁵ ' each independently represent, at each occurrence, H or C _1-4 alkyl, which latter group is optionally substituted by one or more substituents selected from halo, OH and NH ₂ ,

R ^5k independently represents, at each occurrence, H or C^ ₅ alkyl, which latter group is optionally substituted by one or more substituents selected from halo, OH and NH ₂ ,

R ⁷ represents phenyl which is optionally substituted by one or more substituents selected from OH, nitro, C(0)OR ⁵¹ , CN, halo, d_ ₄ alkyl and C _1-4 alkoxy; each independently represents a bond or C _1-4 alkyl; each Hetg to Het _d independently represents a 5- to 12-membered heterocyclic group containing from one to five heteroatoms selected from O, S and N; each n is independently, at each occurrence, 0, 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the treatment of dengue fever or West Nile virus. The first aspect of the invention is disclosed in Claim 1. Embodiments of this aspect are disclosed in Claims 2 to 7. A second aspect relates to a compound, as defined herein, for use in the manufacture of a medicament for the treatment of dengue fever or West Nile virus. The second aspect is disclosed in Claim 8.

A third aspect relates to a method of treating dengue fever comprising administration of a therapeutically effective amount of a compound, as defined herein, to a patient in need of such treatment. The third aspect of the invention is disclosed in Claim 9.

Another aspect relates to a compound of formula (II)

wherein:

R ^1' represents H, ethyl, /-propyl, cyclopentyl or benzyl;

R ⁴ independently represents:

(a) H;

(b) ethyl;

(c) /-propyl;

(d) cyclopentyl;

(e) benzyl;

(f) S(0) ₂ R ^6a' ;

R ² represents H or benzyl;

R ^3' represents:

(a) H;

(b) S(0) ₂ R ^6b' ;

(c) C(0)R ^5k' ;

(d) benzyl, or R ² and R ³ , when taken together with the nitrogen atom to which they are attached, represent an -N=CH(R ^7' ) group;

R ^5k' independently represents, at each occurrence, Ci _-4 alkyl;

R ^6a' represents a phenyl group optionally substituted by one or more substituents selected from C1- alkyl;

R ^6b' represents a phenyl group optionally substituted by one or more substituents selected from OH, nitro, halo, Ci _-4 alkyl and C1.4 alkoxy, or salts and solvates thereof; and

R ^7' independently represents phenyl which is optionally substituted by one or more substituents selected from OH, nitro, halo and C _1-4 alkoxy, provided that:

(a) one of R ¹ to R ^4' is not H;

(b) when R ^1' is H or benzyl, R ² is H and R ³ is H, then R ⁴ is not benzyl;

(c) when R ^r is H or CH ₃ , R ² (or R ³ ) is H and R ^4' is H, then R ³ is not C(0)R ^5f \

The fourth aspect is disclosed in Claim 10. Embodiments of this aspect are disclosed in Claims 1 1 to 13.

A fifth aspect of the invention relates to the use of the compounds of the fourth aspect of the invention for use in medicine. A sixth aspect of the invention relates to a pharmaceutical formulation including a compound of the fourth aspect of the invention in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

BRIEF DESCRIPTION OF FIGURES

Aspects and embodiments will be described with reference to the following figures, wherein:

Figure . illustrates inhibition assay plots a) to y) for example compounds (i) to (xxv). Figure 2. illustrates the 3D structure of example compound (vi) solved by X-Ray crystallography.

Figure 3. illustrates the 3D structure of example compound (xi) solved by X-Ray crystallography.

DETAILED DESCRIPTION

References herein (in any aspect or embodiment of the invention) to compounds of formula (I) and compounds of formula (II) 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.

Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such 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) or formula (II) 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). Salts may also be prepared by exchanging a counter-ion of a compound of formula (I) or formula (II) in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples 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.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene-2- sulfonic, naphthalene-1 ,5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L- aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1 ,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), ooxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L- malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic, 1-hydroxy-2- naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, 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, metaphosphoric, nitric and sulfuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulfonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula (I) and formula (II) are any solvates of the compounds and their salts. Preferred 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). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates 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 (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric 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., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

"Pharmaceutically functional derivatives" of compounds of formula (I) or formula (II) 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) or formula (II). The term "prodrug" of a relevant compound of formula (I) or formula (II) 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)).

Prodrugs of compounds of formula (I) or formula (II) 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. Prodrugs include compounds of formula (I) or formula (II) wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula (I) or formula (II) is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

Examples 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 Prodrugs" p. 1-92, Elsevier, New York-Oxford (1985).

Compounds of formula (I) and formula (II), 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) and formula (II)".

Compounds of formula (I) or formula (II) may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of formula (I) or formula (II) may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. For example, the following tautomers are included within the scope of the invention:

Compounds of formula (I) or formula (II) 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 crysiallisation. 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 'chiral pool' method), by reaction of the appropriate starting material with a 'chiral 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.

For the avoidance of doubt, compounds of formula (I) or formula (II) may contain the stated atoms in any of their isotopic forms. In this respect, embodiments of the invention that may be mentioned include those in which:

(a) the compound of formula (I) or formula (II) is not isotopically enriched or labelled with respect to any atoms of the compound; and

(b) the compound of formula (I) or formula (II) is isotopically enriched or labelled with respect to one or more atoms of the compound. METHOD OF TREATMENT

The compound of formula (I) or formula (II) 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) the use of a compound of formula (I) or formula (II) for the manufacture of a medicament for the treatment of dengue fever or West Nile virus; and

(ii) a method of treating dengue fever or West Nile virus comprising administration of a therapeutically effective amount of a compound of formula (I) or formula (II) to a patient in need of such treatment.

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

(a) A compound of formula (I) or formula (II), as hereinbefore defined, for use in the treatment of a condition or disorder selected from dengue fever or West Nile virus.

(b) Use of a compound of formula (() or formula (II), as hereinbefore defined, for the preparation of a medicament for the treatment of a condition or disorder selected from dengue fever or West Nile virus

(c) A method of treatment of a disorder or condition selected from dengue fever or West Nile virus, which method comprising administration of a therapeutically effective amount of a compound of formula (I) or formula (II) to a patient in need of such treatment.

DEFINITIONS

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.

Unless otherwise specified, 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).

