WO2008117047A1 - Pyrazolo[3, 4-d]pyrimidine derivatives as antibacterial compounds - Google Patents

Abstract

This invention relates to 6-oxy-4-amino-1-(tetrahydrofuranyl)-1H- pyrazolo[3,4-d]pyrimidin-3(2H)-ones in free or salt form, e.g., compounds of formula (I) and formula (II) their use, e.g., in the treatment of bacterial infections and the process of preparing said compounds.

Description

PYRAZOLO [3, 4-D] PYRIMIDINE DERIVATIVES AS ANTIBACTERIAL -COMPOUNDS

Background of the Invention

The international microbiological community continues to express serious concern that the evolution of antibacterial resistance could result in bacterial strains against which currently available antibacterial agents will be ineffective. In general, bacterial pathogens may be classified as either Gram-positive or Gram-negative pathogens. Antibiotic compounds with effective activity against both Gram-positive and Gram-negative pathogens are generally regarded as having a broad spectrum of activity.

Gram-positive pathogens, for example staphylococci, enterococci, streptococci and mycobacteria, are particularly important because of the development of resistant strains that are both difficult to treat and difficult to eradicate from the hospital environment once established. Examples of such strains are methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant coagulase-negative staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiple resistant Enter ococcus faecium. The preferred clinically effective antibiotic of last resort for treatment of such resistant Gram-positive pathogens is vancomycin. Vancomycin is a glycopeptide and is associated with various toxicities, including nephrotoxicity. Furthermore, and most importantly, antibacterial resistance to vancomycin and other glycopeptides is also appearing. This resistance is increasing at a steady rate rendering these agents less effective in the treatment of Gram-positive pathogens. There is also increasing resistance to agents such as β-lactams, quinolones and macrolides used for the treatment of upper respiratory tract infections caused by Gram-negative strains including H. influenzae and M. catarrhalis. Consequently, in order to overcome the threat of widespread multi-drug resistant organisms, there is an on-going need to develop new antibacterials, particularly those with either a novel mechanism of action and/or containing new pharmacophoric groups.

Deoxyribonucleic acid (DNA) ligases catalyze the formation of a phosphodiester linkage at single-strand breaks between adjacent 3'-OΗ and 5'-phosphate termini in double-stranded DNA (Lehman 1974. Science 186: 790-797). This activity plays an indispensable role in DNA replication where it joins Okazaki fragments. DNA ligase also plays a role in repair of damaged DNA and in recombination (Wilkinson 2001. Molecular Microbiology 40: 1241 - 1248). An early report describing conditional lethal mutations in the DNA ligase gene (UgA) of Escherichia coli supportedthe essentiality of this enzyme (Dermody et al. 1979. Journal of Bacteriology 139: 701-704). ^" This was followed by the isolation and characterization of DNA ligase temperature-sensitive or knockout mutants of Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (Park et al. 1989. Journal of Bacteriology 171: 2173-2180, Kaczmarek et al. 2001. Journal of Bacteriology 183: 3016-3024, Petit and Ehrlich. 2000. Nucleic Acids Research 28: 4642-4648). In all species, DNA ligase was shown to be essential.

The DNA ligase family can be divided into two classes: those requiring ATP for adenylation (eukaryotic cells, viruses and bacteriophages), and those requiring NAD⁺ (nicotinamide adenine dinucleotide) for adenylation, which include all known bacterial DNA ligases (Wilkinson 2001, supra). Eukaryotic, bacteriophage, and viral DNA ligases show little sequence homology to DNA ligases from prokaryotes, apart from a conserved KXDG motif located within the central cofactor-binding core of the enzyme. Amino acid sequence comparisons clearly show that NAD⁺-dependent ligases are phylogenically unrelated to the ATP-dependent DNA ligases. The apparent lack of similarity between the DNA ligases of bacteria and those of higher organisms suggests that bacterial DNA ligase is a good target for developing new antibacterials.

In 2003, Brotz-Oesterhelt et al. (Journal of Biological Chemistry 278:39435-39442) reported pyridochromanones as the first example of a selectively potent class of bacterial DNA ligase inhibitors whose mode of action was confirmed. This publication demonstrated proof-of-principle validation of LigA as an antibacterial target.

As a result, there is need for compounds that inhibit LigA and thus that are useful as antibacterial agents.

Summary of the Invention

These and other needs are met by the present invention which is directed to substituted 4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-ones, e.g., a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one., for example, a compound of formula I

or a pharmaceutically acceptable salt thereof, wherein:

R is selected from Ci_-8alkyl, C₂-₈alkenyl, C₂-₈alkynyl, C₃.₈carbocyclyl, aryl, and heterocyclyl, any of which may be optionally substituted on one or more carbon atoms by R';

R^a is hydrogen, Ci-βalkyl, C₂-₆alkenyl, hydroxy(Ci-₆alkyl), and cyano, any of which may be optionally substituted on one or more carbon atoms by R';

R¹, R¹', R², R^2', R³, and R^3' are each independently selected from hydrogen, hydroxy, cyano, azido, Ci_-6alkyl, C₃-₈carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C₂.₆alkenyl, C₂-₆alkynyl, heterocyclyl, -OR⁷, NR⁸R⁹, wherein R¹, R^1', R², R^2', R³, and R^3' may be optionally substituted on one or more carbon atoms by one or more R ; or alternatively,

R¹, and R¹ , R² and R² , or R³ and R^3', taken together with the carbon to which they are attached, form C=O or C=N- 0-R⁶, or an optionally substituted 3, 4, 5, 6, or 7-membered ring containing O, 1, or 2 heteroatoms selected from O, S, NH, or N(Ci.₆alkyl); or alternatively,

