NZ535516A - Inhibitors of nucleoside phosphorylases and nucleosidases - Google Patents

Abstract

Disclosed are compounds of the general formula (I) which are inhibitors of 5'-methylthioadenosine phosphorylases and 5'-methylthioadenosine nucleosidases (MTAP and MTAN), and the use of these compounds in pharmaceutical compositions for the treatment of diseases and infections including cancer, bacterial infections and parasitic infections.

Description

WO 03/080620 PCT/NZ03/00050 INHIBITORS OF NUCLEOSIDE PHOSPHORYLASES AND NUCLEOSIDASES TECHNICAL FIELD This invention relates to certain nucleoside analogues which are inhibitors of 5'-methylthioadenosine phosphorylases and 5'-methylthioadenosine nucleosidases, processes for preparing these compounds, their use in the treatment of diseases and 10 infections, and pharmaceutical compositions containing them.

BACKGROUND US 5,985,848, US 6,066,722 and US 6,228,741 are directed to nucleoside IS analogues that are inhibitors of purine nucleoside phosphorylase (PNP). The analogues are useful in treating parasitic infections, as well as T-cell malignancies and autoimmune diseases.

PCT/NZ00/00048 provides a process for preparing these PNP inhibitor compounds. 20 This application recognises the compounds as PNP inhibitors and addresses a need for simpler methods of preparing them.

PNP catalyses the phosphorolytic cleavage of ribo- and deoxyribonucleosides, for example those of guanine and hypoxanthine, to give the corresponding sugar-1-25 phosphate and guanine, hypoxanthine or other purine bases.

The applicants have now determined that certain of these PNP inhibitor compounds are actually powerful and biologically available inhibitors of 5'-methylthioadenosine phosphorylase (MTAP) and 5'-methylthioadenosine nucleosidase (WITAN). 2 MTAP and MTAN function at or near the crossroads of polyamine biosynthesis, and of purine salvage in mammals and microbes, and of quorum sensing pathways in microbes. They respectively catalyse the reversible phosphorolysis of 5'-methylthioadenosine (MTA) to adenine and 5-methylthio-a-D-ribose-1-phosphate 5 (MTR-1P), and the hydrolysis of MTA to adenine and 5-methylthio-a-D-ribose. The adenine formed is subsequently recycled, converted into nucleotides and is essentially the only source of free adenine in the human cell. The MTR-1P is subsequently converted into methionine by successive enzymatic actions.

Scheme 1 shows the role of MTAP and MTA in polyamine biosynthesis. Scheme 2 shows the reaction catalysed by MTAP (phosphorolysis of MTA to adenine and 5-methylthio-a-D-ribose-1-phosphate) including the proposed transition state structure.

Scheme 1 M jNH2 H2N^S\^N"V HO OH Putrescine ., NH2 Spermidine t «fsVo-vNV HP°4"2 H APRT W HO OH MTA c02 | '-deoxy-S'-methytthioadenosine (MTA) w PRPP PPi AMP MeS Me H2Nv^St nj!hz rr N n-O HO OH S-adenosylmethionine PPI + Pi atp MeS a-keto acid amino ^O" nh2 methionine add o HPO4 02 K.y MeS^A0- > O + hc02" MeS O 2 i-^^X^-opos 3 Scheme 2 MeS HO OH mtap H NH2 MeS~_ Y J MTA is a by-product of the reaction involving the transfer of an aminopropyl group from decarboxylated S-adenosyl methionine to putrescine during the formation of spermidine. The reaction is catalyzed by spermidine synthase. The spermidine synthase is very sensitive to product inhibition by MTA, therefore inhibition of MTAP or MTAN will severely limit the polyamine biosynthesis and the salvage pathway for adenine in the cells.

Inhibition of MTAN may also decrease production of the quorum sensing pathways in bacteria, and thereby decrease the virulence of microbial infections.

In the AI-1 quorum sensing pathway, S-adenosylmethionine (SAM) and specific acyl-acyl carrier proteins are the substrates for homoserine lactone (HSL) biosynthesis. The biosynthesis of HSL results in concomitant release of MTA. Thus, a buildup of MTA due to inhibition of MTAN should result in inhibition of the AI-1 pathway.

WO 03/080620 PCT/NZ03/00050 In the AI-2 quorum sensing pathway, SAM is converted to S-adenosylhomocysteine (SAH), then to S-ribosylhomocysteine, and on via 4,5-dihydroxy-2,3-pentanedione to the AI-2 quorum sensing molecule. The SAH is a substrate for MTAN, so inhibition of MTAN should directly inhibit the AI-2 pathway.

MTAP deficiency due to a genetic deletion has been reported with many malignancies. The loss of MTAP enzyme function in these cells is known to be due to homozygous deletions on chromosome 9 of the closely linked MTAP and p16/MTS1 tumour suppressor gene. As absence of p16MTS1 is probably 10 responsible for the tumour, the lack of MTAP activity is a consequence of the genetic deletion and is not causative for the cancer. However, the absence of MTAP alters the purine metabolism in these cells so that they are mainly dependent on the de novo pathway for their supply of purines. That makes these cells unusually sensitive to inhibitors like methotrexate and azaserine, that block the de novo pathway. 15 Therefore, a combination therapy of methotrexate or azaserine with an MTAP inhibitor will have unusually effective anti-tumour properties.

MTAP inhibitors are may also be effective as radiation sensitizing agents. The inhibition of MTAP could result in a reduced ability to repair damage caused by 20 ionising radiation.

MTAP inhibitors would also be effective against parasitic infection such as malaria that infects red blood cells (RBCs). It has been shown that Plasmodium faicipamm has an active MTAP pathway (Sufrin, J.R., Meshnick, S.R., Spiess, A.J., Garofolo-25 Hannan, J., Pan, X-Q. and Bacchi, C.Y. (1995) Antimicrobial Agents and Chemotherapy, 2511-2515). This is a target for MTAP inhibitors. Such inhibitors may also kill the parasites without having any negative effect on the host RBCs, as RBCs are terminally differentiated cells and they do not synthesize purines, produce polyamines or multiply.

The polyamine pathway is important in cancer development. For example, evidence suggests that the excessive accumulation of putrescine and spermidine favors malignant transformation of cells. (Seiler N., Atanassov C.L., Raul F. Int J. Oncol. 1998 Nov:13(5):993-1006). Thus, inhibition of polyamine formation provides a 5 rational target for drug design. Blocking the polyamine pathway with inhibitors of MTAP is therefore expected to provide reduced growth of cancers.

Genetically modified mice (TRAMP mice, Gupta, S., Ahmad, N., Marengo, S.R., MacLennan, G.T., Greenberg, N.M., Mukhtar, H. (2000) Cancer Res. 60,5125-5133) 10 with a propensity for prostate tumour development have been described. Treatment of these mice with known inhibitors of the polyamine pathway such as a-difluoromethylornithine (DFMO) delays the onset of cancers and prevents metastasis to other tissues. However, the use of DFMO in humans is limited by its ototoxicity (causes deafness).

MTAP inhibitors target a different step in the polyamine pathway to DFMO. Since MTAP inhibitors influence a different step in this pathway, one that is only used in the polyamine pathway in humans, they may act without the side-effects that have limited the application of other polyamine pathway inhibitors.

It is therefore an object of the present invention to provide compounds that are inhibitors of MTAP and/or MTAN, or at least to provide the public with a useful choice.

STATEMENTS OF INVENTION Accordingly, in a first aspect, the present invention provides a compound of the formula (I): 6 OH X wherein: A is selected from N, CH and CR, where R is selected from halogen, alkyl, aralkyl and aryl, OH, NH2, NHR1, NR1R2 and SR3, where R1, R2 and R3are each alkyl, aralkyl or aryl groups; B is selected from NH2 and NHR4, where R4 is an alkyl, aralkyl or aryl group; X is selected from H, OH and halogen; and Z is selected from H, Q, SQ and OQ, where Q is alkyl optionally substituted with OH or halogen; aralkyl optionally substituted with OH, alkyl or halogen; or aryl optionally substituted with OH, alkyl or halogen; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; with the proviso that the stereochemistry of the aza-sugar moiety is D-ribo or 2-deoxy-D-erythro-.

Preferably, A is CH. More preferably Z is SQ when A is CH.

OFFICE OF N-Z 1 I 2 8 MAY 2007 | UecejvJLS- 7 It is also preferred that B is NH2. More preferably Z is SQ when B is NH2. Still more preferably Q is Ci-C5 alkyl when B is NH2 and Z is SQ.

It is further preferred that A is N. More preferably Z is SQ when A is N. Still more preferably Q is Ci-Cs alkyl when A is N and Z is SQ.

Preferably X is OH.

It is also preferred that Z is SQ. More preferably Q is CrC5 alkyl when Z is SQ. Still more preferably Q is an aryl group optionally substituted with OH, alkyl or halogen when Z is SQ.

Preferred compounds of the invention include those where Q is selected from phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 3-methylphenyl, 4-methylphenyl, benzyl, hydroxyethyl, fluoroethyl, naphthyl, methyl and ethyl, when Z is SQ.

Most preferred compounds of the invention include: (1 S)-1 -(9-deazaadenin-9-yl) -1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4,5-trideoxy-1,4-imino-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-0-methyl-D-ribitol; (1 S)-1 -(7-amino-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol; (1 S)-1 -(9-deazaadenin-9-yl)-1,4-dideoxy-5-ethylthio-1,4-imino-D-ribitol; (1 S)-1 -(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-phenylthio-D-ribitol; (1S)-1-(9-deazaadenin-9-yl)-5-benzylthio-1,4-dideoxy-1,4-imino-D-ribitol; (1 S)-1 -(9-deazaadenin-9-yl)-1,4-dideoxy-5-(2-hydroxyethyl)thio-1,4-imino-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(4-methylphenyl)thio-D-ribitol; l QFP'CE OP N, z 28 may 2007 8 (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(3-methylphenyl)thio-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-5-(4-chlorophenyi)thio-1,4-dideoxy-1,4-imino-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-5-(3-chlorophenyl)thio-1,4-dideoxy-1,4-imino-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-5-(4-fluorophenyl)thio-1,4-imino-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(1-naphthyl)thio-D-ribitoi; (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-5-(2-fluoroethyl)thio-1,4-imino-D-ribitol; and (1 S)-1-(9-deazaadenin-9-yl)-1,4,5-trideoxy-5-ethyl-1,4-imino-D-ribitol.

In a second aspect, the invention provides a pharmaceutical composition comprising 15 a pharmaceutically effective amount of a compound of formula (I).

In another aspect, the invention provides a method of treating a disease or condition in which it is desirable to inhibit MTAP, comprising administering a pharmaceutically effective amount of a compound of formula (I) to a non-human patient requiring treatment. The disease includes cancer or a protozoan parasitic infection, such as malaria.

The invention further provides the use of a compound of formula (I) in the manufacture of a medicament for treating a disease or condition in which it is desirable to inhibit MTAP.

In another aspect, the invention provides a method of treating a disease or condition in which it is desirable to inhibit MTAN, comprising administering a pharmaceutically effective amount of a compound of formula (I) to a non-human patient requiring 30 treatment. The disease includes a bacterial infection.