Unless otherwise specified, the term "halo", when used herein, includes references to fluoro, chloro, bromo and iodo.

Unless otherwise specified, the term "alkyl" as used herein refers to a saturated straight chain or branched aliphatic group of 1-12 carbon atoms. Preferably, alkyl groups are Ci. C ₇ alkyl, particularly Ci_C ₄ alkyl. Examples of "alkyl" include, but are not limited to, methyl, ethyl, D-propyl, isopropyl, r?-butyl, /-butyl, sec-butyl, f-butyl, n-pentyl, neopentyl, n-hexyl, n- hepty!, cyclopropyl, especially n-butyl.

Unless otherwise specified, the term "alkoxy" as used herein refers to a CMO alkyl or alkenyl linked to an oxygen atom. Alkoxy is preferably C _1-7 alkoxy, more preferably C _1-4 alkoxy. Examples of alkoxy groups include, but are not limited to, groups such as methoxy, ethoxy, n-butoxy, ferf-butoxy, and allyloxy.

Unless otherwise specified, the term "cycloalkyi" as used herein refers to a saturated or partially saturated (non-aromatic) ring comprising preferably 3 to 15 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The "cycloalkyi" groups preferably contain from 4 to 6 ring carbon atoms.

The terms Het _a , Het _b , Het _c and Het _d as used herein refer to a heterocyclic group. The heterocyclic group may contain up to 5 heteroatom ring members selected from O, N and S, and more particularly up to 4 heteroatom ring members. For example, the heterocyclic group may contain 1 , 2 or 3 heteroatom ring members.

In one embodiment, Het _a , Het _b , Het _c and Het _d may each independently represent a monocyclic, bicyciic or tricyclic 5- to 14- or 3- to 10-membered heterocyclic group, respectively, containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S. Within this subset, Het _a , Het , Het _c or Het _d (where appropriate) may be selected, for example, from (i) monocyclic heterocyclic groups of 5 to 7 ring members containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S; (ii) 6.5 fused bicyciic heterocyclic groups of 9 ring members containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S; (iii) 6.6 fused bicyciic heterocyclic groups of 9 ring members containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S; (iv) 6.5.6 fused tricyclic heterocyclic groups of 13 ring members containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S; (v) 6.6.6 fused tricyclic heterocyclic groups of 14 ring members containing 1 , 2, 3 or 4 heteroatom ring members selected from O, N and S; and (vi) bridged bicyciic heterocyclic groups of 7 or 8 ring members containing 1 or 2 heteroatom ring members selected from O, N and S.

By "bridged ring systems" is meant ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4 ^th Edition, Wiley Interscience, pages 131- 33, 1992.

For example, each of Het _a , Het _b , Het _c and Het _d may be selected from the group comprising of azepinyl, diazepinyl, dihydrofuranyl (e.g. 2,3-dihydrofuranyl, 2,5-dyhdrofuranyl), 4,5- dihydro-1 A7-maleimido, dioxolanyl, furanyl, furazanyl, hydantoinyl, imidazolyl, isothiaziolyl, isoxazolidinyl, isoxazolyl, morpholinyl, oxadiazolyl, 1 ,2- or 1 ,3-oxazinanyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolidinonyl, pyrrolinyl, pyrrolyl, sulfolanyl, 3-sulfolenyl, tetrahydrofuranyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl, thiophenetyl, triazolyl, more particularly, dihydrbpyranyl (e.g. 3,4-dihydropyranyl, 3,6-dihydropyranyl), dioxanyl, hexahydropyrimidinyl, isobenzofuranyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, tetrahydropyranyl, 3,4,5,6- tetrahydropyridinyl, 1 ,2,3,4-tetrahydropyrimidinyl, 3,4,5,6-tetrahydropyrimidinyl, tetrahydrothiophenyl, tetramethylenesulfoxide, thiazolidinyl, triazinanyl and the like. The point of attachment of carbocyclic groups may be via any atom of the ring system. FORMULATIONS

Compounds of formula (I) or formula (II) may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermal^, 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) or formula (II) 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. Such 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. Suitable pharmaceutical formulations may be found in, for example, Remington, The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable 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, Science (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) or formula (II) 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 particular 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 0.0 to 99.99 % (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 contain from 0.0 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) or formula (II) 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 0.01 mg to about 1000 mg per day of a compound of formula (I) or formula (II).

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. BIOLOGICAL TESTS

Optimisation of the enzyme and substrate concentration.

The dengue protease activity assay was conducted by modifying the method of Yusof and co-workers (Yusof, R., Clum, S., Wetzel, M., Murthy, H. M. K. and Padmanabhan, R. (2000). Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence of cleavage of substrate with dibasic amino acids in vitro. J. Biol. Chem., 275, 9963-9969) and comprised using 200 mM Tris-HCI pH 8.5 as the assay buffer, DENV2 NS2B-NS3pro as the protease and Boc-GRR-MCA as the substrate. The protease optimisation assay was executed to ascertain the maximum protease activity at a constant 5 μΜ substrate concentration. The protease concentrations were varied within the range of 0-10 μΜ. The next process including pre-incubation as well as fluorescence intensity measurements was done by using the above mentioned Yusof procedure.

The substrate optimisation assay was determined using a constant 3 μΜ protease at varied substrate concentration (0-50 μΜ). The 200 pL reaction assays contained assay buffer, and constant 3μΜ protease. The same procedure of pre-incubation was applied. Subsequently, the 7-amino-4-methylcoumarin (AMC) produced were measured as fluorescence intensity at λ excitation 340 nm and λ emission 440 nm by using enzyme-linked immunosorbent assay (ELISA) modulus micro plate reader.

Dengue protease inhibition assay.

The procedure of Dengue Protease Inhibition Assay was carried out by preparing the reaction mixture containing assay buffer, test samples (a varied concentration of from 0 to 800 pg/mL), and constant 3μΜ proteases were pre-incubated at 37°C for 10 minutes with centrifugation at 200 rpm. Then after the addition of 25 μΜ substrate, the reaction assays were incubated at 37°C for 60 minutes with centrifugation at 200 rpm. The assays were quadruplicated. The fluorescence intensities of 7-amino-4-methylcoumarin (AMC) product were measured by Modulus Microplate Reader with UV optical kit. Examples

General Procedures

Compounds of formula (I) may be known and/or may be commercially available. Other compounds of formula (I) (e.g. that are not commercially available) may be prepared in accordance with techniques that are well known to those skilled in the art, for example as described hereinafter.