R¹ and R² or R² and R³ taken together with the carbons to which they are attached, form an optionally substituted 3, 4, 5, or 6-membered ring containing 0, 1, or 2 heteroatoms selected from O, S, NH, or N(Ci.₆alkyl);

p is independently at each occurrence 0, 1 or 2;

R⁴ at each occurrence is independently -NR⁸R⁹, C_halky!, C₂-₆alkenyl, Ci-βalkoxy, - A -

C₃-₈cycloalkyl, heterocyclyl, and aryl wherein R⁴ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁵ at each occurrence is independently hydrogen, -NR⁸R⁹, -OR⁷,

C₂_6alkenyl, C_3-8cycloalkyl, C₃_₈cycloalkenyl, heterocyclyl, and aryl wherein each R⁵ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁶ at each occurrence is independently hydrogen, C_halky., C₂-₆alkenyl, C₂-₆alkynyl, C₃_₈cycloalkyl, Cs^cycloalkenyl, heterocyclyl and aryl, wherein each R⁶ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁷ at each occurrence is independently hydrogen, Ci-βalkyl, C₂-6alkenyl, C₃_8cycloalkyl, C₃-₈cycloalkenyl, aryl, S(O)_PR⁴, and heterocyclyl wherein R⁷ may be optionally substituted on one or more carbon atoms by one or more R';

R⁸ and R⁹ are each independently selected from hydrogen, Ci-βalkyl, C_2-6alkenyl, C₂-₆alkynyl, -OR⁷, C₃_₈cycloalkyl, C₃_₈cycloalkenyl, heterocyclyl, and aryl, wherein each R⁸ or R⁹ may be optionally substituted on one or more carbon atoms by one or more R ;

R at each occurrence is independently halo, hydroxy, nitro, -NR⁸R , azido, cyano, isocyano, Ci-βalkyl, C₂-₆alkenyl, C_∑-βalkynyl, aryl, C₃.gcycloalkyl, C₃.₈cycloalkenyl, heterocyclyl, keto(=O), -OR⁷, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴; =N-O-R⁶, -NHC(O)NR⁸R⁹, -N(Ci_-6alkyl)C(O)NR⁸R⁹, -NHC(O)R⁷, -NHCO₂R⁷, -NHSO₂(R⁴), -amidino i.e.-NHC(NH)NH₂, wherein each R may be optionally substituted on one or more carbon atoms by one or more R ;

R^" at each occurrence is independently halo, azido, cyano, Ci-βalkyl, C₂-₆alkenyl, C_2-6alkynyl, aryl, cycloalkyl, cycloalkenyl, heterocyclyl, hydroxy, -OR⁷, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, -NR⁸R⁹, -amidino i.e.-NHC(NH)NH₂. In another embodiment, the invention is directed to compounds of formula I shown in formula II

or a pharmaceutically acceptable salt thereof, wherein R, R^a, R¹' R¹ , R², R , R and R are as defined in formula I.

The invention further provides compounds of Formula I or formula II, in free or salt form, e.g., pharmaceutically acceptable salt form, as follows:

1.1 Compounds of Formula I or formula II, wherein R is selected from a group consisting of Ci_₈alkyl, C₂-₈alkenyl, C₂-₈alkynyl, C₃.₈carbocyclyl, aryl and heterocyclyl wherein R is optionally substituted with one or more R'.

1.2 Compounds of Formula I, formula II or 1.1, wherein R is Ci-salkyl (e.g., ethyl, propyl, pentyl) or Ci-shaloalkyl (e.g., dichloroethyl, 2,2,2-trichloroethyl, 2,2-dichloropropyl or 2,2-dichloropentyl) optionally substituted with one or more R'.

1.3 Compounds of Formula I, formula II, 1.1 or 1.2, wherein R is selected from a group consisting of neopentyl, 2,2-dichloroethyl, 2,2,2-trichloroethyl, 2,2-dichloropropyl and 2,2-dichloropentyl.

1.4 Compounds of Formula I, formula II or any of 1.1 - 1.3, wherein R is neopentyl.

1.5 Compounds of Formula I, formula II or 1.1 , wherein R is C_3-8carbocyclyl (e.g., cyclohexyl, bicyclo[3.1.0]hexan-3-yl or bicyclo[4.1.0]heptan-3-yl) or C3_8carbocyclylalkyl (e.g., cyclobutylmethyl, cyclopentylmethyl or bicyclo[3.1.0]hexan- 3-ylmethyl) wherein R is optionally substituted with one or more R'.

1.6 Compounds of Formula I, formula II, 1.1 , 1.4 or 1.5, wherein R is selected from a group consisting of cyclobutyl, cyclohexyl, (methylcyclopropyl)methyl, cyclobutylmethyl, methylcyclohexyl, spiro[2.2]pentan-l-ylmethyl, bicyclo[3.1.0]hexan-3-yl and bicyclo[4.1.0]heptan-3-yl wherein R is optionally substituted with one or more R'.

1.7 Compounds of Formula I, formula II, 1.1 or any of 1.4- 1.6, wherein R is cyclobutylmethyl.

1.8 Compounds of Formula I, formula II, 1.1 or any of 1.4- 1.6, wherein R is 4-methylcyclohex- 1 -yl.

1.9 Compounds of Formula I, formula II, 1.1 or any of 1.4- 1.6, wherein R is ( 1 -methylcyclopropyOmethyl.

1.10 Compounds of Formula I, formula II, 1.1 or any of 1.4- 1.6, wherein R is ?ra«5-4-methylcyclohex- 1 -yl.

1.11 Compounds of Formula I, formula II, or any of 1.1 - 1.10, wherein R^a is selected from a group consisting of hydrogen, Ci-βalkyl, C₂-₆alkenyl, hydroxy(Ci_₆alkyl), and cyano, wherein R^a is optionally substituted with R'.