INTELLECTUAL PROPERTY OFFICE OF N.Z 1 1 apr 2007 received 9 The invention further provides the use of a compound of formula (I) in the manufacture of a medicament for treating a disease or condition in which it is desirable to inhibit MTAN.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the inhibition of MTAP by 5'-methylthio-lmmA at varying concentrations. (MTA at 150 nM; Km = 2.5 nM, Ki = 107 pM).

Figure 2 shows the effect of methylthioadenosine (MTA) alone, 5'-methylthio-lmmA alone and a combination of MTA and 5'-methylthio-lmmA on the irradiation of Lewis Lung carcinoma cells.

Figure 3 shows the effect of 5'-methylthio-lmmA on MTAP activity in mouse blood.

Figure 4 shows inhibition of mouse liver MTAP by 5'-methylthio-lmmA.

DETAILED DESCRIPTION OF THE INVENTION This invention provides compounds of the formula (I) as defined above, which are potent inhibitors of MTAP and MTAN. The compounds of the invention are therefore expected to have clinical utility in treating diseases such as cancer, bacterial infections and protozoan parasitic infections (such as malaria).

The compounds of the invention are useful in both free base form and in the form of salts. The term "pharmaceutically acceptable salts" is intended to include non-toxic salts derived from inorganic or organic acids, including, for example, the following acids: hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric, succinic, tartaric, gluconic, citric, methanesulfonic and p-toluenesulfonic acids.

As used herein, the term "aza-sugar moiety" means a fragment of general structure: Z OH X where Z and X are as defined above for a compound of formula (I).

General synthetic methods for preparing the compounds of the invention are given below.

Method (A): (5'-thio-lmmucillin-A derivatives) reacting a compound of formula (II) Z«—CH2 [wherein Z' is a trialkylsilyloxy, alkyIdiarylsilyloxy or optionally substituted 15 triarylmethoxy group] (typically Z' is a tert-butyldimethylsilyloxy, trityloxy or similar group) sequentially with N-chlorosuccinimide then a sterically hindered base (such as lithium tetramethylpiperadide) to form an imine, then with the anion of acetonitrile 20 (typically made by treatment of acetonitrile with n-butyllithium). The resulting 3,6- nh (H) 11 dideoxy-3,6-iminoheptononitrile derivative is then 7-0-deprotected. in the case of a trialkyisilyl or alkyidiarylsiiyl protecting group, this is typically achieved by treatment with a fluoride ion source, conveniently tetrabutyiammonium fluoride in tetrahydrofuran. In the case of an optionally substituted triarylmethyl protecting 5 group this is typically achieved by use of an acidic reagent, typically boron trifluoride in methanol, or aqueous acetic acid. The resulting 7-hydroxy-derivative is then N-protected by reaction with di-tert-butyl dicarbonate to generate the compound of formula (III) C02Bul HO CH2 | CH2CN X (Hi) which is then elaborated by displacement of the 7-hydroxy group, conveniently by sulfonate displacement with a thiolate anion, for example by conversion first to a 7-O-methanesulfonate with methanesulfonyl chloride and base (e.g. triethylamine) and displacement with sodium methanethiolate (e.g. NaSMe in dimethylformamide), to give a compound of formula (IV) z ch2 ccj2but fi, ch2cn (iv) [wherein Z is SQ as defined for formula (I)] 12 which is then elaborated either by condensation with ethyl formate in the prescence of a base, typically sodium hydride, or by condensation with (Me2N)2CHOBut (Brederek's reagent) and hydrolysis under weakly acidic conditions, to give a compound of formula (V) [wherein Z is SQ as defined for formula (I)] which is then reacted with aminoacetonitriie under mildly basic conditions, and cyclized by reaction with a simple ester of chloroformic acid (typically benzyl chloroformate or methyl chloroformate) to give a compound of formula (VI) C02R COjBut J COjjBut OH Z— [wherein Z is SQ as defined for formula (I) and R is an alkyl or aralkyl group] PCT/N Z03/00050 13 which is then deprotected on the pyrrole nitrogen by hydrogenolysis in the presence of a noble metal catalyst (e.g. Pd/C) in the case of a benzyl group or under mildly basic conditions in the case of a simple alkyl group such as a methyl group, and then condensed with formamidine acetate to give a compound of formula (VII) which is then fully deprotected under acidic conditions, e.g. by treatment with trifluoroacetic acid or with hydrochloric acid in methanol.

This method follows the approach used to prepare 9-deazaadenosine and its analogues [Lim and Klein, Tetrahedron Lett., 22 (1981) 25, and Xiang et al., Nucleosides Nucleotides 15 (1996) 1821], including Immucillin-A [Evans et al., Tetrahedron 56 (2000) 3053].

Methods for the preparation of a compound of formula (II) [wherein Z* is a tert-butyldimethylsilyloxy group] are detailed in Furneaux et al., Tetrahedron 53 (1997) 2915 and references therein.

An alternative method of making the compound of formula (III) is described in Preparative Example A. 14 Method (B): (5'-0-substituted Immucillin-A derivatives) reacting the compound of formula (III) with an optionally substituted alkylating or aralkylating agent in the presence of a base to give a compound of formula (IV) 5 [wherein Z is OQ as defined for formula (I)]. For methylation, a typical reagent combination would be methyl iodide and sodium hydride in a solvent such as tetrahydrofuran, dimethylsulfoxide or dimethylformamide. The resulting compound of formula (IV) [wherein Z is OQ as defined for formula (I)] is then converted to a compound of formula (I) as described for the corresponding conversion of a 10 compound of formula (IV) in Method A.

Method (C): (5'-deoxy-lmmucillin-A derivatives) 7-deoxygenating the compound of formula (III), then converting the resulting 15 compound of formula (IV) [wherein Z is hydrogen] to a compound of formula (I) as described for the corresponding conversion of a compound of formula (IV) in Method A. Deoxygenation can be achieved by various Barton radical deoxygenation methods, or preferably by formation and dehalogenation of a 7-deoxy-7-halogeno-intermediate. Conveniently this would be the 7-deoxy-7-iodo-derivative, formed by 20 sulfonation of the compound of formula (III), typically with methanesulfonyl chloride and base (e.g. diisopropylethylamine), then displacement of the sulfonate group with a source of halide ion, typically sodium iodide in acetone. Dehalogenation would then be affected either by catalytic hydrogenolysis, typically with hydrogen over a palladium catalyst, or preferably with a radical dehalogenation reagent such as 25 tributyltin hydride in benzerie.

Method (D): (8-aza-5'-thio-lmmuciilin-A derivatives - Daves' methodology) reacting a compound of formula (II) (as defined where first shown above) 30 sequentially with /V-chlorosuccinimide and a hindered base (such as lithium tetramethylpiperidide) to form an imine, then condensing this with the anion produced by abstraction of the bromine or iodine atom from a compound of formula [wherein R3 is a bromine atom, R4 is a tetrahydropyran-2-yl group, and R5 is a methyl group] typically using butyllithium or magnesium, the coupling preferably being catalyzed by a Lewis acid catalyst, typically tin(IV) chloride at low temperature, typically in the range -30 to -80 °C, preferably -78 °C, to give a product which is then sequentially (i) N-protected preferably as the tert-butoxycarbonyl derivative, typically by treatment with di-tert-butyl dicarbonate, preferably in methanol at room temperature; (ii) subjected to a displacement of the methoxy group (introduced as the RsO of a compound of formula (VIII)) with ammonia, typically with concentrated ammonia in methanol at 100 °C; (iii) N-protected on the primary amino group, preferably as the N-mono- . benzoate, typically by treatment with benzoylimidazole and a catalytic amount of 4-N,N-dimethylaminopyridine in acetonitrile at 65 °C; (iv) 5'-0-deprotected; in the case of a trialkylsilyloxy or aikyIdiaryisilyloxy this is typically achieved by treatment with a fluoride ion source, conveniently tetrabutylammonium fluoride in tetrahydrofuran; in the case of an optionally substituted triarylmethoxy group this is typically achieved by use of an acidic reagent, typically boron trifluoride in methanol, or aqueous acetic acid; (VIII) ORS (VIII) 16 (v) subjected to displacement of the 5'-hydroxy group with thiolacetic acid, preferably under Mitsunobu reaction conditions, typically with a combination of triphenylphosphine and diisopropyl azodicarboxylate in tetrahydrofuran, then thiolacetic acid; (vi) 5'-S-deprotected then 5'-S-alkylated or 5'-S-aralkylated by sequential reaction with sodium methanethiolate then an alkylating or alkylating agent, conveniently methyl iodide or benzyl bromide in methanol where the 5'-S-methyl or 5'-S-benzyl-derivative is required; and finally (vii) full N.O-deprotection by acidic treatment, conveniently with concentrated aqueous hydrochloric acid in methanol, to give the compound of formula (I) as the dihydrochloride salt.

Methods for preparing compounds of formula (VIII) are described in Stone et al., J. Org. Chem., 44 (1979) 505, and references therein. It will be appreciated that while 15 the tetrahydropyran-2-yl and methyl groups are favoured as the protecting group for this reaction, other 0,N-protecting groups can be used.

Method (E): (5'-alkyl-5'-deoxy-lmmucillin derivatives) reacting a compund of formula (X) (X) [wherein R6 is an N-protecting group, R7 is an alkoxycarbonyl or aralkyloxycarbonyl group, B' is selected from N(R8)2, R8 is an N-protecting group and D is H] 17 with an oxidizing agent capable to converting the 5-hydroxy group into a 5'-aldehydo group. There are many such reagents, but conveniently this may be conducted using the Dess-Martin periodinane reagent, or a chromium(VI) oxidant such as Collins reagent (Cr03 in pyridine) or pyridinium dichromate catalyzed by molecular 5 sieves and pyridinium trifluoroacetate; reacting the resulting aldehyde with a Wittig or Horner-Wittig reagent, chosen depending upon the alkyl substituent required; hydrogenating the resulting alkene, conveniently using palladium on charcoal as the catalyst; and finally fully N,0,S-deprotecting the resulting 5-C-alkyl derivative by acid-, alkali-or fluoride ion-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysis as 15 required for the 0-, N- and S-protecting groups in use.

Compounds of formula (X) can be prepared as described in US 5,985,848.