Compounds of formula (I) include example compounds (i) to (xxv) presented below:

(i) 2-amino-9H-purine-6-thiol;

(ii) 9-ethyl-6-ethylsulfanyl-purin-2-amine;

(iii) 9-isopropyl-6-isopropylsulfanyl-purin-2-amine;

(iv) 9-[(2S)-2-methylhexyl]-6-[(2S)-2-methylhexyl]sulfanyl-purin- 2-amine/9-[(2R)-2- methylhexyl]-6-[(2R)-2-methylhexyl]sulfanyl-purin-2-amine;

(v) 9-[(2R)-2-methylhexyl]-6-[(2S)-2-methylhexyl]sulfanyl-purin- 2-amine/9-[(2S)-2- methylhexyl]-6-[(2R)-2-methylhexyl]sulfanyl-purin-2 -amine;

( i) 6-cyc(opentylsuifany(-9H-purin-2-amine;

(vii) 9-cyclopentyl-6-cyclopentylsulfanyl-purin-2-amine;

(viii) methyl 2-[2-amino-9-(2-methoxy-2-oxo-ethyl)purin-6-yl]sulfanylaceta te;

(ix) 6-(2-pyridylmethylsulfanyl)-9H-purin-2-amine;

(x) 6-benzylsulfanyl-9H-purin-2 -amine;

(xi) 9-benzyl-6-benzylsulfanyl-purin-2-amine;

(xii) N,9-dibenzyl-6-benzylsulfanyl-purin-2-amine;

(xiii) N,N,9-tribenzyl-6-benzylsulfanyl-purin-2-amine;

(xiv) 2-[(4-fluorophenyl)methyleneamino]-9H-purine-6-thiol;

(xv) 2-methoxy-4-[(E)-(6-sulfanyl-9H-purin-2-yl)iminomethyl]pheno l;

(xvi) 5-[(E)-(6-sulfanyi-9H-purin-2-yl)iminomethy(]benzene-1 ,3-dioi;

(xvii) 2-[(4-nitrophenyl)methyleneamino]-9H-purine-6-thiol;

(xviii) 4-[(E)-(6-sulfanyl-9H-purin-2-yl)iminomethyl]benzoic acid;

(xix) 2-methoxy-6-nitro-4-[(E)-(6-sulfanyl-9H-purin-2-yl)iminometh yl]phenol;

(xx) 3-methyl-N-[6-(m-tolylsulfonylsulfanyi)-9H-purin-2yl]benzene sulfonamide;

(xxi) 3-methoxy-N-(6-sulfanyl-9H-purin-2-yl)benzenesulfonamide;

(xxii) 4-methoxy-N-(6-sulfany!-9H-purin-2-yl)benzenesulfonamide;

(xxiii) N-(6-sulfanyl-9H-purin-2-yl)acetamide; (xxiv) N-(9-cyclopentyl-6-sulfanyl-purin-2-yl)acetamide;

(xxv) N-(6-sulfanyl-9H-purin-2-yl)hexadecanamide.

Preparation 1

General procedure for the preparation of example compounds (ii) - (xiii)

A mixture containing the corresponding alkyl bromide (1.315 mmol for monoalkylation; 2.630 mmol for dialkylation; 3.945 mmol for triple/quadruple alkylation), tetrabutylammonium iodide (TBAI) (present in an equal to the number of alkylations required) in 3.5 mL of dimethylformamide (DMF) was freshly prepared. In a separate flask, 6-thiouguanine (0.598 mmol) was mixed with caesium carbonate (Cs ₂ C0 ₃ ) (present in an equal to the number of aikyfations required) in 3.5 mL of dimethylformamide (DMF) and then stirred vigorously for 15 minutes. The first mixture was added into the second mixture and the stirring was continued at room temperature for six hours. The reaction progress was monitored by TLC using solvent system of /?-hexane:ethyl acetate (EtOAc) (1 :3). After the product formed, the reaction mixture was diluted with 70 mL of water and then extracted using 3 x 70 mL of EtOAc. The organic phase was collected, washed with 3 x 70 mL of water and then dried over anhydrous magnesium sulfate. The resultant organic phase was then evaporated in vacuo and then purified using PLC with the same solvent system used in the monitoring of the reaction progress.

Similar procedures can be found in in Salvatore, R. N., Smith, R. A., Nischwitz, A. K. and Gavin, T. (2005). A mild and highly convenient chemoselective alkylation of thiols using Cs ₂ C0 ₃ -TBAI. Tetrahedron Lett., 46, 8931 -8935 which can also be used for the preparation of the compounds of formula (I).

Example compound (ii): 9-ethyl-6-ethylsulfanyl-purin-2-amine.

Colorless semi solid; Yield 19%; δ _Η (CDCI ₃ ) 1.41 (3H, t, J = 7.5 Hz, SR-CH ₃ ), 1.48 (3H, t, J = 7.5 Hz, WR-CH ₃ ), 3.31 (2H, q, J = 7.5 Hz, A/-CH ₂ -R), 4.1 1 (2H, q, J = 7.5 Hz, S-CH _Z -R), 4.87 (2H, br, s, NH ₂ ), 7.65 (1 H, s, HC=); 5 _C -i ₃ 15.55 (SCH ₃ ), 15.61 (A/CH ₃ ), 22.37 (SCH ₂ ), 38.39 (NC ₂ ), 124.69 (C=N), 140.70 (HC=N), 151 .18 (NC=C), 159.77 (NC=N), 159.96 (SC=N).

Example compound (iii): 9-isopropyi-6-isopropylsulfanyl-purin-2-amine.

Colorless semi solid; Yield 26%; δ _Η 1.45 (CDCI ₃ ) (6H, d, J = 6.5Hz, SR-(CH ₃ ) ₂ ), 1.55 (6H, d, J = 7 Hz, A/R-(CH ₃ ) ₂ ), 4.28 (1 H, m, J = 7 Hz, /V-CH-R), 4.67 (1 H, m, = 7 Hz, S-CH-R), 4.83 (2H, br, s, NH ₂ ), 7.69 (1 H, s, CH=); 5 _C -i ₃ 22.55 (SCH ₃ ), 23.34 (A/CH ₃ ), 30.93 (SCH), 34.29 (Λ/CH), 46.55 (C=N), 125.93 (C=N), 137.78 (C=C), 158.30 (C=N), 167.32 (C=N).