1.12 Compounds of Formula I, formula II, or any of 1.1 - 1.11 , wherein R^a is hydrogen.

1.13 Compounds of Formula I, formula II, or any of 1.1 - 1.12, wherein R¹ and R¹ are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., acetonitrile), azido, Ci-βalkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C₂.₆alkenyl, C₂-₆alkynyl, heterocyclyl, -OR⁷ and NR⁸R⁹, wherein R¹ and R¹ are optionally substituted with one or more R'.

1.14 Compounds of Formula I, formula II, or any of 1.1 - 1.13 , wherein one of R¹ or R¹ is hydroxy.

1.15 Compounds of Formula I, formula II, or any of 1.1 - 1.15 , wherein one of R¹ or R¹ is hydroxy and the other is hydrogen.

1.16 Compounds of Formula I, formula II, or any of 1.1 - 1.15, wherein one of R¹ or R¹ is hydroxy and said hydroxy group is cis to R^a. 1.17 Compounds of Formula I, formula II, or any of 1.1 - 1.15, wherein one of R¹ or R¹ is hydrogen and the other is hydroxy wherein said hydroxy group is cis to R^a.

1.18 Compounds of Formula I, formula II, or any of 1.1 - 1.17, wherein R² and R² are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., acetonitrile), azido, C^aHcyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C_2-6alkenyl, C₂-₆alkynyl, heterocyclyl, -OR⁷ and NR⁸R⁹, wherein R² and R² are optionally substituted with one or more R'.

1.19 Compounds of Formula I, formula II, or any of 1.1-1.18, wherein one of R² or R² is selected from a group consisting of fluoro, chloro, azide, cyano and cyanomethyl.

1.20 Compounds of Formula I, formula II, or any of 1.1 - 1.19, wherein one of R² or R² is hydroxy.

1.21 Compounds of Formula I, formula II, or any of 1.1-1.20, wherein one of R² or R² is hydroxy and the other is hydrogen.

1.22 Compounds of Formula I, formula II, or any of 1.1 - 1.20, wherein one of R¹ or R¹ and another of R² or R² are cώ-diol wherein said cw-diol is cis to R^a.

1.23 Compounds of Formula I, formula II, or any of 1.1 - 1.22, wherein R³ and R³ are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., acetonitrile), azido, Ci-₆alkyl, C₃_₈carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C₂.₆alkenyl, C₂-₆alkynyl, heterocyclyl, -OR⁷ and NR⁸R⁹, wherein R³ and R³ are optionally substituted with one or more R'.

1.24 Compounds of Formula I, formula II, or any of 1.1-1.23, wherein R³ and R³ are independently hydrogen or methyl.

1.25 CCoommppoouunndds of Formula I, formula II, or any of 1.1- 1.24, wherein R³ and R³ are hydrogen.

1.26 Compounds of Formula I, formula II, or any of 1.1 - 1.24, wherein one of R or R³ is methyl.

1.27 Compounds of Formula I, formula II, or any of 1.1-1.24 or 1.26, wherein one of R³ or R³ is methyl and said methyl group is trans to R^a 1.28 Compounds of Formula I, formula II, or any of 1.1-1.24 or 1.26, wherein one of R or R³ is hydrogen and the other is methyl and said methyl group is trans to R^a.

In one embodiment, the compound of Formula I or formula II is selected from the group consisting of

in free or salt form.

In another embodiment, the compound of Formula I or formula II is

in free or salt form. For example, in one embodiment, the compound of Formula I or formula II is

in free or salt form.

The invention also provides a method for producing an antibacterial effect in a warm blooded animal, such as man, in need of such treatment, comprising administering to said animal an effective amount of a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form.

The invention also provides a method for inhibition of bacterial DNA ligase in a warm-blooded animal, such as a human being, in need of such treatment comprising administering to said animal an effective amount of a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form.

The invention also provides a method of treating a bacterial infection in a warm-blooded animal, such as a human being, in need of such treatment comprising administering to said animal an effective amount of a 6-oxy-4-amino-l-

(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form.

The invention also provides a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, or a pharmaceutically acceptable salt thereof for use as a medicament.

The invention also provides the use of a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form in the manufacture of a medicament for use in the production of an anti-bacterial effect in a warm-blooded animal.

The invention also provides the use of a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1 - 1.28, in free or pharmaceutically acceptable salt form in the manufacture of a medicament for use in inhibition of bacterial DNA ligase in a warm-blooded animal, such as a human being.

The invention also provides the use of a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form in the manufacture of a medicament for use in the treatment of a bacterial infection in a warm-blooded animal such as a human being.

The invention also provides a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form for use in the production of an anti-bacterial effect in a warm-blooded animal such as a human being.

The invention also provides a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form for use in inhibition of bacterial DNA ligase in a warm-blooded animal such as a human being.

The invention also provides a 6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, or a pharmaceutically acceptable salt thereof for use in the treatment of a bacterial infection in a warm-blooded animal such as a human being.

The invention also provides a pharmaceutical formulation comprising a

6-oxy-4-amino-l-(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form and a pharmaceutically acceptable diluent or carrier.

The invention also provides a pharmaceutical composition comprising a

6-oxy-4-amino-l-(tetrahydrofuranyl)-/H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, e.g., a compound of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable excipient or carrier for use in the production of an anti-bacterial effect in a warm-blooded animal, such as a human being.

In one embodiment, the invention also provides a process (Process I) for preparing 6-oxy- 4-amino-l-(tetrahydrofuranyl)-/H- pyrazolo[3,4-d]pyrimidin-3(2H)-ones in free or pharmaceutically acceptable salt form, which process comprising the step of treating 6-oxy-l-(tetrahydrofuranyl)-3-methoxy-7H-pyrazolo[3,4-d]pyrimidin-4-amines with a hydride, e.g., lithium triethylborohydride.