The N-protecting group R8 in the compound of formula (X) may conveniently be an 20 alkoxymethyl group (such as benzyloxymethyl) or a tetrahydropyranyl group. It will be appreciated that protection of a pyrazolo[4,3-d]primidine moiety can result in one or both of a pair of isomers depending upon which of the nitrogen atoms in the pyrazoles moiety is protected, and that either isomer is satisfactory for the purposes of making a 5'-substituted derivative. The N-protecting group R8 and the S-25 protecting group R9 in the compound of formula (X) may conveniently be an alkoxymethyl group (such as benzyloxymethyl), a silyl group (such as tert-butyldimethylsilyl) or an arylmethyl group (such as benzyl). Each N-protecting group R8 may conveniently be independently an arylmethyl group (such as benzyl or 4-methoxylbenzyl), or the two R8 groups together may form the 2,4-hexadien-2,5-yl 30 group. 18 Preparative method (A): {Compound (III)} A compound of formula (III) can be prepared by reacting a compound of formula (II) [as defined where first shown above] with an oxidizing agent, such as meta-5 chloroperbenzoic acid, or preferably the combination of hydrogen peroxide and selenium dioxide, to give a nitrone of formula (XI) (XI) [wherein Z is is a trialkylsilyloxy, alkyldiarylsilyloxy or optionally substituted 10 triarylmethoxy group] which is then reacted in sequence with: (a) the anion of acetonitrile (typically made by treatment of acetonitrile with n-. butyllithium), conveniently in tetrahydrofuran; and IS (b) a reagent capable of reducing the resulting N-hydroxy group to an amine, conveniently with zinc in acetic acid; and di-tert-butyl dicarbonate, typically in chloroform; then deprotected at 0-5; in the case of a trialkylsilyloxy or alkyldiarylsilyloxy this is 20 typically achieved by treatment with a fluoride ion source, conveniently tetrabutylammonium fluoride in tetrahydrofuran; in the case of an optionally substituted triarylmethoxy group this is typically achieved by use of an acidic reagent, typically boron trifluoride in methanol, or aqueous acetic acid.

O Q .0 19 Inhibition of MTAP and MTAN Inhibition constants for selected compounds of the invention are collected in Tables 1 and 2. Table 1 shows inhibition constants for MTAN and Table 2 shows inhibition 5 constants for MTAP.

Ki as shown in Tables 1 and 2 is the initial inhibition constant formed by the enzyme-inhibitor complex, and Ki* is the equilibrium dissociation constant for inhibition that is observed following a period of slow-onset, tight binding inhibition. Ki* is the 10 biologically effective constant.

The compounds of the invention are potent inhibitors of MTAP and MTAN. For example, 5'-methylthio-lmmA has Ki* in the pM range for both enzymes. In contrast, methylthio-lmmH, which does not fall within the selected class of compounds, shows 15 no inhibition of MTAN.

Furthermore, Immucillin A which also does not fall within the selected class of 20 compounds, shows no inhibition of MTAP.

Methylthio-lmmH HO OH HO Immucillin-A HO OH Table 1: Inhibition Constants for MTAN Inhibitors Inhibitor Name Structure K, K* '-Phenylthio-ImmA h fh2 ho oh 46 ± 3 pM 32 ± 2 pM '-methylthio-ImmA h ?h2 /v*" v hu ^ ho oh 130 ±12 pM 77 ±20 pM '-Ethylthio-lmmA u nh s-^ hyin^ vN7 ho oh 73 ±11 pM 27 ±0.3 pM '-deoxy-5'-ethyl-ImmA h yh2 s h rv vN7 ho oh 121 + 5 pM 38 ± 5 pM '-methylthio-8-aza-lmmA h nh2 n'NT^N hv^ ^ ho oh 55 + 3 pM 26 ±0.3 pM '- Hydroxyethylthio-ImmA h nh2 ho , /"y^n h va j vN7 ho oh 407 ±16 pM n. d.

PCT/N Z03/00050 '- Fluoroethylthio-ImmA H ^2 vv hvo HO OH 103 ±11 pM ± 3 pM '-deoxy-lmmA H r* /nY^n v hyv' vnv HO OH 13 ± 1 nM N. D.

'-Methoxy-lmmA H /vs ch,°\s HO OH ± 1.0 nM N. D. '-(p-Fluoro-phenyl-thio)-ImmA H JfH2 HO OH 82.0 ±7.0 pM ±4.0 pM '-(p-Chloro-Phenyl-thio)-ImmA H JJH2 HO OH 6.0 ±0.3 pM No late onset. Ki* is same as Ki i.e. 6 pM '-(m-Chloro-Phenyl-thio)-ImmA CI H NH2 HO OH 44.0 ±4 pM ±2.0 pM '-Benzylthio-ImmA /=\ H ^2 qs £rs HO OH 38.0 ± 3.0 pM 12.0 ±1.0 pM '-(m-tolylthio)-ImmA /={ H NH2 HO OH .0 ±0.6 pM 9.0 ±1.0 pM '-(p-tolylthio)-ImmA )=\ h t2 vnv HO OH 18.0 ±1.0 pM 8.0 ±1.0 pM '-Napthylthio-ImmA /=\ H NH2 /k i j i vn7 HO OH 750 ±33 pM ND PCT/N Z03/00050 Table 2: Inhibition Constants for MTAP Inhibitors Inhibitor Name Structure K, K,* '-Phenylthio-ImmA h ?h2 .n^an < ho oh 890.0 ± 120 pM 82 ± 9 pM -Methylthio-ImmA h nh2 vnv ho oh .0 ± 0.5 nM 90 ±18 pM '-Ethylthio-lmmA AX j vn7 ho oh 18.0 ±2.0 nM 260 ± 15 pM '-Methylthio-8-aza-lmmA h f"2 \s N;j ,n vn7 ho oh 7.0 ±1.0 nM 760 ±140 pM '- (Hydroxyethylthio )-lmmA h nh2 H ya J vm7 ho oh 22.0 ±2.0 nM Ki* not determina ble '- (Fluoroethylthio)-ImmA h nh2 fv/v nyo1 . ho oh 7.0 ±0.5 nM 1.5±0.17 nM PCT/N Z03/00050 24 '-deoxy-lmmA « r , Hv-\,ry n HO OH 220 ±14 nM K/* not determina ble '-Methoxy-lmmA H f» /nY^N -o--, Hw vn7 HO OH 70 ± 5 nM Ki* not determina ble '-(p-Fluoro-phenyl-thio)-ImmA H J"H2 HO OH 3.0 ±0.2 nM ND '-(p-Chloro-phenyl-thio)-ImmA H NH* HO OH 334 ±36 pM 90 ±20 pM '-(m-Chloro- phenyl-thio)- IrnmA CI |m* HO OH 4.0 ± 0.4 nM ND '-Benzylthio-ImmA /=\ H NH2 vn7 HO OH 12 ±1 nM ND '-(n>Tolylthio)-ImmA /=( H t2 HO OH 602 ± 32 pM 146 ±48 pM '-(p-T olylthio)-ImmA . )=\ h l"2 vn7 HO OH 1.6 ±0.2 nM 252 ±23 pM '-Napthylthio-ImmA /=\ H N"2 fil co HO OH 52 ± 5 nM ND Figure 1 shows the inhibition of MTAP by 5'-methylthio-lmmA at varying concentrations. (MTA at 150 pM; Km = 2.5 pM, K,- = 107 pM).

Demonstration of Radiation Sensitizing Effect of 5'-methylthio-immA Figure 2 shows the effect of methylthioadenosine (MTA) alone, 5'-methylthio-lmmA alone and a combination of MTA and 5'-methylthio-lmmA on the irradiation of Lewis 10 Lung carcinoma cells. These data show that the combination of MTA and 5'-methylthio-lmmA acts as a radiation sensitizer, lowering cell numbers after irradiation.

Further Aspects The active compounds can be administered in combination with one or more conventional pharmaceutical carriers or excipients, and may be administered by a 26 variety of routes, including oral administration, injection, or topical administration. The amount of compound to be administered will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range less than 1 to 1000 milligrams, 5 preferably 0.1 to 100 milligrams.

For oral administration the compounds can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. Such preparations are well known in the art as are other oral dosage 10 regimes not listed here. In the tablet form the compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid and the lubricant may be magnesium stearate. Other components such as colourings or IS flavourings may be added.

Liquid forms include carriers such as water and ethanoi, with or without other agents such as a pharmaceutically acceptable surfactant or suspending agent.

The compounds may also be administered by injection in a physiologically acceptable diluent such as water, or saline. The diluent may comprise one or more other ingredients such as ethanoi, propylene glycol, an oil or a pharmaceutically acceptable surfactant.

The compounds may be present as ingredients in creams, for topical administration to skin or mucous membranes. Preferably the creams include a pharmaceutically acceptable solvent to assist passage through the skin or mucous membranes. Suitable creams are well known to those skilled in the art. 27 The compounds may further be administered by means of sustained release systems. For example, they may be incorporated into a slowly dissolving tablet or capsule.

The invention will be described in more detail with reference to the following non-limiting examples.

EXAMPLES Example 1: Preparation of (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-fmino-5-methylthio-D-ribitol (5'-methylthio-lmmucillin-A, Scheme 1) Scheme 1 Reagents and conditions: (a) CH3CN, nBuLI, THF, -78°C, 36%; (b) (I) TBAF, THF, r.t. (II) B0C2O, MeOH, r.t. (iii) MsCI, Et3N, DCM, r.t., 48% far 3 steps; (c) MeS"Na+, DMF, r.t 88% (d) (i) Brederecks reagent, DMF, 70°C; (II) THF, HOAc, H20, r.t.; (iii) aminacetonltrlle hydrochloride salt, NaOAc, MeOH, r.t; (iv) Methyl chloroformate, DBU, DCM, D; (v) MeOH, r.t. 69% for 5 steps (e) (0 formamidlne acetate, EtOH, D; (ii) MeOH, cHCI, r.t., 90%. 28 Example 1.1: N-fe/t-Butoxycarbonyl-3,6-imino-4,5-0-isopropylidene-7-0-methanesulfony)-2,3,6-trideoxy-D-a//o-heptononitrile (3). - TBAF (5 mL, 1M in THF, 5.0 mmol) was added dropwise to a stirred solution of 7-O-tert-butyldimethylsilyl-3,6-imino-415-0-isopropylidene-2,3,6-trideoxy-D-a//o-heptononitrile 5 (2) (1.40 g, 4.3 mmol) in THF (20 mL) at room temperature. After 1 h the reaction was complete by TLC The solution was diluted with water (200 mL) and extracted with chloroform (3 x 50 mL). The organic layers were combined, dried (MgS04) and concentrated in vacuo. The crude residue (0.92 g, 4.3 mmol) was dissolved in methanol (20 mL) and di-fert-butyl dicarbonate (1.00 g, 4.6 mmol) was added 10 portionwise at room temperature and the resulting solution left to stir for 1 h. The reaction was concentrated in vacuo and chromatography of the residue presumably afforded N-te/f-butoxycarbonyl-3,6-imino-4,5-0-isopropylidene-7-0-methanesulfonyl-2,3,6-trideoxy-D-a//o-heptononitrile (1.1 g) as an oil. A solution of the oil in anhydrous dichloromethane (20 mL) and N-ethyldiisopropylamine (2 mL, 11.4 mmol) 15 was treated with methanesulfonyl chloride (0.5 mL, 6.5 mmol). After 0.5 h the solution was diluted with chloroform (100 mL), washed with 10% HCI (50 mL), water (50 mL) and brine (50 mL). The organic phase was dried (MgS04) and concentrated in vacuo. Chromatography afforded N-fe/f-butoxycarbonyl-3,6-imino-4,5-0-isopropylidene-7-0-methanesulfonyl-2,3,6-trideoxy-D-a//o-heptononitrile (3) (800 mg, 20 48% overall yield) as a syrup. 1H NMR (CDCI3) 5 4.75 (dd, J = 5.7,1.4 Hz, 1H), 4.61 (brs, 1H), 4.38 (brd, J * 5.7 Hz, 2H), 4.19 (m, 2H), 3.07 (s, 3H), 2.81 (m, 2H), 1.50 (s, 12H), 1.35 (s, 3H); 13C NMR S 154.0, 117.5, 113.3, 83.3, 82.4, 81.3, 68.6, 64.2, 61.5, 60.7, 37.7, 28.6, 27.6, 25.6, 21.8. HRMS (MH*) calc. for Ci6H27N207S:. 391.1539. Found: 391.1539.