Example compound (iv): 9-[(2S)-2-methylhexyl]-6-[(2S)-2-methylhexyl]sulfanyl-purin- 2-amine / 9-[(2R)-2-methylhexyl]-6-[(2R)-2-methylhexyl]sulfanyl-purin- 2-amine (racemic mixture).

Colorless semi solid; Yield 31 %; δ _Η (CDCI ₃ ) 0.90 (24H, m, A R-|R ₂ R3-C4H ₁₂ ), 1.30 (24H, m, SR^ a-CsH^), 1.45 (4H, m, /V ^-CHz^ s, SR ₁ R ₂ -CH ₂ -R ₄ R ₅ ), 1.70 (2H, m, NR^CH- R2-R4R5), 1 .90 (2H, m, SRiCH-R ₂ -R ₄ R ₅ ), 3.34 (4H, m, NC ₂ - R2R3R4), 3.93 (4H, d, SCH ₂ - R ₂ R ₃ R ₄ ), 4.81 (4H, br, s, NH ₂ ), 7.58 (2H, s, CH=). 5 _C 10.45 (A/R ₁ R2R3-(CH ₂ ) ₃ CH ₃ ), 11.05 (SR ₁ R ₂ R ₃ -(CH2)3CH ₃ 14.01 (A/R^Rs-CC^jaC^CHs), 14.1 1 (SR ₁ R2R3-(CH2) ₃ CH ₃ ) _i 22.92 (A/R-i R2R ₃ -CH ₂ CH ₂ C ₂ H ₅ ), 22.96 25.83 28.42 (AfR ₁ R ₂ CH ₂ CH ₃ -R ₄ ), 28.97 (SR ₁ R ₂ CH ₂ CH3-R ₄ ), 30.29 (A/R ₁ R ₂ CH ₂ CH ₃ -R ₄ ), 30.92 (SR ₁ R ₂ CH ₂ CH ₃ -R ₄ ), 32.29 (A/R ₁ CHR ₃ -R ₄ ), 32.61 (SRiCHR ₃ -R4), 39.36 ( yCH ₂ R ₂ R ₃ -R4), 39.56 (SCH ₂ R ₂ R3-R4), 125.75 (C=C), 140.29 (C=N), 150.73 (NC=C), 158.77 (C-NH ₂ ), 161.96 (C=CSH).

Example compound (v): 9-[(2R)-2-methylhexyl]-6-[(2S)-2-methylhexyl]sulfanyl-purin- 2-amine / 9-f(2S)-2-methylhexyl]-6-[(2R)-2-met ylhexyl]sulfanyl-purin-2-amine (racemic mixture).

White powder, Yield 12%, m.p. ; o _max / cm ^"1 (KBr) 3305, 3179 (NH ₂ ), 2533 (CN), 1556 (C=N); δ _Η (CDCIs) 0.91 (24H, m, A/R ₁ R ₂ R ₃ -C ₅ H ₁₂ ), 1 .29 (24H, m, SRiR ₂ R ₃ -C ₅ H ₁₂ ), 1.44 (4H, m, /VR ₁ R2-CH ₂ -R ₄ R _5, SR ₁ R ₂ -CH ₂ -R ₄ R ₅ ), 1.68 (2H, m, A/R ₁ CH-R ₂ -R ₄ R ₅ ), 1.95 (2H, m, SR ₁ CH- R ₂ -R ₄ R ₅ ), 3.34 (4H, m, A/CH ₂ - R ₂ R ₃ R ₄ ), 4.15 (4H, m, SCH ₂ - R ₂ R ₃ R ₄ ), 4.83 (4H, br, s, NH ₂ ), 7.73 (2H, s, CH=).

Example compound (vi): 6-cyclopentylsulfanyl-9H-purin-2-amine. White powder, Yield 62%, m.p. 160-162 ^° C ; o _max / cm ^"1 (KBr) 3300, 3220 (NH ₂ ), 2853 (CN), 1565 (C=N); δ _Η (CDCI ₃ ) 1.69 (4H, m, C ₂ H ₄ ), 1.81 (2H, m, CH ₂ ), 2.02 (2H, m, CH ₂ ), 4.32 (1 H, m, CH), 4.92 (2H, br, s, NH ₂ ), 7.78 (1 H, s, HC=); 5 _C 24.89 (CH ₂ ), 33.64 (CH ₂ ), 42.01 (CH), 124.53 (), 137.52 (C=N), 150.84 (C=C), 158.92 (C=N), 162.99 (C=N).

Example compound (vii): 9-cyclopentyl-6-cyclopentylsulfanyl-purin-2-amine.

White powder, Yield 72%, m.p. 129-131 ^° C ; v _m cm ^"1 (KBr) 3334, 3215 (NH ₂ ), 2868 (CN), 1560 (C=N); δ _Η (CDCI ₃ ) 1.70 (4H, m, A/R^-C^Rs), 1.78 (4H, m, SR^-C^Rs), 1.92 (4H, m, ₁ -C ₂ H ₄ -R ₃ R4), 2.23 (4H, m, SR C ₂ H ₄ -R ₃ R ₄ ), 4.30 ( H, m, NCH- R ₂ R ₃ R ₄ R ₅ ), 4.79 (1 H, m, SCH- R ₂ R ₃ R ₄ R5), 4.83 (2H, s, NH ₂ ), 7.67 (1 H, s, HC=). 6 _C 23.87 (A/CH- CH(CH ₂ ) ₂ (CH ₂ ) ₂ ), 24.89 (SCH-CH(CH ₂ ) ₂ (CH ₂ ) ₂ ), 32.64 (A/CH~CH(CH ₂ ) ₂ (CH ₂ ) ₂ ), 33.67 (SCH-CH(CH ₂ ) ₂ (CH ₂ ) ₂ ), 41.82 (A/CH-CHC ₄ H ₈ ), 55.35 (SCH-CHC ₄ H _S ), 126.01 (C=C), 138.03 (C=N), 150.49 (NC=C), 158.65 (C-NH ₂ ), 162.46 (C=CS).