In a further embodiment, the invention provides Process I further comprising the step of heating the reaction mixture from Process I.

In another embodiment, the invention also provides a process (Process II) for preparing compounds of formula I, formula II, or any of 1.1 - 1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of treating a 6-oxy-3-methoxy- /H-pyrazolo[3,4-d]pyrimidin-4-amine with a hydride, e.g., lithium triethylborohydride.

In another embodiment, the invention also provides Process II, further comprising the step of heating the reaction mixture of 6-oxy-3-methoxy-7H-pyrazolo[3,4-d]- pyrimidin-4-amine and hydride.

In a further embodiment, the invention provides a process (Process III) for preparing compounds of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of:

(i) treating a compound of the general formula of Η-O-R with base, e.g., sodium hydride or potassium hydride; and

(ii) adding 3-methoxy-6-(methylsulfonyl)- 1 -(tetrahydrofuranyl)-iH-pyrazolo [3,4-d]pyrimidin-4-amine to step (i).

Therefore, in one embodiment, the invention provides a process for preparing compounds of formula I, formula II, or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of

(i) treating Η-O-R, e.g., 4-methylcyclohexanol with sodium hydride and

(ii) (ii) adding 3-methoxy-6-(methylsulfonyl)-l-(tetrahydrofuranyl)- 7H-pyrazolo[3,4-d]pyrimidin-4-amine to step (i).

In a further embodiment, the invention provides a process (Process IV) for preparing compounds of formula I, formula II or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of treating 3-methoxy-6- (methylsulfonyl)- lH-pyrazolo[3,4-d]pyrimidin-4-amine with:

(i) a tetrahydrofϊiranyl electrophile of formula (2):

Formula (2) wherein L is a leaving group (e.g., acetate, methoxy, benzoyl or halo); and

(ii) a reagent selected from a group consisting of (a) a base, (b) a Lewis Acid (e.g., tin (VI) chloride, aluminum chloride), (c) palladium and (d) triphenylphosphine and diethylazodicarboxylate.

In a particular embodiment, therefore, the invention provides Process IV, which process comprises the step of treating a 3-methoxy-6-(methylsulfonyl)-lH-pyrazolo- [3,4-d]pyrimidin-4-amine with, for example, (i) l,2,3-tri-0-acetyl-5-deoxy-β-D- ribofuranose and (ii) SnCl₄.

In another embodiment, the invention provides a process (Process V) for preparing compounds of formula I, formula II or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of treating a 3-methoxy-4,6-bis(methylsulfonyl)pyrazolo[3,4-d]pyrimidine with ammonia.

In another embodiment, the invention provides a process (Process VI) for preparing compounds of formula I, formula II or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises the step of treating

3-methoxy-4,6-bis(methylthio)pyrazolo[3,4-d]pyrimidine with persulfuric acid sold under the tradename Oxone®.

In another embodiment, the invention provides a process (Process VII) for preparing compounds of formula I, formula II or any of 1.1-1.28, in free or pharmaceutically acceptable salt form, which process comprises performing the steps of:

(i) Process VI

(ii) Process V

(iii) Process IV;

(iv) Process III;

(v) Process II; and

(vi) Process I.

Detailed Description of the Invention

The definitions set forth herein are intended to clarify terms used throughout this application unless specifically indicated otherwise. The term "herein" means the entire application. The term "carbocyclyl" refers to saturated, partially saturated and unsaturated, mono, bi or polycyclic carbon rings. These may include fused or bridged bi- or polycyclic systems. Carbocyclyls may have from 3 to 12 carbon atoms in their ring structure, i.e. C₃-i₂carbocyclyl, and in a particular embodiment are monocyclic rings have 3 to 7 carbon atoms or bicyclic rings having 7 to 10 carbon atoms in the ring structure. Examples of suitable carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclopentadienyl, indanyl, phenyl and naphthyl.

The term "hydrocarbon" used alone or as a suffix or prefix, refers to any structure comprising only carbon and hydrogen atoms and containing up to 12 carbon atoms.

In this specification the term alkyl, used alone or as a suffix or prefix, includes both monovalent straight and branched chain hydrocarbon radicals but references to individual alkyl radicals such as propyl are specific for the straight chain version only. An analogous convention applies to other generic terms. Unless otherwise specifically stated, the term alkyl refers to hydrocarbon radicals comprising 1 to 12 carbon atoms, in another embodiment 1 to 10 carbon atoms, and in a still further embodiment, 1 to 6 carbon atoms.

The term "alkenyl" used alone or as suffix or prefix, refers to a monovalent straight or branched chain hydrocarbon radical having at least one carbon-carbon double bond which, unless otherwise specifically stated, comprises at least 2 up to 12 carbon atoms, in another embodiment 2-10 carbon atoms and in a still further embodiment 2-6 carbon atoms.

The term "alkynyl" used alone or as suffix or prefix, refers to a monovalent straight or branched chain hydrocarbon radical having at least one carbon-carbon triple bond which, unless otherwise specifically stated, comprises at least 2 up to 12 carbon atoms, in another embodiment 2-10 carbon atoms and in a still further embodiment 2-6 carbon atoms.

In this specification, the terms alkenyl and cycloalkenyl include all positional and geometrical isomers.