Example 1.2: (1S)-N-fe/f-Butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-methylthio-D-ribitol (4). - Sodium thiomethoxide (0.75 g, 10.7 mmol) was added to a solution of N-ferf-butoxycarbonyl-3,6-imino-4,5-0-isopropyiidene-7-0-methanesulfonyl-2,3,6-trideoxy-D-a//oheptononitrile (3) (0.85 30 g, 2.2 mmol) in DMF (10 mL) at room temperature. After stirring overnight the reaction was diluted with toluene (100 mL), washed with water (50 mL), brine (50 29 mL), dried (MgS04) and concentrated in vacuo. Chromatography afforded (1S)- 1-N-ferf-butoxycarbonyl-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-methylthio-D-ribitol (4) (0.66 g, 3.06 mmol, 88%) as a colourless foam. 1H NMR 8 4.68 (m, 2H), 4.13 (m, 2H), 2.79 (m, 3H), 2.59 (dd, J = 13.5, 10.6 Hz, 1H), 2.18 (s, 5 3H), 1.50 (s, 3H), 1.48 (s, 9H), 1.35 (s, 3H); 13C NMR 5 154.2, 118.0, 113.1, 83.6, 82.6, 81.7, 63.9, 62.2, 37.0, 28.7, 27.6, 25.6, 21.9, 16.1. HRMS (MH*) calc. for CiaH^NzCUS: 343.1692. Found: 343.1700.

Example 1.3: (1 S)-1-(3-Amino-2-cyanopyrrol-4-yl)-N-tert-butoxycarbonyl-1,4-10 dideoxy-1,4-imino-2,3-0-isopropylidene-5-methylthio-D-ribitol (5). - Brederecks reagent (1.5 mL) was added dropwise to a stirred solution of (1 S)-N-fert-butoxycarbony 1-1 -C-cyanomethyi-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-methylthio-D-ribitol (4) (0.66 g, 1.9 mmol) in DMF (20 mL) under an inert atmosphere at room temperature. The resulting solution was heated at 70 °C for 18 h and then 15 cooled to room temperature, diluted with toluene (100 mL), washed water (50 mL), brine (50 mL), dried (MgS04) and concentrated in vacuo. The crude residue was dissolved in THF/acetic acid/water (1:1:1, v/v/v, 10 mL) at room temperature and stirred for 2 h. The reaction was then diluted with chloroform (100 mL) and the resulting mixture washed with water (2 x 25 mL), saturated aqueous sodium 20 bicarbonate and then dried and concentrated in vacuo. The crude residue was redissolved in methanol (5 mL) and sodium acetate (500 mg, 6.1 mmol) and aminoacetonitriie hydrochloride (200 mg, 2.2 mmol) were added consecutively at room temperature and the resulting suspension left to stir for 16h. The reaction was then concentrated in vacuo and partitioned between chloroform (100 mL) and water 25 (50 mL). The organic layer was separated, washed with water (25 mL), brine (25 mL), dried and concentrated in vacuo. The crude residue was redissolved in dichloromethane (5 mL) and treated dropwise with DBU (2.25 mL, 20 mmol) and methylchloroformate (1.0 mL, 12.7 mmol) and the resulting solution heated under reflux for 1 h. The reaction was then cooled and diluted with methanol (20 mL) and 30 left for a further 1 h. The resulting solution was diluted with chloroform (250 mL), washed with dilute aqueous HCI, aqueous sodium bicarbonate, dried and concentrated in vacuo. Chromatography of the resultant residue afforded (1S)-1-(3-amino-2-cyanopyrrol-4-yl)-N-fert-butoxycarbonyi-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-methylthio-D-ribitol (5) (380 mg, 69%) as an oil. 1H NMR S 7.80 (s, 1H), 5.68 (s, 1H), 5.22 (s, 1H), 4.63 (d, J = 5.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.31 5 (brs, 1H), 2.51 (dd, J = 13.6, 3.7 Hz, 1H), 2.14 (m, 1H), 1.87 (s, 3H)', 1.45 (s, 3H), 1.35 (s, 9H), 1.23 (s, 3H); 13C NMR (CeDe) 8 155.2 (C), 128.3, 120.2 (CH), 115.2, 112.6, 112.0, 87.2 (C), 84.6, 84.2 (CH), 80.5, (C), 64.6, 59.4 (CH), 37.0 (CH2), 28.3, 27.4, 25.5, 14.5 (CH3). HRMS (MH*) calc. for C19H28N4O4S: 408.1831. Found: 408.1842 Example 1.4: (1 S)-1 -(9-Deazaadenin-9-yI) -1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol (6). - (1S)-1-(3-Amino -2-cyanopyrrol-4-yl)- N-fert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-D-isopropylidene-5-methylthio-D-ribitol (5) (90 mg, 0.22 mmol) was dissolved in ethanoi (5 mL), formamidine acetate (45 mg, 0.43 mmol) was 15 added and the resulting suspension heated at reflux for 16h. The crude reaction mixture was preabsorbed onto silica and chromatography afforded an oil which was not characterised but redissolved in methanol (1.5 mL) and stirred with concentrated HCI (1.5 mL) for 2h. The crude reaction was concentrated in vacuo to afford (1 S)-1-(9-deazaadenin-9-yl) -1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol (6) (65 mg, 90%) 20 as a hydrochloride salt which decomposed between 223-225°C without melting. 1H NMR (D20) 8 8.33 (s, 1H), 7.97 (s, 1H), 4.90 (d, J = 8.4 Hz, 1H), 4.75 (m, 1H), 4.38 (t, J = 4.2 Hz, 1H), 3.87 (quintet, J = 4.8 Hz, 1H), 3.05 (dd, J = 14.4, 5.7 Hz, 1H), 2.91 (dd, J = 14.4, 9.3 Hz, 1H ), 2.11 (s, 3H); 13C NMR (D20) 8 149.4 (C), 143.6 (CH), 139.1 (C), 133.0 (CH), 113.0, 105.6 (C), 73.2, 72.5 63.6, 56.3 (CH), 33.5 25 (CH2), 14.6 (CH3). HRMS (MH*) calc. for C12Hi8Ns02S: 296.1181. Found: 296.1171. Anal. calc. for Ci2Hi8N502.HCI C, 39.14; H, 5.20; CI, 19.25; N, 19.02; S, 8.71. Found C, 38.96; H, 5.28; CI, 19.25; N, 18.82; S, 8.61. 31 Example 2: Preparation of (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1l4-imino-5-O-methyl-D-ribitol hydrochloride (S'-O-methyl-lmmucillin-A, Scheme 2) H3c 5°5~cn £ vii,viii,xv,xvi H " .N CN 7 x,xi H NH2 Reagents: i, SeO 2, H 20 2; ii. LiCH 2CN; iii, Zn, HOAc; iv, (Boc) 20; v, Bu 4NF; vi, NaH, Mel; vii, NaH, EtOCH viii, NaOAc, H2NCH2CN.HCI; ix, DBU, MeOCOCI, then MeOH; x, formamidine acetate; xi, aq. HCI; xii, MsCI, 'Pr2NEt; xiii, Nai; xiv, BujSnH; xv, DBU, MeOCOCI; xvi, EtjN, MeOH. 32 Example 2.1: 5-0-fert-Butyldimethylsilyl-1 ,W-dehydro-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol W-oxide (2). - Selenium dioxide (0.6 g) was added to a solution of 5-0-ferf-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (1) (Horenstein, B.A.; Zabinski, R.F.; Schramm, V.L.

Tetrahedron Lett., 1993, 34, 7213) (30 g) in acetone (50 mL) and the solution was cooled to 0 °C. 30% Hydrogen peroxide (~ 40 mL) was added slowly keeping the solution at <4 °C until t.l.c. indicated that the reaction was complete, then chloroform (250 mL) was added and the mixture was washed with water (500 mL). The organic phase was dried and concentrated to dryness. Chromatography of the residue 10 afforded 5-0-ferf-Butyldimethylsilyl-1 ,A/-dehydro-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol N-oxide (2) (18.3 g) as a solid with m.p. 121-124 °C. Anal, calc. for C14H27N04Si: C, 55.78; H, 9.03; N, 4.65; found: C, 55.81; H, 8.88; N, 4.75. 1H NMR (CDCb) 8 6.84 (s, 1H), 5.09 (m, 1H), 4.79 (d, 1H, J = 6.2 Hz), 4.20 (dd, 1H, J = 2.0, 11.0 Hz), 3.99 (d, 1H, J = 0.7 Hz), 3.80 (dd, 1H, J = 2.1, 11.0 Hz), 1.33 (s, 15 3H), 1.38 (s, 3H), 0.81 (s, 9H), 0.02, (s, 3H), 0.00, (s, 3H); 13C NMR 8 133.48, 111.97, 81.11, 79.49, 77.48,60.21, 27.72, 26.25, 26.10,18.50.

Example 2.2: W-fert-Butoxycarbonyl-7-0-fert-butyldlmethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-0-isopropylidene-D-affo-heptononitrile (3). - Butyl lithium (32.5 20 mL, 2.3 M, 74.8 mmol) was added to THF (300 mL) and the solution was cooled to -70 °C, then acetonitrile (4.2 mL, 80.2 mmoi) was added slowly keeping the reaction temperature <-65 °C. After 30 min. a solution of 5-0-fert-butyldimethylsilyi-1 ,N-dehydro-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol N-oxide (2) (15 g, 49.8 mmol) in THF (30 mL) was added. The resulting solution was stirred at -70 °C for 30 25 min. then quenched with water. Petroleum ether (500 mL) was added and the mixture was washed with water, and processed normally to give a syrup. A solution of this material in acetic acid (100 mL) was stirred while zinc dust (20 g) was added. Cooling was applied as necessary to keep the reaction temperature <30 °C. After stirring for 6 h the mixture was filtered and the filtrate was concentrated to a syrup. A 30 solution of this in chloroform (200 mL) was washed with aq. NaHC03, dried, and then di-fert-butyl dicarbonate (11.5 g) was added. After standing overnight the solution 33 was concentrated to dryness and chromatography afforded /V-ferf-Butoxycarbonyl-7-0-ferf-butyldimethylsilyl-2I3,6-trideoxy-3,6-imino-4,5-0-isopropylidene-D-a//o-heptononitrile (3) (15.9 g) as a syrup with identical NMR spectra to that reported (Evans, G.B.; Furneaux, R.H.; Gainsford, G.J.; Schramm, V.L.; Tyler, P.C.

Tetrahedron 2000, 56, 3053).