Example compound (viii): methyl 2-[2-aminc~9-(2-methoxy-2-oxo-ethyl)purin-6- yljsulfanylacetate.

White powder, Yield 58%, m.p. 139-141 ^° C; v _m cm ^"1 (KBr) 3416, 3334 (NH ₂ ), 2917 (CN), 1556 (C=N); δ _Η (CDCI ₃ ) 3.75 (3H, s, A/R^COOCHa), 3.79 (3H, s, SR^OOC^), 4.09 (2H, s _f A/CH ₂ COOR ₂ ), 4.82 (2H, s, SCH ₂ COOR ₂ ), 4.89 (2H, br, s, NH ₂ ), 7.69 (1 H, s, CH=). 6 _C 30.74 (A CH ₂ ), 43.72 ( CH ₂ ), 52.74 (A/CH ₂ COCH ₃ ) _; 52.97 (SCH ₂ COCH ₃ ), 124.92 (C=C), 140.37 (C=N), 151.02 (NC=C), 158.92 (C-NH ₂ ), 159.57 (C=CS), 167.58 (A/CH ₂ COCH ₃ ), 169.74 (SCHzCOCHs).

Example compound (ix): 6-(2-pyridylmethylsulfanyl)-9H-purin-2-amine.

White powder, Yield 53%, m.p. >250 ^° C; o _max / cm ^"1 (KBr) 3318, 3199 (NH ₂ , NH), 2978 (CN), 1548 (C=N); δ _Η (DMSO-D6) 4.65 (2H, s, CH ₂ ), 6.46 (2H, s, NH ₂ ), 7.26 (1H, s, HC=), 7.60 (1 H, s, HC=), 7.73 (1 H, s, HC=), 7.91 (1 H, s, HC=), 8.51 (1 H, s, HC=), 12.57 (1 H, s, NH). δ ₀ 33.60 (CH ₂ ), 122.71 (HC=), 123.94 (HC=), 137.22 (HC=), 137.62 (HC=N), 139.37 (HC=), 141.91 (C=C), 149.63 (C=N), 152.34 (NC=C), 158.34 (C-NH ₂ ), 160.02 (C=CS)

Example compound (x): N,9-dibenzyl-6-benzylsulfanyl-purin-2-amine.

White powder, yield 74%, m.p. 193-195 ^° C; u _max / cm ^"1 (KBr) 3432, 3240 (NH ₂ , NH), 2913 (CN), 1556 (C=N); δ _Η (DMSO-D6) 5.44 (2H, s, CH ₂ ), 5.67 (2H, s, NH ₂ )' 7.33 (6H, m, HC=), 9.65 (1H, s, NH); 6 _C (D SO-D6) 32.76 (CH ₂ ), 114.03 (HC=), 134.16 (HC=), 34.56 (HC=), 137.47 (C=N), 139.76 (C=C), 141.27 (C=N), 149.99 (C=C), 157.56 (C=N), 159.79 (C=N). Example compound (xi): 9-benzyl-6-benzylsulfanyl-purin-2-amine.

Needles crystal from methanol, yield 68%, m.p. 155-157Ό; o _ma x cm ^'1 (KBr) 3301 , 3187 (NH ₂ ), 3081 (CN), 1556 (C=N); δ _Η (CDCI ₃ ) 4.50 (2H, s, NH ₂ ), 4.85 (2H, s, CH ₂ )' 5.16 (2H, s, CH ₂ ), 7 (10H, m, HC=), 7.52 (1 H, s, HC=); 6 _C 32.62 (SCH ₂ ), 46.69 (A/CH ₂ ), 127.16 (HC=), 127.59 (HC=), 128.29 (HC=), 128.47 (HC=), 129.01 (HC=), 129.10 (HC=), 135.63 (C=C), 137.78 (C=C), 139.96 (C=N), 150.84 (C=C), 158.98 (C=N), 161.69 (C=N).

Example compound (xii): N,9-dibenzyl-6-benzylsulfanyl-purin-2-amine.

White powder, yield 25%, m.p. 132-134 ^° C; o _max / cm ^"1 (KBr) 3399 (NH), 3027 (CN), 1540 (C=N); δ _Η (CDCI ₃ ) 4.45 (2H, s, CH ₂ ), 4.63 (2H, s, CH ₂ )' 5.14 (2H, s, CH ₂ ), 5.34 (1 H, s, NH), 7.25 (15H, m, HC=), 7.51 (1 H, s, HC=); 5 _C 32.50 (SCH ₂ ), 46.05 (A HCH ₂ ), 46.78 (A/CH ₂ ), 127.49 (HC=), 127.82 (HC=), 128.21 (HC=), 128.44 (HC=), 128.47 (HC=), 128.52 (HC=), 128.54 (HC=), 128.57 (HC=), 128.94 (HC=), 129.00 (C=C), 135.87 (C=C), 137.93 (C=C), 139.46 (C=C), 150.96 (C=N), 158.76 (C=C), 160.66 (C=N), 165.02 (C=N).

Example compound (xiii): N,N,9-tribenzyl-6-benzylsulfanyl-purin-2-amine.

Brown liquid, yield 70%; δ _Η (CDCI ₃ ) 4.45 (8H, s, CH ₂ ), 7.21 -7.38 (21 H, m, CH=); 5 _C 33.59 (SCH ₂ ), 65.41 (N, N-CHz), 72.13 (/VCH ₂ ), 127.59 (HC=), 127.80 (HC≡), 127.90 (HC=), 128. 42 (HC=), 128.74 (HC=), 128.84 (HC=), 129.77 (C=C), 129.94 (C=C), 131.67 (C=C), ), 133.28 (C=C), 134.48 (C=N), 137.79 (C=C), 138.28 (C=N), 139.29 (C=N).

Preparation 2

General procedure for the preparation of example compounds (xiv) - (xix)

The general procedure for the preparation of example compounds (xiv) - (xix) was modified from the Schiff base reaction published by Pannerselavam and co-workers (Panneerselvam, P., Nair, R. R., Vijayalakshmi, G., Subramaniam, E. H. and Sridhar, S. K. (2005). Synthesis of schiff bases of 4-(4-aminopheny()-morpholine as potential antimicrobial agents. Eur. J. Med. Chem., 40, 225-229).