The term "cycloalkyl," used alone or as suffix or prefix, refers to a monovalent ring-containing hydrocarbon radical which, unless otherwise specifically stated, comprises at least 3 up to 12 carbon atoms, in another embodiment 3 up to 10 carbon atoms and includes monocyclic as well as bicyclic and polycyclic ring systems. When a cycloalkyl ring contains more than one ring, the rings may be fused or unfused. Fused rings generally refer to at least two rings sharing two atoms there between. Spiro rings generally refer to at least two rings sharing one atom there between. Suitable examples include C₃-Cio cycloalkyl rings, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclooctyl radicals, adamantanyl, norbomyl, decahydronapthyl, octahydro-lH-indenyl, spiro [2.2] pentanyl, and bicyclo [3.1.0] hexanyl.

The term "cycloalkenyl" used alone or as suffix or prefix, refers to a monovalent ring-containing hydrocarbon radical having at least one carbon-carbon double bond and unless otherwise specifically stated comprising at least 3 up to 12 carbon atoms, in another embodiment 3 up to 10 carbon atoms. Suitable examples include cyclopentenyl and cyclohexenyl.

The term "aryl" used alone or as suffix or prefix, refers to a hydrocarbon radical having one or more polyunsaturated carbon rings having aromatic character, (e.g., 4n + 2 delocalized electrons) and comprising 5 up to 14 carbon atoms, wherein the radical is located on a carbon of the aromatic ring. Examples of suitable aryl radicals include phenyl, napthyl, and indanyl.

The term "alkoxy" used alone or as a suffix or prefix, refers to radicals of the general formula -O-R, wherein -R is selected from an optionally substituted hydrocarbon radical. Exemplary alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, cyclopropylmethoxy, allyloxy, and propargyloxy.

The terms "heterocyclic radical" or "heterocyclyl" (both referred to herein as "heterocyclyl") used alone or as a suffix or prefix, refer to a ring-containing structure or molecule having one or more multivalent heteroatoms, independently selected from N, O, and S, as a part of the ring structure and, unless otherwise specifically stated, including at least 3 and up to 14 atoms in the ring(s), or from 3 - 10 atoms in the ring, or from 3 - 6 atoms in the ring. Heterocyclyl groups may be saturated or unsaturated, containing one or more double bonds, and heterocyclyl groups may contain more than one ring. When a heterocyclyl contains more than one ring, the rings may be fused or unfused. Fused rings generally refer to at least two rings sharing two atoms therebetween. Heterocycle groups also include those having aromatic character. Examples of suitable heterocycles include, but are not limited to, indazole, pyrrolidonyl, dithiazinyl, pyrrolyl, indolyl, piperidonyl, carbazolyl, quinolizinyl, thiadiazinyl, acridinyl, azepane, azetidine, aziridine, azocinyl, benzimidazolyl, benzofuran, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazole, benzoxazolyl, benzthiophene, benzthiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzthiazole, benzisothiazolyl, benzimidazoles, benzimidazalonyl, carbazolyl, β-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, dioxanyl, dioxolanyl, furyl, dihydrofuranyl, tetrahydrothiopyranyl, furanyl, furazanyl, homopiperidinyl, imidazole, imidazolidine, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, keto, moφholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxirane, oxazolidinylperimidinyl, oxetanyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidine, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, purinyl, pyranyl, pyrrolidine, pyrroline, pyrrolidine, pyrrolidine-2-onyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, N-oxide-pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, pyridine, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, thiophane, thiotetrahydroquinolinyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, thiirane, triazinyl, triazolyl, and xanthenyl. It is further to be understood that heterocyclyl may be optionally substituted on carbon as indicated hereinbefore. Finally, if a heterocyclyl contains an -NH- moiety, the nitrogen of that moiety may be optionally substituted by a group selected from Ci-βalkyl, C₂-₆alkenyl, C₂-₆alkynyl, aryl, C₃_₈cycloalkyl, C₃_8cycloalkenyl, heterocyclyl, C(O)R⁵, S(O)_pCi-₆alkyl, -C(O)NR⁸R⁹, wherein the variables are as defined hereinbefore.

"Halo" includes fluorine, chlorine, bromine and iodine.

As used herein, the term "optionally substituted," means that substitution is optional and therefore it is possible for the designated substituent to be unsubstituted. In the event a substitution is desired then such substitution means that any number of hydrogens on the designated substituent is replaced with a selection from the indicated group, provided that the normal valency of the atoms on a particular substituent is not exceeded, and that the substitution results in a stable compound. For example when a substituent is keto (i.e., =0), then 2 hydrogens on the atom are replaced. In the case of cyclic substituents, e.g. cycloalkyl and aryl, two hydrogens may be replaced to form a second ring resulting in an overall fused or spiro ring system which may be partially or fully saturated, unsaturated or aromatic. Suitable substituents include alkylamido, e.g. acetamido, propionamido; alkyl; alkylhydroxy; alkenyl; alkenyloxy; alkynyl; alkoxy; halo; haloalkyl; hydroxy; cycloalkyl; alkylcycloalkyl; acyl; aryl; acyloxy; amino; amido; carboxy; carboxy derivatives e.g. -CONH₂, -CO₂H, -COalkyl, -COaryl, -COcycloalkyl, -COcycloalkenyl, -COheterocyclyl; substituted -NH₂; aryloxy; nitro; cyano; azido, heterocyclyl; thiol; imine; sulfonic acid; sulfate; sulfonyl; sulfinyl; sulfanyl; sulfamoyl; thioester; thioether; acid halide; anhydride; oxime i.e. =N-0H; hydrazine; carbamate; or any other viable functional group provided that the resulting compound is stable and exhibits bacterial DNA ligase inhibitory activity. These optional substituents may themselves be optionally substituted again as long as the resulting compound is stable and exhibits a bacterial DNA ligase inhibitory effect.

When a particular substituent is indicated to be "substituted", then that substituent can be substituted with any of the optional substituents listed above provided the resulting compound is stable and exhibits a bacterial DNA ligase inhibitory effect. Moreover, it is to be understood that combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. Finally, when any variable occurs more than one time in any formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence.