Example 2.3: (1S)-1-(3-Amino-2-cyanopyrrol-4-yl)-yV-fert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-0-methyl-D-ribltol (4).

Tetrabutylammonium fluoride (2.5 mL, 1M in THF) was added to a solution of N-tert-10 butoxycarbonyl-7-0-?e/f-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-0- isopropylidene-D-a//o-heptononitrile (3) (0.5 g) in THF (2.5 mL). After 1 h chloroform (20 mL) was added and the solution was washed with water, dried and concentrated to dryness. A solution of the residue in THF (10 mL) and methyl iodide (0.25 mL) was stirred while sodium hydride (0.1 g, 60 %) was added and the resulting mixture 15 was stirred for 2 h. After quenching with ethanoi, chloroform was added and the mixture was washed with water, dried and concentrated to dryness. A solution of the crude product in THF (5 mL) and ethyl formate (1.2 mL) was stirred with sodium hydride (0.25 g, 60 %) for 2 h. Acetic acid (0.6 mL) was added followed by chloroform and the mixture was washed with water, dried and concentrated to 20 dryness. A solution of the crude product in methanol (10 mL) containing sodium acetate (1.2 g) and aminoacetonitriie hydrochloride (0.7 g) was heated under reflux for 1 h. Chloroform was added and the solution was washed with water, dried and concentrated to dryness. A solution of the residue in methylene chloride (10 mL) containing DBU (0.54 mL) and methyl chloroformate (0.14 mL) was heated under 25 reflux for 0.5 h. Methanol (5 mL) was added to the cooled solution and after 1 h the solution was processed normally to give, after chromatography, (1S)-1-(3-amino-2-cyanopyrrol-4-yl)-A/-fert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-O-methyl-D-ribitol (4) (0.091 g) as a syrup. 1H NMR (CDCI3) 5 8.70 (s, 1H), 6.42 (d, J = 3.2 Hz, 1H), 4.79 (bs, 1H), 4.67 (m, 2H), 4.14-3.98 (m. 3H), 3.35 (m, 2H), 30 3.25 (s, 3H), 1.45 (s, 3H), 1.36 (s, 9H), 1.27 (s, 3H); 13C NMR 5 154.2, 141.5 (C), 34 120.4 (CH), 114.2, 111.4, 111.0, 85.6 (C), 83.2, 81.3 (CH), 79.7 (C), 71.4 (CH2), 63.1, 59.2 (CH), 57.9,27.4, 26.5, 24.6 (CH3).

Exam pie 2.4: (1 S)-1 -(9-Deazaadenin-9-yl)-1,4-dldeoxy-1,4-imi no-5-O-methyl-D-5 ribitol hydrochloride (5). - A solution of (1 S)-1 -(3-amino-2-cyanopyrrol-4-yl)-W-ferf-butoxycarbonyH ,4-dideoxy-1,4-imino-2,3-0-isopropylidene-5-0-methyl-D-ribitol (4) (0.09 g) in ethanoi (5 mL) containing formamidine acetate (0.048 g) was heated under reflux for 3 h and then concentrated to dryness. Chromatography of the residue gave the product which was dissolved in methanol (5 mL) and conc. HCI (5 10 mL), allowed to stand overnight, and then concentrated to dryness to give (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-0-methyl-D-ribitol hydrochloride (5) as a solid (0.068 g). 1H NMR (DzO) 5 8.44 (s, 1H), 8.05 (s, 1H), 5.01 (d, J = 8.9 Hz, 1H), 4.81 (dd, J = 8.9,4.8 Hz, 1H), 4.48 (dd, J = 4.8, 3.4 Hz, 1H), 4.03-3.98 (m, 1H), 3.85 (dd, J = 11.2, 5.4 Hz, 1H), 3.79 (dd, J = 11.2, 3.9 Hz, 1H), 3.43 (s, 3H); 13C NMR 5 15 149.8 (C), 143.9 (CH), 138.6 (C), 132.9 (CH), 113.0,105.5 (C), 73.8, 71.2 (CH), 68.9-(CH2), 64.3 (CH), 59.1 (CH3), 55.9 (CH).

Example 3: Preparation of (1S)-1-(9-deazaadenin-9-yI)-1,4,5-trideoxy-1,4-imino-D-ribitol hydrochloride (5'-deoxy-lmmucillin-A, Scheme 2) Example 3.1: A/-fert-Butoxycarbonyl-2,3,6,7-tetradeoxy-3,6-imino-4,5-0-isopropylidene-D-a//o-heptononitrile (6). - Tetrabutylammonium fluoride (4 mL, 1M in THF) was added to a solution of /V-tert-Butoxycarbonyl-7-0-ferf-butyldimethylsilyl-2,3,6-trideoxy-3,6-imino-4,5-0-isopropylidene-D-a//o-heptononitrile 25 (3) (0.75 g) in THF (4mL). After 1 h chloroform (20 mL) was added and the solution was washed with water, dried and concentrated to dryness. A solution of the residue in dry methylene chloride (10 mL) was treated with diisopropylethylamine (0.92 mL) and then methanesuifonyl chloride (0.2 mL). After 0.5 h, the solution was washed with 2M aq. HCI, aq. NaHC03, dried and concentrated to dryness. A solution of the 30 product in acetone (10 mL) containing sodium iodide (1.3 g) was heated under reflux for 24 h, and then concentrated to dryness. Chloroform was added and the mixture was washed with water, dried and concentrated to dryness. Tributyitin hydride (1.0 mL) was added to a solution of the crude product in benzene (10 mL) and the solution was heated under reflux. After 0.5 h more tributyitin hydride (0.5 mL) was added and refluxing was continued for a further 1 h. The solution was concentrated 5 to dryness and the residue was redissolved in ether. This solution was stirred with 10 % aq. KF for 1 h, then the organic layer was collected, dried and concentrated to dryness. Chromatography of the residue afforded /V-terf-Butoxycarbonyl-2,3,6,7-tetradeoxy-3,6-imino-4,5-0-isopropylidene-D-a//o-heptononitrile (6) (0.34 g) as a syrup. 1H NMR (CDCfe) 5 4.66 (dd, J = 5.6, 2.4 Hz, 1H), 4.44 (dd, J = 5.6, 1.3 Hz, 10 1H), 4.104.05 (m, 2H), 2.90-2.72 (m, 2H), 1.48 (s, 12H), 1.33 (s, 3H), 1.32 (d, J = 7.0 Hz, 3H); 13C NMR 5 154.3,117.9,1.12.9 (C), 85.4, 82.9 (CH), 81.1 (C), 61.8, 60.2 (CH), 28.7, 27.7, 25.7 (CH3), 22.4 (CH2), 20.3 (CH3).

Example 3.2: (1 S)-1 -{9-Deazaadenin-9-yl)-1,4,5-trideoxy-1,4-imino-D-ribitol 15 hydrochloride (8). - A solution of N-fert-butoxycarbonyl-2,3,6,7-tetradeoxy-3,6-imino-4,5-0-isopropylidene-D-a//o-heptononitrile (6) (0.33 g) in THF (10 mL) containing ethyl formate (0.9 mL) was stirred with sodium hydride (0.18 g, 60 %) for 3 h. Acetic acid (0.5 mL) was added followed by chloroform and the mixture was washed with water, dried and concentrated to dryness. A solution of the crude 20 product in methanol (15 mL) containing sodium acetate (0.91 g) and aminoacetonitriie hydrochloride (0.52 g) was stirred at room temperature for 3 days. Chloroform was added and the solution was washed with water, dried and concentrated to dryness. A solution of the residue in methylene chloride (20 mL) containing DBU (0.85 mL) and methyl chloroformate (0.15 mL) was heated under 25 reflux for 1 h. The cooled solution was washed with 2M aq. HCI, aq. NaHC03 dried and concentrated to dryness. Triethylamine (1 mL) was added to a solution of this material in methanol (10 mL) and after 3 h the solution was concentrated to dryness. Chromatography afforded (1S)-1-(3-amino-2-cyanopyrrol-4-yl)-A/-ferf-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (7) (0.36 g). 30 A solution of this material in ethanoi (10 mL) containing formamidine acetate (0.207 g) was heated under reflux for 3 h and then concentrated to dryness. After 36 chromatography of the residue the product was dissolved in methanol (5 mL) and conc. aq. HCI (5 mL), the solution was allowed to stand at room temperature for 3 h and then concentrated to dryness. Trituration of the residue with ethanoi afforded (1 S)-1-(9-deazaadenin-9-yl)-1,4,5-trideoxy-1,4-imino-D-ribitol hydrochloride (8) 5 (0.218 g) as a white solid. 1H NMR (D20) 5 8.41 (s, 1H), 8.04 (s, 1H), 4.96 (d, J = 8.5 Hz, 1H), 4.88 (dd, J = 8.5, 4.8 Hz, 1H), 4.31 (t, J = 4.5 Hz, 1H), 3.87 (dq, J = 7.1, 4.2 Hz, 1H), 1.54 (d, J = 7.1 Hz, 3H); 13C NMR 6 149.5 (C), 143.7 (CH), 139.2 (C), 132.8 (CH), 113.2,106.2 (C), 74.5, 73.2, 60.8, 56.2 (CH), 16.0 (CH3). 37 Example 4: Preparation of (1S)-1-(7-Amino-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol.2HCI (8-aza-5'-methylthio- Immucillin-A, Scheme 3) Scheme 3 Example 4.1: (1 S)-5-0-tert-Butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-1-(7-methoxy-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-D-ribitol (1). - n-BuLi (1.6M, 8.2 mL, 13.1 mmol) was added 10 dropwise to a stirred solution of 3-bromo-7-methoxy-2-(tetrahydropyran-2-yl)-pyrazolo[4,3-d]pyrimidine (4.3 g, 13.7 mmol) and anhydrous THF (30 mL) at -78°C until no starting material remains. A THF (10 mL) of imine (3.5 g, 12.3 mmol), 38 dissolved in THF (30 mL), was added dropwise via cannula followed by SnCI4 (0.51 ml, 4.4 mmol) at such a rate that the reaction temperature was maintained below -70°C. The reaction mixture was allowed to warm to r.t. and then quenched by addition of 15% NaOH (30 ml). Diethyl ether (200 ml) was added and the organic 5 phase separated, dried (MgS04) and concentrated in vacuo. Chromatography afforded (1S)-5-0-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-1 -(7-methoxy-2-tetrahydropyran-2-yl-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-D-ribitol (1) (2.8 g, 44%) as a clear oil. This exists as a diastereomeric mixture because of the THP-group. 1H-NMR (CDCI3): 5 8.37 (1H, s), 5.96, 5.86 (1H, m), 5.31, 4.98 (1H, m), 10 4.75 (2H, m), 4.15 (3H, s), 4.02 - 3.70 (4H, m), 3.33, 3.26 (1H, m), 2.60 (1H, m), 2.08 (2H, m)1.70 (1H, m), 1.59, 1.56 (3H, s), 1.34, 1.32 (3H, s), 0.88, 0.85 (9H, s), 0.05, 0.03, 0.00 (6H, s). "C-NMR (CDCI3) 8 162.3, 151.4, 139.6, (135.5, 135.3), 131.7, (114.9, 114.4), (87.6, 87.1), (86.4, 85.9), (83.1, 82.8), (68.3, 68.0), (66.9, 66.4), (63.0, 62.3), (61.7, 61.5), 54.3, (30.0, 29.7), (28.0, 28.0), 26.2, (25.9, 25.8), 15 25.2,(22.8,22.6), 18.6,-4.9.