6-thioguanine (0.598 mmol) was mixed with one molar equivalent of the corresponding aromatic aldehyde and stirred for up to 15 minutes. A liquid mixture of ethanol-NaOH 10% (1.5 mL (1 :1 )) was added to the first mixture and stirred until a yellowish precipitate formed. A second volume of ethanol-NaOH 10% was then added and the stirring was continued until the product was completely formed (monitored by TLC with CHCI ₃ - Methanol (2:2) as a solvent). The mixture was neutralized using 6M HCI and the solid product was collected by filtration. The product was then washed with water, ethyl acetate and then recrystallized from a hot methanol to afford the pure product.

Example compound (xiv): 2-[(4-fluorophenyl)methyleneamino]-9H-purine-6-thiol.

Yellow crystal from methanol-ethyl acetate (2:2), yield 75%, m.p. 149-151 ^° C; D _max / cm ^"1 (KBr) 3399 (NH), 3027 (CN), 1540 (C=N); δ _Η (DMSO-D ₆ ) 7.32 (2H, dd, J _orth0 = 8.5 Hz, J _meta = 4Hz, HC=), 7.34 (1 H, s, HC= imidazole), 7.78 (1 H, s, N=CH imine), 7.87 (2H, m, HC=); 5c (CDCI ₃ ) 116.09 (C=C), 116.27 (C=N imidazole), 125.8 (HC=), 130.27 (C=C), 130.33 (=C-N), 131.00 (HC=), 142.13 (NC=CN), 163.08 (N=CH imine), 165.08 (=CH), 188.50 (N=CS).

Example compound (xv): 2-methoxy-4-[(E)-(6-sulfanyl-9H-purin-2- yl)iminomethyfJphenof.

White powder, yield 45%, m.p. >250 ^° C; v _m cm ^"1 (KBr) 3375 (NH), 2905 (CN), 1585 (C=N); δ _Η (DMSO-D ₆ ) 3.85 (3H, s, OCH ₃ ), 7.126 (3H, br, s, HC=), 8.16 (1 H, s, N=CH imidazole), 8.28 (1 H, s, N=CH imine), 12.59 (1 H, br, s, NH). δ ₀ 84.10 (OCH ₃ ), 127.50 (HC=), 133.14 (HC=), 140.31 (HC=), 155.52 (C=C), 155.07 (C=C imidazole), 155.12 (C=N imidazole), 161.08 (=C-OH), 161.36 (=C-OCH ₃ ), 163.21 (C=CN), 168.48 (C-N=), 172.42 (C=N imine), 174.55 (C=CS).

Example compound (xvi): 5-[(E)-(6-sulfanyl-9H-purin-2-yl)iminomethyl]benzene-1 ,3- diol.

Dark brown powder, yield 29%, m.p. >250 ^° C; v _m cm ^"1 (KBr) 3391 (NH), 3162 (OH), 2770 (CN), 1593 (C=N); δ _Η (DMSO-D ₆ ) 7.02 (3H, br, s, HC=), 8.14 (1 H, s, N=CH imidazole), 8.74 (1 H, s, N=CH imine), 12.51 (1 H, br, s, NH). δ ₀ 95.60 (HC=), 97.35 (HC=), 120.98 (C=C imidazole), 140.98 (C=C), 147.58 (C=N imine), 148.92 (C=CN), 154.66 (C-N=), 160.23 (C- OH), 170.07 (C=N imine), 171.96 (C=CS). Example compound (xvii): 2-[(4-nitrophenyl)methyleneamino]-9H-purine-6-thiol.

Yellow powder, yield 52%, m.p. >250 ^° C; v _m cm ^'1 (KBr) 3375 (NH), 2905 (CN), 1610 (C=N); δ _Η (DMSO-D ₆ ) 7.07 (2H, br, s, HC=), 8.18 (1 H, d, J _ortho = 9 Hz, HC=), 8.33 (8.791 H, d, J _or tho = 9 Hz, HC=), (1H, s, N=CH imine), 12.55 (1 H, br, s, NH). 6 _C 120.84 (HC=), 124.21 (HC=), 131.18 (C=C imidazole), 136.83 (C=C), 140.80 (C=N imine), 148.73 (C=CN), 150.52 (C-N=), 154.69 (C-N0 ₂ ), 166.28 (C=N imine), 172.00 (C=CS).

Example compound (xviii): 4-[(E)-(6-sulfanyl-9H-purin-2-yl)iminomethyl]benzoic acid.

Yellow powder, yield 47%, m.p. >250 ^° C; o _max / cm ^"1 (KBr) 2946 (NH), 2831 (CN), 1687 (C=0); δ _Η (CDCIs) 5.46 (1 H, s, SH), 7.57 (2H, d, J _ortho = 8Hz, HC=), 8.00 (1 H, d, J _ortho = 8.5 Hz, HC=N imidazole), 8.12 (2H, d, J _ortho = 8.5 Hz, HC=), 8.26 ( (1 H, d, J _ortho = 8.5 Hz, N=CH imine), 10.13 (1 H, s, COOH). 6 _C 95.56 (HC=), 196.09 (HC=), 98.06 (C=C imidazole), 126.94 (C=C), 129.57 (C=N imine), 130.12 (C=CN), 130.71 (C-N=), 134.46 (C-COOH), 139.52 (C=N imine), 168.32 (C=CS), 191.67 (COOH).

Example compound (xix): 2-methoxy-6-nitro-4-[(E)-(6-sulfanyl-9H-purin-2- yl)iminomethyl]phenol.

Light yeiiow powder, yield 81 %, m.p. >250 ^° C: c _max / cm ^"1 (KBr) 2946 (NH), 2831 (CN), 1687 (C=0); δ _Η (D SO-De) 3.67 (3H, s, OCH ₃ ), 6.83 (1 H, d, J _meta = 2.5 Hz, HC=), 7.96 (1 H, d, J _meta = 2Hz, HC=), 8.32 (1 H, s, HC=N imidazole), 9.39 (1 H, s, N=CH imine); 5 _C (DMSO-D ₆ ) 55.59 (OCH ₃ ), 104.80 (HC=), 1 16.22 (HC=), 129.74 (C=CH), 135.41 (C-N0 ₂ ), 137.36 (C- OH), 156.28 (NC=CN), 165.46 (N=CH imine), 188.34 (=C-SH).