Where R¹ and R² or R² and R³ together with the carbons to which they are attached form an optionally substituted cyclic ring containing 3-6 atoms, the cyclic ring can be a carbocyclic or heterocyclic ring. Suitable optionally substituted carbocyclic and heterocyclic rings include, cyclic ethers e.g. epoxide, oxetanyl, dioxanyl, e.g. 2,2-dimethyl-l,3-dioxanyl; cycloalkyl rings e.g. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, cyclohexanonyl rings; heterocyclyl rings e.g. azetidinyl, oxazolidonyl ring, oxathiolanyl ring, oxazinonyl ring, pyranonyl ring, piperidinonyl, tetrahydrothiophenyl ring, pyrrolidinyl ring, dioxolanyl ring, dioxanonyl ring, triazolyl ring, tetrazolyl ring, morpholinyl ring, l,3,2-dioxathiolane-2,2-dioxidyl ring and piperdinyl ring.

The terms "cis" and "trans" are well known in the art and generally refer to the relative orientation of two substituents on a double bond or a cyclic compound. As applied to the compounds of the present invention, they refer to the orientation of R^a, R¹, R¹ , R², R² , R³ and R³ relative to each other on the tetrahydrofuran ring. Therefore, "cis" refers to two substituents that are on the same side of the plane of the ring (e.g., both above or both below the plane of the ring) while "trans" refers to two substituents that are on different side of the plane of the ring (e.g., one above and one below the plane of the ring). For example, since R¹ and R¹ have not been designated a specific orientation, the phrase "wherein one of R¹ or R¹ is hydroxy and said hydroxy group is cis to R^a" refers to compounds wherein the hydroxy group is on the same side of the plane of the ring as R^a. Similarly, "wherein one of R¹ or R¹ and another of R² or R² are cis-dioV refers to compounds wherein the two hydroxy groups (i.e., diol) on the b-carbon and c-carbon are on the same side of the plane of the ring. Therefore, c/s-diol may be illustrated below as an example only:

Furthermore, the phrase "wherein one of R³ or R³ is methyl and said methyl group is trans to R^a" refers to compounds wherein the methyl group on the d-carbon is on a different side of the plane of the ring as R^a on the a-carbon as illustrated below by way of examples only:

wherein R³ or R³' is methyl

"Pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Compounds of the foregoing formulas I may form stable acid or basic salts, and in such cases administration of a compound as a salt may be appropriate, and pharmaceutically acceptable salts may be made by conventional methods well-known in the art.

Suitable pharmaceutically-acceptable salts include acid addition salts such as methanesulfonate, trifluoroacetate, tosylate, α-glycerophosphate fumarate, hydrochloride, citrate, maleate, tartrate and hydrobromide. Also suitable are salts formed with phosphoric and sulfuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium, an alkaline earth metal salt for example calcium or magnesium, an organic amine salt for example triethylamine, morpholine, N-methylpiperidine, N-ethylpiperidine, procaine, dibenzylamine, N,N-dibenzylethylamine, frø-(2-hydroxyethyl)amine, N-methyl D-glucamine and amino acids such as lysine. There may be more than one cation or anion depending on the number of charged functions and the valency of the cations or anions.

However, to facilitate isolation of the salt during preparation, salts which are less soluble in the chosen solvent may be preferred whether pharmaceutically-acceptable or not.

Within the present invention it is to be understood that a compound of formula I, formula II or a salt thereof may exhibit the phenomenon of tautomerism and that the formula drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the invention encompasses all tautomeric forms that inhibit bacterial DΝA ligase and is not to be limited merely to any one tautomeric form utilized within the formula drawings.

It will be appreciated by those skilled in the art that in addition to the asymmetric carbon atoms specifically indicated in formula I, the compounds of the of the invention may contain additional asymmetrically substituted carbon and/or sulphur atoms, and accordingly may exist in, and be isolated in, optically-active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic or stereoisomeric form, or mixtures thereof, which possesses properties useful in the inhibition of bacterial DΝA ligase, it being well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, by enzymatic resolution, by biotransformation, or by chromatographic separation using a chiral stationary phase) and how to determine efficacy for the inhibition of bacterial DΝA ligase by the standard tests described hereinafter.

When an optically active form of a compound of the invention is required, it may be obtained as specifically exemplified above or by carrying out one of the above procedures for racemic compounds but using an optically active starting material (formed, for example, by asymmetric induction of a suitable reaction step), or by resolution of a racemic form of the compound or intermediate using a standard procedure, or by chromatographic separation of diastereoisomers (when produced). Enzymatic techniques may also be useful for the preparation of optically active compounds and/or intermediates. Similarly, when a pure regioisomer of a compound of the invention is required, it may be obtained by carrying out one of the above procedures using a pure regioisomer as a starting material, or by resolution of a mixture of the regioisomers or intermediates using a standard procedure.

It is also to be understood that compounds of formula I, formula II and salts thereof can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that inhibit bacterial DNA ligase.

The removal of any protecting groups and the formation of pharmaceutically acceptable salts are within the skill of an ordinary organic chemist using standard techniques.

The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl /?-hydroxybenzoate; and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art. Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finely powdered form or in the form of nano or micronized particles together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives such as ethyl or propyl β-hydroxybenzoate; anti-oxidants such as ascorbic acid); colouring agents; flavouring agents; and/or sweetening agents such as sucrose, saccharine or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil or in a mineral oil such as liquid paraffin. The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavoring and/or coloring agent.

The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1 ,3-butanediol.