Example 4.2: (1S)-1-(7-Amino-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-/¥-(fert-butoxycarbonyl)-5-0-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (2). ■ Di-tert-butyl dicarbonate 20 (1.4 g, 6.5 mmol) was added portionwise to a stirred solution of (1S)-5-0-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-1 -(7-methoxy-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-D-ribitol (1) (2.8 g, 5.4 mmol) in methanol (40 mL) at room temperature. After 30 min the reaction was complete and purification by flash chromatography provided two separate diastereomers (2.2 25 g, 66%) as yellow oils. The faster running of the two diasteromers (900 mg, 1.45 mmol) was redissolved in 7N NH3 in methanol and the resulting solution heated in a sealed tube at 100 °C overnight. The reaction was concentrated in vacuo and purification of the resulting residue by chromatography afforded (1S)-1-(7-Amino-2-tetrahydropyran-2-yl-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-/V-(fert-butoxycarbonyl)-5-0-30 tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (2) (0.52 g, 59%) as an oil. 13C-NMR (CDCI3) 5 156.5, 155.5, 152.9, 136.4, 133.8, 131.7, 39 112.0, 85.9, 83.9, 80.8, 68.9, 62.7, 59.4, 31.5, 28.7, 27.7, 26.2, 25.8, 25.2, 23.2, 18.6, -4.9.

Example 4.3: (1 S)-1 -{7-/V-Benzoyl-(7-amino-2-tetrahydropyran-2-yl-1 H-5 pyrazolo[4,3-d] pyrimidin-3-yl)}-W-(fert-butoxycarbonyl)-1,4-dideoxy-1,4-imlno-2,3-O-isopropylidene-D-ribitol (3). - (1S)-1-(7-Amino-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)-A/-(fe/f-butoxycarbonyl)-5-0-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (2) (200 mg, 0.33 mmol) was dissolved in acetonitrile and then benzoyl tetrazole (130 mg, 0.74 mmol) and DMAP 10 (45 mg, 0.36 mmol) were added consecutively. The resulting solution was stirred at reflux for 0.5 h. and then cooled to r.t.. The reaction was diluted with ethyl acetate and the organic layer washed with 10% HCI, saturated NaHC03 and brine, the organic layer was then dried (MgSO<) filtered and concentrated in vacuo. The crude residue was redissolved in THF (5 mL) and treated with acetic acid (60 DL) and n-15 tetrabutylammonium fluoride (700 DL, 1M in THF) and allowed to stir for 48 h at r.t.. The reaction was preabsorbed onto flash silica gel (5 g) and purified by chromatography to afford (1 S)-1-{7-A/-benzoyl-(7-amino-2-tetrahydropyran-2-yl-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)}-A/-(ferf-butoxycarbonyl)-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (3) (190 mg, 97%) as an oil. 13C-NMR (CDCI3) 5 154.1, 20 152.1,138.5,137.5,134.9, 133.2, 132.1,129.0, 128.8, 112.6, 88.4, 87.1, 85.6, 84.9, 83.6, £3.0, 80.8, 68.9, 68.1, 66.0, 63.0, 62.5, 60.7, 30.1, 28.6, 28.4, 26.0, 25.1, 22.9, 21.3.

Example 4.4: (1 S)-5-acetylthio-1 -{7-W-Benzoyl-(7-amino-2-tetrahydropyran-2-yl-25 1H-pyrazoIo[4,3-d]pyrimidin-3-yl)}-/V-(fert-butoxycarbonyl)-1,4-dideoxy-1,4- imino-2,3-0-isopropylidene-D-n'bitol (4). - DIAD (0.13 mL, 0.65 mmol, 95%) was added dropwise to a THF (5 mL) solution of triphenylphosphine (0.17 g, 0.65 mmol) at 0 °C and left to stir. After 0.5 h a THF (5 mL) solution of (1S)-{N-benzoyl-(7-amino-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)}-A/-(ferf-30 butoxycarbonyl)-1,4-dideoxy-1,4-imino-2,3-0-isopropylidene-1-D-ribitol (3) (190 mg, 0.32 mmol) and thiolacetic acid (50 pL) was added dropwise maintaining the reaction 40 at 0 °C and then the resulting solution was allowed to warm to r.t.. After 1 h at r.t., the reaction was concentrated in vacuo and the resulting residue purified by chromatography to afford (1S)-5-acetylthio-1-{7-W-Benzoyl-(7-amino-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yl)}-W-(fe/f-butoxycarbanyl)-1,4- dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (4) (130 mg, 62%). "C-NMR (CDCI3) 8 194.8, 155.0, 152.0, 135.3, 133.0, 128.9, 112.7, 86.6, 84.9, 84.1, 81.4, 68.7, 66.3, 59.6, 30.8, 28.7, 27.7, 25.7, 25.1, 23.0, 22.3.

Example 4.5: (1 S)-1 -(7-Amino-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-10 1,4-imino-5-methylthio-D-ribitol.2HCI (5). - A methanolic solution of sodium thiomethoxide (2 mL, 0.1 M) was added dropwise to a solution of (1S)-5-acetylthio-1-{/V-benzoyl-(7-amino-2-tetrahydropyran-2-yl-1H-pyrazolo[4,3-d]pyrimidin-3-yi)}-A/-(ferf-butoxycarbonyl)-l ,4-dideoxy-1,4-imino-2,3-0-isopropylidene-D-ribitol (4) (80 mg, 0.12 mol) in anhydrous methanol and cooled to 0 °C. The reaction was allowed 15 to warm to r.t. and then left for 0.5 h after which time the reaction was quenched with methyl iodide (0.2 mL, xs) and the resulting reaction left to stir overnight. The reaction mixture was partitioned between chloroform and water, the organic layer separated and dried (MgS04) and the residue purified by chromatography. The resulting residue was dissolved in 1:1 cHCI:MeOH and left to stand overnight. 20 Concentration in vacuo followed by trituration with methanol and diethyl ether yielded (1 S)-1 -(7-amino-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol.2HCI (5).2HCI. 1H-NMR (D20) 5 8.44 (1H, s), 5.22 (1H, d, J 8.8 Hz), 4.80 (1H, t, J 4.9 Hz), 4.52 (1H, t, J 5.7 Hz), 3.99 (1H, dt, J 9.6, 5.7 Hz), 3.09 (2H, m), 2.19 (3H, s). 13C-NMR (DzOyS 152.11 (CH), 151.75, 138.8, 138.5, 123.3 25 (C), 74.3, 74.0, 62.3, 58.6 (CH), 35.0 (CH2), 14.9 (CH3). 41 Example 5: Preparation of (ISJ-l-^-deazaadenin-S-ylJ-l^dideoxy-IAiinino-S-(substituted)thio-D-ribitols (Scheme 4) Scheme 4 TBDMSO—i gOC cn H0 BOC CN RS BOC CN h ~ " h iuii ' w 3£ Reagents: i, b114nf; ii, MsCI, !Pr2NEt; iii, NaH, RSH, DMF; iv, NaH, EtOCHO, THF; v, H2NCH2CN, NaOAc, MeOH; vi, MeOCOCI, DBU; vii, MeOH, E^N; viii, formamidlne acetate EtOH; ix, MeOH, aq HCI.

Example 5.1: A/-7erf-butoxycarbonyl-3,6-imino-4,5-0-isopropylidene-2,3,6-trldeoxy-D-a//o-heptononitrile (2). - Tetrabutylammonium fluoride (75 mL, 1M in 10 THF) was added to a solution of the product 1 (Scheme 4.3, prepared as described in Example 2.2) (19.1 g, 44.8 mmol) in THF (50 mL) and the solution was allowed to stand for 1 h. Chloroform (350 mL) was added and the solution was washed twice with water, dried and concentrated to dryness. Chromatography of the residue afforded A/-ferf-butoxycarbonyl-316-imino-4,5-0-isopropylidene-213,6-trideoxy-D-a//o-15 heptononitrile (2) as a syrup (13.8 g, 98 %). 1H NMR (CDCI3) 5 4.6-4.05 (m, 4H), 3.61 (brs, 1H), 3.40 (brs, 1H), 2.7-2.2 (m, 3H), 1.35, (s, 12H), 1.14 (s, 3H). 42 Example 5.2: JV-Teft-butoxycarbonyl-5-ethylthio-3,6-imino-4,&-C>- isopropylidene-2,3,6-trideoxy-D-a//o-heptononitrile (3, R = Et). - A solution of the product from example 5.1 (0.51 g) in dichloromethane (8 mL) was treated with diisopropylethylamine (0.56 mL) and methanesulfonyl chloride (0.185 mL). After 1 h, 5 the solution was washed with dil HCI, aq NaHC03l dried and concentrated to dryness. A solution of the residue in DMF (2 mL) was added to a solution resulting from the addition of ethanethiol (0.24 mL) to a mixture of sodium hydride (0.13 g, 60 % dispersion) in DMF (5 mL). The resulting mixture was stirred for 1 h, then toluene was added and the mixture was washed twice with water, dried and concentrated to 10 dryness. Chromatography of the residue afforded syrupy title compound 3 (R = Et) (0.55 g).

Example 5.3: (1S)-1-(3-Amino-2»cyanopyrrol-4-yl)-AWerf-butoxycarbonyl-1,4-dideoxy-5-ethylthio-1,4-imino-2,3-0-isopropylidene-D-ribitol (4, R = Et). - Ethyl 15 formate (1.1 mL) was added to a solution of 3 (R = Et) (0.48 g) in THF (10 mL) followed by sodium hydride (0.22 g, 60 % dispersion). The mixture was stirred for 2 h, then acetic acid (0.6 mL) was added and the solution was partitioned between chloroform and water, the organic phase was dried and concentrated to dryness. A solution of the residue in methanol (15 mL) was treated with sodium acetate (1.1 g), 20 and aminoacetonitriie hydrochloride (0.62 g) and the mixture was stirred for 16 h and then was heated under reflux for 0.5 h. The resulting mixture was partitioned between chloroform and water, the organic phase was dried and concentrated to dryness. A solution of the residue in dichloromethane (20 mL) was treated with DBU (1.24 mL) and methyl chloroformate (0.3 mL) and the solution was stirred for 16 h, 25 then was washed with dil HCI and aq NaHC03, dried and concentrated to dryness. The residue in methanol (10 mL) was treated with triethylamine (1 mL). After 2 h at room temperature the solution was concentrated to dryness. Chromatography of the residue gave syrupy 4 (R = Et) (0.49 g).