Preparation 3

General procedure for the preparation of example compounds (xx) - (xxii)

The general procedure for the preparation of example compounds (xx) - (xxii) was modified from the procedures found in Ramezanain et al., 1993, US Patent 5,194,651.

6-thioguanine (0.598 mmol) was dissolved in 5 mL of NaOH 10% whilst cooling using an ice bath. A corresponding benzenesulfonyl chloride (0.718 mmol) was carefully added dropwise and the mixture was stirred at room temperature while monitoring the reaction progress using TLC (n-hexane-ethyl acetate (2:2)). After the reaction completed, 6M HCI was added until the pH of the mixture became neutral. The solid product was collected by filtration, washed with water, followed by washing with cold methanol before drying at 50°C to afford the crude product. The pure product was then afforded by recrystallizing the crude product from hot ethanol.

Example compound (xx): 3-methyl-N-[6-(m-tolylsulfonylsulfanyl)-9H-purin- 2yl]benzenesulfonamide.

Yellow powder, yield 66%, m.p. >250 ^° C; o _max / cm ^"1 (KBr) 3309 (NH), 2991 (CN), 1569 (C=N); δ _Η (DMSO-D ₆ ) 2.31 (3H, s, CH ₃ ), 2.43 (3H, s, , CH ₃ ), 6.65 (1 H, s, NH-S0 ₂ ), 7.11 (2H, d, J _ortho = 6.5 Hz, HC=), 7.20 (1 H, r, J _ortho = 7.5 Hz, HC=), 7.39 (1 H, f, J _ortho = 8 Hz, HC=), 7.58 (2H, m, HC=), 7.64 (2H, d, Jortho = 7.5 Hz, HC=), 8.45 (1 H, s, HC=N imidazole), 12.1 1 (1 H, br, s, NH imidazole); 5 _C (D SO-D ₆ ) 21.16 (CH ₃ ), 21.42 (CH ₃ ), 123.09 (HC=), 123.94 (HC=), 125.66 (HC=), 126.54 (HC=), 127.94 (=CCH ₃ ), 128.58 (=CCH ₃ ), 129.35 (HC=), 130.15 (HC=), 136.8 (HC=), 137.17(HC=), 138.35 (C=CN), 140.45 (C=CN), 140.66(C=CS), 153.38 (N=CN), 153.75 (N=CN), 155.45 (C=CN), 160.33 (N=CS). Preparation 4

General preparation for example compounds (xxi) and (xxii)

Example compound (xxi): 3-methoxy-N-(6-sulfanyl-9H-purin-2- yl)benzenesulfonamide.

Yellow powder, yield 51 %, m.p. >250 ^° C; v _m cm ^"1 (KBr) 3309 (NH), 2991 (CN), 1565 (C=N); δ _Η (DMSO-D ₆ ) 3.86 (3H, s, OCH ₃ ), 6.42 (1 H, s, NH-S0 ₂ ), 7.1 1 (1 H, s, HC=), 7.13 (1 H, s, HC=N imidazole), 7.37 (1 H, dd, J _orth0 = 6 Hz, J _meta = 2Hz, HC=), 7.20 (1 H, t, J _ort ho = 7.5 Hz, HC=), 7.39 (1 H, t, J _ortho = 8 Hz, HC=), 7.58 (2H, m, HC=), 7.64 (2H, d, J _ortho = 7.5 Hz, HC=), 8.45 (1 H, s, HC=N imidazole), 12.11 (1 H, br, s, NH imidazole); δ ₀ (DMSO-D ₆ ) 55.53 (OCH ₃ ), 93.45 (HC=), 108.77 (HC=), 111.11 (HC=), 114.82 (HC=), 1 18.33 (C=C imidazole), 129.19 (C=CS0 ₂ ), 138.05 (C=N imidazole), 153.37 (C=CN), 159.05 (C-NH ₂ ), 160.33 (C-OCH ₃ ), 168.23 (C=CSH).

Example compound (xxii): 4-methoxy-N-(6-sulfanyl-9H-purin-2- yl)benzenesulfonamide.

Yellow powder, yield %, m.p. >250 ^° C; o _m cm ^-1 (KBr) 3354 (NH), 2795 (CN), 1581 (C=N); δ _Η (DMSO-D ₆ ) 3.75 (3H, s, OCH ₃ ), 7.08 (1 H, s, NH-S0 ₂ ), 8.58 (1 H, s, HC=N imidazole), 8.78 (1 H, s, HC=), 8.89 (1 H, s, HC=), 9.3 (1 H, d, J _meta = 1.5 Hz, CH=), 1 1 .54 (1 H, br, s, NH imidazole); 5 _C (DMSO-D ₆ ) 55.59 (OCH ₃ ), 84.32 (HC=), 1 15.22 (HC=), 124.73 (HC=), 128.73 (HC=), 141.56 (C=C imidazole), 153.44 (C=CS0 ₂ ), 154.20 (C=N imidazole), 157.57 (C=CN), 161 .18 (C-NH ₂ ), 163.05 (C-OCH ₃ ), 188.92 (C=CSH).

Preparation 5

General procedure for the preparation of example compound (xxiii)

The general procedure for the preparation of example compound (xxiii) was modified from the procedures found in Hu, Y. L, Liu, X., Lu, M., Ge, Q. and Liu, X. B. (2010). Synthesis of some biologically actives halogenopurines. J. Kor. Chem. Soc, 54, 429-43.6

R XI , pyridine, rt, 1 hr

6-thioguanine (0.598 mmol) was mixed with anhydride acetic acid (Ac ₂ 0) (1.2 mL) in 1.2 mL of glacial acetic acid (GAA). The mixture was then refluxed at 135°C for 7.5 hours and the reaction progress was monitored by TLC using chloroform (CHCI ₃ ) : acetone (AcO) (2:2) as the solvent system. After the product formed, the reaction mixture was diluted with 70 mL of ice water and then extracted using 3 x 70 mL of EtOAc. The organic phase was then collected, washed with 3 x 70 mL of water and then dried over anhydrous magnesium sulfate. The organic phase was then evaporated in vacuo to afford the product as off-white powder.