Compositions for administration by inhalation may be in the form of a conventional pressurized aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

For further information on formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

In addition to the compounds of the present invention, the pharmaceutical composition of this invention may also contain or be co-administered (simultaneously, sequentially or separately) with one or more known drugs selected from other clinically useful antibacterial agents (for example, macrolides, quinolones, β-lactams or aminoglycosides) and/or other anti-infective agents (for example, an antifungal triazole or amphotericin). These may include carbapenems, for example meropenem or imipenem, to broaden the therapeutic effectiveness. Compounds of this invention may also contain or be co-administered with bactericidal/permeability-increasing protein (BPI) products or efflux pump inhibitors to improve activity against gram negative bacteria and bacteria resistant to antimicrobial agents.

As stated above the size of the dose required for the therapeutic or prophylactic treatment of a particular disease state will necessarily be varied depending on the host treated, the route of administration and the severity of the illness being treated. Preferably a daily dose in the range of 1-50 mg/kg is employed. Accordingly, the optimum dosage may be determined by the practitioner who is treating any particular patient.

In addition to its use in therapeutic medicine, compounds of formula I, formula II and their pharmaceutically acceptable salts are also useful as pharmacological tools in the development and standardization of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of DNA ligase in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.

In any of the above-mentioned pharmaceutical composition, process, method, use, medicament, and manufacturing features of the instant invention, any of the alternate embodiments of the compounds of the invention described herein also apply.

Enzyme Potency Testing Methods

Compounds will be tested for inhibition of DNA ligase using a Fluorescence Resonance Energy Transfer (FRET) detection assay as previously described (Chen et al. 2002. Analytical Biochemistry 309: 232-240; Benson et al. 2004. Analytical Biochemistry 324:298-300). Assays will be performed in 384- well polystyrene flat-bottom black plates in 30 μl reactions containing 3 μl compound dissolved in dimethylsulfoxide, 20 μl 1.5X Enzyme Working Solution (25% glycerol, 45 mM potassium chloride, 45 mM ammonium sulfate, 15 mM dithiothreitol, 1.5 mM ethylenediaminetetraacetic acid (EDTA), 0.003% Brij 35, 75 mM MOPS pH 7.5, 150 nM bovine serum albumin, 1.5 μM NAD⁺, 60 nM DNA substrate, 0.375 nM enzyme in water) and 7 μl 70 mM magnesium chlorine solution (96 mM magnesium chloride, 20% glycerol in water) to initiate the reaction. The DNA substrate is similar to that described in Benson et al. (2004. Analytical Biochemistry 324:298-300). The assay reactions will be incubated at room temperature for approximately 20 minutes before being terminated by the addition of 30 μl Quench reagent (8 M Urea, 1 M Trizma base, 20 mM EDTA in water). Plates will be read in a Tecan Ultra plate reader at two separate wavelengths - Read 1 : excitation 485, emission 535, Read 2: excitation 485, emission 595. Data is initially expressed as a ratio of the 595/535 emission values and percent inhibition values were calculated using 0.2 % dimethylsulfoxide (no compound) as the 0% inhibition and EDTA-containing (50 mM) reactions as 100% inhibition controls. Compound potency will be based on IC₅₀ measurements determined from reactions performed in the presence of ten different compound concentrations.

The compounds are predicted to have an IC₅₀ in this assay against at least one isozyme (S. pneumoniae, S. aureus, H. influenzae, E. coli, or M. pneumoniae) of <400 μM or the compounds inhibited the ligation reaction by >20% at the limit of their solubility in the assay medium. Solubility will be determined under assay conditions using a nephelometer to detect a change in turbidity as the concentration of compound increases. The limit of solubility will be defined as the maximum concentration before a detectable increase in turbidity is measured.

Bacterial Susceptibility Testing Methods

Compounds will be tested for antimicrobial activity by susceptibility testing using microbroth dilution methods recommended by NCCLS. Compounds will be dissolved in dimethylsulfoxide and tested in 10 doubling dilutions in the susceptibility assays such that the final dimethylsulfoxide concentration in the assay was 2 % (v/v). The organisms used in the assay ae grown overnight on appropriate agar media and then suspended in the NCCLS-recommended liquid susceptibility-testing media. The turbidity of each suspension is adjusted to be equal to a 0.5 McFarland standard, a further 1 -in- 10 dilution was made into the same liquid medium to prepare the final organism suspension, and 100 μL of this suspension was added to each well of a microtiter plate containing compound dissolved in 2 μL of dimethylsulfoxide. Plates will be incubated under appropriate conditions of atmosphere and temperature and for times according to NCCLS standard methods prior to being read. The Minimum Inhibitory Concentration (MIC) is the lowest drug concentration able to reduce growth by 80 % or more.

Process

If not commercially available, the necessary starting materials for the procedures such as those described herein may be made by procedures which are selected from standard organic chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the described procedure or the procedures described in the Examples.

It is noted that many of the starting materials for synthetic methods as described herein are commercially available and/or widely reported in the scientific literature, or could be made from commercially available compounds using adaptations of processes reported in the scientific literature. The reader is further referred to Advanced Organic Chemistry, 4^th Edition, by Jerry March, published by John Wiley & Sons 1992, for general guidance on reaction conditions and reagents.

It will also be appreciated that in some of the reactions mentioned herein it may be necessary/desirable to protect any sensitive groups in compounds. The instances where protection is necessary or desirable are known to those skilled in the art, as are suitable methods for such protection. Conventional protecting groups may be used in accordance with standard practice (for illustration see T.W. Greene, Protective Groups in Organic Synthesis, published by John Wiley and Sons, 1991).