Example 5.4: (1S)-1-(9-Deazaadenin-9-yl)-1,4~dideoxy-5-ethylthio-1,4-imino-D-ribitol. - A solution of the product from Example 5.3 in ethanoi (15 mL) containing 43 formamidine acetate (0.25 g) was heated under reflux for 3 h and then concentrated to dryness. Chromatography afforded 0.47 g of material which was dissolved in methanol (10 mL) and 4M HCI (10 mL). After 6 h at room temperature the solution was concentrated to dryness. Trituration with ethanoi or propan-2-ol gave the title 5 compound as a bis hydrochloride salt, white solid (0.275 g) with m.p. 204-212 °C (dec).

The following compounds were prepared by the same sequence of reactions as above except that the appropriate thiol replaced ethanethiol. (1S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-phenylthio-D-ribitol bis hydrochloride. M.p. 180-182 °C; 13C NMR (D20) 8 149.1, 143.4, 139.5, 133.0, 132.9,130.9,130.0, 128.1,113.0, 105.9, 73.2, 72.6, 63.8, 56.7, 33.8. (1S)-1-(9-Deazaadenin-9-yl)-5-benzylthio-1,4-dideoxy-1,4-imino-D-ribitol bis hydrochloride. 13C NMR (DzO) S 149.2, 143.5, 138.1, 133.0, 129.4, 128.0, 113.0, 105.9, 73.2, 72.6, 63.9, 56.6, 35.6, 30.8. (1S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(2-hydroxyethyl)thio-1,4-lmino-D-20 ribitol bis hydrochloride. 13C NMR (D20) 5 149.4, 143.6, 139.4, 133.0, 113.1, 105.8, 73.2, 72.6, 64.3, 60.8, 56.5, 34.2, 31.7. (1S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(4-methylphenyl)thio-D-ribitol bis hydrochloride. 13C NMR (DzO) 8 149.0, 143.3, 139.6, 139.0, 133.0, 25 131.6,130.6,129.0,113.0,105.9, 73.3, 72.6, 63.9, 56.8, 34.4, 20.5. (1 S)-1 -(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(3-methylphenyl)thio-D-ribitol bis hydrochloride. 13C NMR (D20) 8 149.00, 143.3, 140.4, 139.6, 133.0, 132.7,131.2, 129.8,128.8, 127.8, 113.0, 105.9, 73.3, 72.6, 63.8, 56.7, 33.8, 20.8.

PCT/N Z03/00050 44 (1 S)-1 -{9-Deazaadenin-9-y l)-5-(4-chlorophenyl)thio-1,4-dldeoxy-1,4-imi no-D-ribitol bis hydrochloride. 13C NMR (D20) 8 149.0, 143.3, 139.7, 133.6, 133.0, 132.4,131.6,129.8,113.0,105.9, 73.3, 72.6, 63.6, 56.7, 34.0. (1S)-1-(9-Deazaadenin-9-yl)-5-(3-chlorophenyl)thio-1,4-dideoxy-1,4-imino-D-rlbitol bis hydrochloride. 13C NMR (DzO) 8 148.9, 143.3, 139.6, 135.1, 134.8, 133.1,131.1,129.9,128.8,128.0,113.0, 105.9, 73.3, 72.6, 63.6, 56.8, 33.6. (1 S)-1 -(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(4-fluorophenyl)thio-1,4-imino-D-10 ribitol bis hydrochloride. 13C NMR (D20) 8 162.8 (d, Jc.f = 245 Hz), 149.1,143.4, 139.6, 134.1 (d, Jqf = 8.4 Hz), 133.0, 127.9, 116.9 (d, Jc,f = 22 Hz), 113.0, 105.8, 73.2, 72.5, 63.7, 56.6, 35.0. (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(1 -naphthyl)thio-D-ribitol 15 bis hydrochloride. 13C NMR (D20) 8 142.7, 132.9, 130.3, 130.1, 129.2, 127.6, 127.3,126.4,124.6, 73.2, 72.8, 63.9, 57.2, 33.5.

Example 5.5: (1S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(2-fluoroethyJ)thio-1,4-imino-D-ribitol. - A solution of N-ferf-butoxycarbonyl-5-(2-hydroxyethyl)thio-3,6-20 imino-4,5-0-isopropylidene-2,3,6-trideoxy-D-a//o-heptononitrile (3, R = CH2CH2OH) (1.0 g) in dry chloroform (10 mL) was treated with DAST (0.71 mL) and the solution was allowed to stand for 16 h, then was washed with water, aq NaHCOs, dried and concentrated to dryness. Chromatography afforded syrupy A/-tert-butoxycarbonyl-5-(2-fluoroethyl)thio-3,6-imino-4,5-0-isopropylidene-2,3,6-trideoxy-D-a//o-heptononitrile 25 (3, R = CH2CH2F) (0.558 g). This material was converted into the title compound by the same sequence of reactions as above in Examples 5.3 and 5.4 to give a solid bis-hydrochloride salt (0.307 g). 13C NMR (D20) 8 149.4,143.6,139.5,133.1,113.1, 105.8, 84.2 (d, Jc,f= 164 Hz), 73.2, 72.6, 64.2, 56.5, 31.9, 31.9 (d, Jc.f = 20 Hz). 45 Example 6: Preparation of (1S)-1-(9-deazaadenin-9-yl)-1,4,5-trideoxy-5-C-ethyl-1,4-imino-D-ribitol (Scheme 5) Scheme 5 h°-| n°/-GN °HC ®oc- w HO OH Reagents: i, Dess-Marfin periodinane; ii, Ph 3P=CHCH 3; iii, H 2, Pd/C; iv, NaH, EtOCHO, THF; H2NCH2CN, NaOAc, MeOH; vi, MeOCOCI, DBU; vii, MeOH, Et3N; viii, fbrmamidine acetate EtOH; ix, MeOH, aq HCI.

Example 6.1: W-7ert-butoxycarbonyl-3,6-imino-4,5-0-lsopropylidene- 2,3,6,7I8,9-hexadeoxy-D-a//o-nonononitrile. - Dess-Martin periodinane (1.42 g) 10 was added to a solution of A/-ferf-butoxycarbonyl-3,6-imino-4,5-0-isopropylidene-2,3,6-trideoxy-D-a//o-heptononitrile (Example 5.2, Compound (2) of Scheme 4) (0.7 g) in dichloromethane (20 mL) and the resulting mixture was stirred for 1 h. After concentrating to dryness, ether (20 mL) was added to the residue and the mixture was washed twice with 10 % aq Na^CVsat. aq NaHC03 (1:1 v/v), dried and 15 concentrated to dryness. A solution of this residue in THF (8 mL) was added to the red solution resulting from addition of n-butyllithium (3.4 mL, 1.6 M) to a suspension of ethyltriphenylphosphonium iodode (2.44 g) in THF (25 mL). After 0.5 h, the mixture was diluted with petroleum ether (100 mL) and washed with water, dried and concentrated to dryness. Chromatography afforded a syrup (0.34 g). This material 46 in ethanoi (10 mL) containing 10 % Pd/C (0.05 g) was stirred in an atmosphere of hydrogen for 2.5 h, then the solids and solvent were removed. Chromatography afforded syrupy title compound (0.282 g).

Example 6.2: (1S)-1-(9-Deazaadenin-9-yl)-1,4,5-trideoxy-5-ethyl-1,4-imino-D-ribitol bis hydrochloride. The material from example 6.1 was treated with the same sequence of reactions as in examples 5.3 and 5.4 above to give title compound as a white solid bis-hydrochloride salt (0.095 g) with m.p. 206-215 °C. 13C NMR (DzO) 5 149.7, 143.8, 138.8, 132.9, 113.1, 105.6, 73.0, 72.9, 65.1, 55.9, 10 32.8,19.4,13.2.

Example 7: Enzyme Inhibition Results Enzyme assays were conducted to assess the effectiveness of selected compounds 15 of the invention as inhibitors of MTAP and MTAN. The results are collected in Tables 1 and 2 and shown in Figure 1.

Enzymes. - The human MTAP protein was cloned into pQE32 expression vector and transformed into E. coli. Induction cultures containing 1 L (25 mg/L) and 50 ng/mL 20 ampicillin were inoculated with 1 mL overnight grown starter-culture and incubated at 37°C. When the cultures reached an OD of «0.6 they were induced with 1.5 mM IPTG for 6 to 8 hours. Cells were harvested by centrifugation at 5000 rpm for 30 min, and subsequently resuspended in a buffer (20 mM imidazole, 300 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM Tris, pH 8.0) containing a small 25 amount of lysozyme to weaken the cell membrane. Cells were lysed with a French press. The insoluble material was removed by high-speed centrifugation. The cell extract was further clarified with 35% ammonium sulfate precipitation followed by high-speed centrifugation. The clarified cell extract was then applied to a 5 mL Ni-NTA column that had previously been equilibrated with the binding buffer. Further 30 chromatographic steps were carried out by FPLC. The column was washed with 10 volumes of 50 mM imidazole, 300 mM NaCl, and 100 mM Tris, pH 8.0, and the 47 protein was eluted with a buffer containing a 50-250 mM gradient of imidazole, 300 mM NaCl, 1 M Tris, pH 8.0. The purity of the protein was verified by running polyacrylamide gel followed by Coomassie staining. The protein was subsequently dialyzed in 50 mM NaCl, 2 mM dithiothreitol (DTT) and 50 mM Tris, pH 7.4, and was 5 concentrated to 10 mg/mL. The purified protein was stored at -80 °C in 100 |J.L to 150p.L aliquots.

Inhibitors. - Inhibitor concentrations were determined by the UV absorbance spectrum using the published millimolar extinction coefficients for 9-dazadenine of 10 8.5 at 275 nm at pH 7.0 .

Assays. - The direct spectrophotometry assay for the conversion of MTA into adenine was measured as the decrease in absorbance at 274 nm. At 274 nm, Ae between MTA and adenine is at a maximum and produces a spectral change of 1.6 15 absorbance units/cm/mM (DeWolf, W.E.Jr., Fullin, F.A., and Schramm, V.L. (1979) J. Biol. Chem. 254,10868-10875) Slow-Onset Inhibition and Inhibition Constants. - The kinetics for slow onset inhibition and the K} measurement was carried out by adding a known concentration 20 of enzyme (1-5 nM) to a reaction mixture containing a substrate concentration of 200 |j.M. This concentration corresponds to an OD of 0.7-1.1 at 274 nM. The formation of product was monitored as a decrease in absorbance at 274 nm. Conditions for K* determination used high concentrations of substrate. Two controls, one having no inhibitor and the other no enzyme were included in the experiment. The Kt values 25 MTAP for the various compounds were calculated by fitting in the ratio of initial rates in the presence of inhibitor to without any inhibitor versus the inhibitor concentration, for the known Km and substrate concentration into the following expression. 48 V0' Kr,+ [S] V0 Km* [S] + Km [1] K, Where V0' is the rate in the presence of inhibitor V0 rate in the absence of inhibitor [I] inhibitor concentration 10 And [S] is the substrate concentration And the Ki* was calculated by fitting to the following expression V,' Km+ [S] Vs Km+ [S] + Km [I] Kr Where Vs' is the steady-state rate following attainment of equilibrium in the presence inhibitor, and Vs is the steady-state rate in the control having no inhibitor. Both these equations are valid for competitive type inhibition.