Example compound (xxiii): N-(6-sulfanyl-9H-purin-2-yl)acetamide.

White powder, yield 87%, m.p. >250 ^° C ; o _max / cm ^"1 (KBr) 3109 (NH), 2933 (CN), 1552 (C=0); δ _Η (D SO-D ₆ ) 2.21 (3H, s, CH ₃ ), 8.39 (1H, s, HC= imidazole), 1.85 (1 H, br, s, NH imidazole), 13.41 (1 H, br, s, NHCO); 5 _C 21.52 (CH ₃ ), 147.45 (C=CN), 153.32 (NC=N imidazole), 165.52 (N=CNH), 168.46 (NC-NH), 172.49 (C=0), 174.53 (N=CS). Preparation 6

General procedure for the preparation of example compound (xxiv)

The general procedure for the preparation of example compound (xxiv) was modified from the procedures found in Salvatore, R. N., Nagle, A. S. and Jung, K. W. . (2002). Cesium effect: high chemoselectivity in direct N-alkylation of amines. J. Org. Chem., 67, 674-683.

138.23 mg of 4 A Molecular Sieves (MS) were suspended in 3 mL of DMF. Cesium hydroxide monohydrate (CsOH.H ₂ 0) (140 μΙ_) was added into that mixture before stirring for 10 minutes at a room temperature. The stirring was continued while adding Λ/-(6- mercapto-9H-puhn-2-yl) acetamide (example compound (xxiii); 0.47 mmol) into the mixture for 30 minutes at the same temperature. Cyclopentyl bromide (0.564 mmol) was then added to the mixture and the stirring was continued while monitoring the reaction progress using TLC with CHCI ₃ : acetone (3:1 ) as the solvent system. After the reaction completed, the mixture was diluted with 70 mL of water and then extracted using 3 x 70 mL of EtOAc. The organic phase was collected, washed with 3 x 70 mL of water and then dried over anhydrous magnesium sulfate. The organic phase was then evaporated in vacuo and purified using PLC to afford the product as an off-white powder.

Example compound (xxiv): N-(9-cyclopentyl-6-sulfanyl-purin-2-yl)acetamide.

White powder, yield 90%, m.p. 154-156 ^° C ; v _m J cm ^"1 (KBr) 3211 (NH), 2946 (CN), 1585 (C=0); δ _Η (DMSO-D ₆ ) 1.73 (4H, m, CH ₂ ), 2.19 (3H, s, CH ₃ ), 2.26 (4H, m, CH ₂ ), 2.65 (1 H. m, CH), 8.03 (1 H, s, HC=N imidazole), 12.85 (1 H, br, s, NHCO);

Preparation 7

General procedure for the preparation of example compound (xxv)

The general procedure for the preparation of example compound (xxv) was modified from the procedures found in Saqib, A., Karigar, C. S., Pasha, M. A. and Harish, M. S. R. (2012). Synthesis, characterization and pharmacological evaluation of palmitic acid derivatives of salicylic acid and anthranilic acid. JPRO, 4, 35-38.

6-thioguanine (0.598 mmol) was mixed with palmitoyi chloride and before the addition of pyridine. The mixture was stirred for 1 hour at a room temperature followed by neutralization using cold 6M HCI. The solid product was then collected by filtration, washed with water, cold methanol and then evaporated to dryness under reduced pressure. Example compound (xxv): N-(6-sulfanyl-9H-purin-2-yl)hexadecanamide.

White powder, yield 70%; m.p. 180-185 ^° C; δ _Η (DMSO-D ₆ ) 0.85 (3H, t, J = 6.5Hz, CH ₃ ), 1.23 (23H, s, -(CH ₂ ) ₁₂ ), 1.47 (2H, m, CH ₂ ), 2.18 (2H, t, J = 7.5Hz, COCH ₂ ), 6.53 (1 H, s, SH), 8.13 (1 H, s, HC=N), 1 1.97 (1 H, s, NH imidazole). δ ₀ -ι ₃ 14.42 (CH ₃ ), 22.54 (CH ₂ CH ₃ ), 24.94 (COCH ₂ CH ₂ ), 31.74 (CH ₂ CH ₂ CH ₃ ), 34.11 (COCH ₂ ), 39.37- 40.46 (-(CH ₂ ) ₁₀ ), 138.14 (C=C), 153.36 (C=N), 158.90 (NC=C), 165.49 (C-NH ₂ ), 171.24 (C=CS), 175.04 (CO).

Example compounds (i) to (xxv) were found to possess activity in the biological test described above. Biological activity determined by the above mentioned biological test includes IC ₅₀ and % inhibition values for NS2B-NS3 protease. Example compounds (i) to (xxv) were found to have IC ₅₀ values as illustrated in the table below:

Biological Data

Example compound % Inhibition at 200 g/mL ICso (μΜ)

(i) 56 753

(ii) 17 >1000

(iii) 20 >1000

(iv) 41 741

(v) 68 212

(vi) 33 >1000

(vii) 18 >1000

(viii) 60 >1000

(ix) 35 >1000

(x) 99 245

(xi) 42 426

(xii) 42 at 50 pg/mL 256

(xiii) 63 55 Example compound % Inhibition at 200 g/mL ICso (μΜ)

(xiv) 58 461

(XV) 37 >1000

(xvi) 90 69

(xvii) 25 >1000

(xviii) 35 >1000

(xix) 87 28

(XX) 71 63

(xxi) 75 78

(xxii) 53 370

(xxiii) 66 151

(xxiv) 42 at 50 Mg/mL 556

(xxv) 44 132

Acetone

Acetic Anhydride

7-Amino-4-Methylcoumarin t-Butyloxycarbonyl-Glycyl-L-Arginyl-L-Arginine-4- Methylcoumary!-7-Amide

Chloroform

Cesium hydroxide DMF Dimethylformamide

ELISA Enzyme-linked immunosorbent assay

EtOAc Ethyl Acetate

GAA Glacial Acetic Acid

HCI Hydrochloric Acid

NaOH Sodium Hydroxide

PLC Preparative Layer Chromatography

TBAI Tetrabutylammonium iodide

TLC Thin Layer Chromatography

Tris-HCI Trisaminomethane Hydrochloride

Prefixes n-, s-, /- tert- have their usual meanings: normal, secondary, iso and tertiary.