Inhibitor Release Studies. - The enzymes and the inhibitor were preincubated at 25 the indicated concentrations for 3-4 h in 50 mM potassium phosphate, pH 7.4. At the indicated times the samples were diluted by the factors of 1:10000 to 1:1000000 into assay mixtures, and the rate of product formation was determined as a function of time. Control incubations had all components but inhibitors. To accommodate slow dissociation of enzyme and inhibitor, very high concentration of substrates and low 30 concentration of enzymes were used. 49 Competitive Inhibition. - The nature of inhibition is established by constructing a four by four-double reciprocal plot. The substrate concentrations were chosen such that they were both below and above the Km value of the enzyme, and the inhibitor concentrations were around the dissociation constant of the enzyme. The reaction is 5 started by adding the enzyme solution to each of the 16 reaction mixtures containing above-mentioned substrate and inhibitor concentrations. The initial rates were calculated. The reciprocal of initial rates were plotted as a function of inverse of substrate concentration to get Lineweaver-Burke plot. For competitive inhibition the point of intersection with the y-axis give us "\Nmax. The slope of the curve is a KJ 10 Vmax, where a = 1 + [l]/Ki.

Example 8: Demonstration of Radiation Sensitizing Effect of 5'-Methyithio-ImmA IS Lewis lung carcinoma cells (IxlO6) were plated and allowed to adhere overnight in 2 mL of DMEL medium substituted with 10% fetal bovine serum, 1% Pen-Strep, 2.5% Na-Pyruvate, 1% non-essential amino acids in 6 well plates. 50 jiM MTA, 50 p.M MTA + 2 n-M 5'-methylthio-lmmA or 2 |iM 5'-methylthio-lmmA was then added in 1 mL of the same medium as indicated. Control wells were treated with medium 20 without any additions. This treatment was allowed to continue for 6 hours. In each experiment one set of treated cells were subjected to 10Gy of X-ray irradiation and a control set received no irridation. Both irradiated and unirradiated cells were then cultured for another 48 hours in the presence of MTA ± 5'-methylthio-lmmA. Manual counting of living and dead cells was done by Trypan Blue dye exclusion following 25 48 hrs of growth. (Approximate doubling time for Lewis Lung cells was 24 hours.) The results are shown in Figure 2, and indicate: Control—irradiation reduces cell numbers by 50% in the absence of additives. MTA at 50 nM reduces growth of cells, but slightly protects from irradiation damage. 30 5'-methylthio-lmmA at 2 p.M acts in a similar manner to 50 pM MTA. 50 '-methylthio-lmmA + MTA is an irradiation sensitizer, lowering cancer cell number following irradiation.

Example 9: Demonstration of Tissue Availability of 5'-Methylthio-lmmA in Mice '-Methylthio-lmmucillin-A (5'-methylthio-lmmA) (10 micromoles) was administered to mice orally, by interperitoneal injection or by intravenous injection. Following 30 to 60 min, biood was collected or mice were sacrificed and the liver removed for tissue analysis. MTAP activity in mouse blood was measured by the conversion of 10 radioactive MTA to radioactive 5-methylthio-a-D-ribose 1-phosphate (MTR-1P). The assay mixture contained 50 mM phosphate buffer, 1 mM dithiothreitol, 26 (iM [5 -14C]MTA with specific radioactivity of 2 nCi/nmole, 0.5% triton X-100, and the desired amount of tissue sample, in a total volume of 100 p.L. Reactions were stopped at various times by the addition of perchloric acid to decrease the pH to 2.0. The 15 protein precipitate was removed by centrifugation, and the supernatant was neutralized to near pH 7 before being placed on a charcoal column. The column was eluted with buffer near pH 7. The product MTR-1P elutes, while unreacted [5-14C]MTA remains on the column. The amount of MTAP activity from control and treated mice is compared.

Figure 3 shows the effect of 5'-methylthio-lmmA on MTAP activity in mouse blood.

Liver protein extracts from control mice converted MTA to products at a rate of 1.0 nMole/min/mg of liver protein extract. Following oral administration of 10 nmoles of 25 5'-methylthio-lmmA, the MTAP in extracts of liver converted MTA to products at a rate of 0.09 nMole/min/mg of liver protein extract treatment, corresponding to 90% inhibition. Therefore 5'-methylthio-lmmA is orally available to the MTAP present inside tissues. In a similar experiment where 5'-methylthio-lmmA was provided by intravenous injection, there was no detectable MTAP activity in liver extracts, 30 indicating that >95% of inhibiton occurred. To estimate the sensitivity of the liver tissue MTAP to the administration of 5'-methylthio-lmmA, mice were injected by 51 intraperitoneal injection with 0.1 or 1.0 micromoles of MT-lmm-A. In this protocol injection of 0.1 micromole of 5-methylthio-lmmA reduced the MTAP activity of liver extract by 70% and injection of 1.0 micromole of 5'-methylthio-lmmA reduced the MTAP activity of liver extract by 77%. Interperitoneal injection of 10 micromoles of 5 5'-methylthio-lmmA also inhibited the activity of MTAP found in mouse blood. Blood sampled 30 min following 5'-methylthio-lmmA injection was >90% inhibited - compared to control blood.

Figure 4 shows inhibition of mouse liver MTAP by 5'-methylthio-lmmA.

Although the invention has been described by way of examples, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the 15 specification.

INDUSTRIAL APPLICABILITY The present invention relates to compounds that are inhibitors of MTAP and MTAN. 20 The compounds are therefore expected to be useful in the treatment of diseases in which the inhibition of MTAP and MTAN is desirable. Such diseases include cancer, bacterial infections and protozoan parasitic infections. 52

Claims (33)

1. A compound of the formula (I): OH X wherein: A is selected from N, CH and CR, where R is selected from halogen, alkyl, aralkyl and aryl, OH, NH2, NHR1, NR1R2 and SR3, where R1, R2 and R3 are each alkyl, aralkyl or aryl groups; B is selected from NH2 and NHR4, where R4 is an alkyl, aralkyl or aryl group; X is selected from H, OH and halogen; and Z is selected from H, Q, SQ and OQ, where Q is alkyl optionally substituted with OH or halogen; aralkyl optionally substituted with OH, alkyl or halogen; or aryl optionally substituted with OH, alkyl or halogen; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; 2 $ MAX 2007 RECEIVES- WO 03/080620 PCT/NZ03/00050 53 with the proviso that the stereochemistry of the aza-sugar moiety is D-ribo or 2'-deoxy-D-erythro-.

2. A compound as claimed in claim 1, where A is CH.

3. A compound as claimed in claim 2, where Z is SQ.

4. A compound as claimed in any one of claims 1 to 3, where B is NH2.

5. A compound as claimed in claim 4, where Z is SQ.

6. A compound as claimed in claim 5, where Q is C^Cs alkyl.

7. A compound as claimed in any one of claims 1, 4, 5 or 6 where A is N.

8. A compound as claimed in claim 7, where Z is SQ.

9. A compound as claimed in claim 8, where Q is CrC5 alkyl.

10. A compound as claimed in any one of claims 1 to 9, where X is OH.

11. A compound as claimed in any one of claims 1 to 10, where Z is SQ.

12. A compound as claimed in claim 11, where Q is CrC5 alkyl.

13. A compound as claimed in claim 11, where Q is an aryl group optionally substituted with OH, alkyl or halogen.

14. A compound as claimed in claim 11, where Q is selected from phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-fluorophenyl, 3-methylphenyl, 4- WO 03/080620 PCT/NZ03/00050 54 methylphenyl, benzyl, hydroxyethyl, fluoroethyl, naphthyl, methyl and ethyl.

15. A compound as claimed in claim 1, selected from 5 (1 S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol; (1 S)-1-(9-deazaadenin-9-yl)-1,4,5-trideoxy-1,4-imino-D-ribitol; (1 S)-1 -(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-0-methyl-D-ribitol; (1 S)-1-(7-Amino-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-10 methylthio-D-ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-5-ethylthio-1,4-imino-D-ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-phenylthio-D-ribitol; (1 S)-1 -(9-Deazaadenin-9-yl)-5-benzylthio-1,4-dideoxy-1,4-imino-D-ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(2-hydroxyethyl)thio-1,4-imino-D-15 ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(4-methylphenyl)thio-D-ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(3-methylphenyl)thio-D-ribitol; 20 (1 S)-1 -(9-Deazaadenin-9-yl)-5-(4-chlorophenyl)thio-1,4-dideoxy-1,4-imino-D- ribitol; (1 S)-1 -(9-Deazaadenin-9-yl)-5-(3-chlorophenyl)thio-1,4-dideoxy-1,4-imino-D-ribitol; (1 S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(4-fluorophenyl)thio-1,4-imino-D-25 ribitol; (1S)-1-(9-Deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-(1-naphthyl)thio-D-ribitol; (1 S)-1 -(9-Deazaadenin-9-yl)-1,4-dideoxy-5-(2-fluoroethyl)thio-1,4-imino-D-ribitol; and (1 S)-1 -(9-Deazaadenin-9-yl)-1,4,5-trideoxy-5-ethyl-1,4-imino-D-ribitol; WO 03/080620 PCT/NZ03/00050 55 or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof.

16. A pharmaceutical composition comprising a pharmaceutically effective amount 5 of a compound as claimed in any one of claims 1 to 15.

17. A method of treating a disease or condition in which it is desirable to inhibit MTAP comprising administering a pharmaceutically effective amount of a compound as claimed in any one of claims 1 to 15 to a non-human patient 10 requiring treatment.

18. The method as claimed in claim 17 where the disease is cancer.

19. The method as claimed in claim 17 where the disease is a protozoan parasitic 15 infection.

20. The method as claimed in claim 19 where the disease is malaria.

21. The use of a compound as claimed in any one of claims 1 to 15 in the 20 manufacture of a medicament for treating a disease or condition in which it is desirable to inhibit MTAP.

22. The use as claimed in claim 21 where the disease is cancer. 25

23. The use as claimed in claim 21 where the disease is a protozoan parasitic infection.

24. The use as claimed in claim 23 where the disease is malaria. 30

25. A method of treating a disease or condition in which it is desirable to inhibit MTAN comprising administering a pharmaceutically effective amount of a INTELLECTUAL PROPERTY OFFICE OF N.Z 11 apr 2007 received WO 03/080620 PCT/NZ03/00050 56 compound as claimed in any one of claims 1 to 15 to a non-human patient requiring treatment.

26. The method as claimed in claim 25 where the disease is a bacterial infection. 5

27. The use of a compound as claimed in any one of claims 1 to 15 in the manufacture of a medicament for treating a disease or condition in which it is desirable to inhibit MTAN. 10

28. The use as claimed in claim 27 where the disease is a bacterial infection.

29. A compound as claimed in claim 1 as specifically set forth herein.

30. A compound as claimed in claim 1 substantially as herein described with 15 reference to any one of the Examples.

31. A pharmaceutical composition as claimed in claim 16 substantially as herein described with reference to any example thereof. 20

32. A method as claimed in claim 17 or claim 25 substantially as herein described with reference to any example thereof.

33. A use as claimed in claim 21 or claim 27 substantially as herein described with reference to any example thereof. 25 intellectual property OFHCF OF N.Z 11 apr 2007 received