Examples of suitable protecting groups for a hydroxy group are, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, a silyl group such as trimethylsilyl or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively a silyl group such as trimethylsilyl may be removed, for example, by fluoride or by aqueous acid; or an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation in the presence of a catalyst such as palladium-on-carbon. A suitable protecting group for an amino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or f-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a ?-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric, phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid, for example boron frø(trifiuoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group, which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine or 2-hydroxyethylamine, or with hydrazine. Another suitable protecting group for an amine is, for example, a cyclic ether such as tetrahydrofuran, which may be removed by treatment with a suitable acid such as trifluoroacetic acid.

The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art, or they may be removed during a later reaction step or work-up.

The skilled organic chemist will be able to use and adapt the information contained and referenced within the above references, and accompanying Examples therein and also the Examples herein, to obtain necessary starting materials and products.

Another aspect of the present invention provides a process for preparing a compound of formula I, formula II or a pharmaceutically acceptable salt thereof which process (wherein R, R^a, R¹, R¹ , R², R² , R³ and R³ are, unless otherwise specified, as defined in formula I) comprises: a) reacting a heterocycle of formula ( 1 ) :

(1) or a suitably protected derivative thereof; with an electrophile of formula (2) wherein L is a suitable leaving group such as acetate, methoxy, benzoyl, or chloro; or

(2) reacting a heterocycle of formula (3):

(3) (4) wherein A is Cl, SO₂Me, NH₂, or a suitably protected amino group and W is halo or SO₂Me, with an electrophile of formula (2) followed by reaction with a compound of formula (4), and if A is a leaving group, a subsequent reaction with the appropriate amine, such as ammonia; and thereafter if necessary: converting a compound of formula I, formula II or any of 1.1-1.28 into another compound of formula I, formula II or any of 1.1 - 1.28; removing any protecting groups; and optionally forming a pharmaceutically acceptable salt.

Specific reaction conditions for the above reactions are as follows:

Heterocycles of formula (1) and electrophiles of formula (2) may be coupled together using standard coupling conditions known in the art. These include, but are not limited to glycosylation conditions such as those described in Vorbrueggen, H. and Bennua, B. Chem. Ber., 1981, 114, 1279-1286, and Dudycz, L.V. and Wright, G.E. Nucleosides and Nucleotides, 1984, 3, 33-44. Other coupling methods include but are not limited to nucleophilic substitution reactions catalyzed by, for example bases, Lewis acids or palladium, and substitution using reagents such as triphenylphosphine and diethylazodicarboxylate.

Displacement of W by the appropriate alcohol can be done either neat or in a suitable solvent such as dioxane, tetrahydrofuran, DCM, DMF, or N-methylpyrrolidinone in temperatures ranging from 40-200⁰C. Bases such as sodium hydroxide, potassium carbonate, w-butyl lithium, potassium tert-butoxide, or sodium hydride can be used as necessary according to one skilled in the art. Compounds of formula (2) are prepared by processes known in the art using procedures found in the literature such as those modifying an appropriately protected ribose derivative. The reader is referred to Preparative Carbohydrate Chemistry, edited by S. Hanessian, published by Marcel Dekker, 1997 for general guidance on transformations and reaction conditions. For example, one method to synthesize compound (11) is shown in Scheme 1. The reaction of compound (8) or other suitably protected ribose derivative with a displaceable group can be carried out by a number of fluorinating reagents such as tetrabutylammonium fluoride, (diethylamino)sulfur trifluoride (DAST), potassium fluoride, or Amberlyst A-26 (F^" 40nm) to give compound (9). Following deprotection and reprotection, compound (11) is obtained and can be coupled with a compound of formula (1). A similar method to synthesize a different compound of formula 2 is shown in Scheme 2.

Scheme 1

(11)

Scheme 2

chromatography

Alternatively, compounds of formula I, formula II or any of 1.1 - 1.28 can be prepared by converting a particular compound of formula I to a different compound of formula I (or by converting a particular compound of formula II to a different compound of formula II) using the appropriate protecting groups, reactions, and deprotections using methods known to one skilled in the art. One non-limiting example of how the 5'-position of the ribose can be modified is shown in Scheme 3, and one non-limiting example of how the T- and 3'-positions of the ribose can be modified is shown in Scheme 4. Appropriate chemistry can be applied to modify the 5' and 2' and 3 '-positions of the ribose, in each case using the appropriate combination of protecting groups. Further manipulations can be made using techniques known to one skilled in the art.

Scheme 3

1. TsCtf Pyr 22.. TTBBAAFF oOrr ILiEt₃BH or appropriate reagents

Scheme 4

The alcohols used in the displacement of the leaving group on the heterocycle may be commercially available. Those that aren't can be synthesized by methods well known to those of skill in the art. One non-limiting example is shown in Scheme 5.

Scheme 5

KOH (5%), water, THF OsO₄, Acetone, water separation

OCOPh OCOPh 6 : 4

Compounds of formula I and formula II can be made from commercially available starting materials by first synthesizing the heterocycle followed by coupling to the appropriately protected sugar derivative. Two non-limiting examples of how this can be accomplished are shown in Schemes 6 and 7.

Scheme 6

references: J. Heterocyclic Chβm. 1990, 27, 439. J. Heterocyclic Chβm. 1986, 23, 1869. Scheme 7

Formic Acid, Water

Alternatively, compounds of Formula I, formula II or any of 1.1-1.28 may be prepared as shown in Scheme 8 by first treating 3-Methoxy-4,6-bis(methylthio)pyrazolo- [3,4-d]pyrimidine with Oxone^® followed by ammonia. The resulting 3-methoxy-6-

(methylsulfonyl)-lH-pyrazolo[3,4-(/]pyrimidin-4-amine may be coupled with a tetrahydrofuranyl acetate derivative by adding SnCU- The -OR group may then be added to the resulting (tetrahydrofuranyl)-3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-c(]- pyrimidin-4- amine by first treating the Η-OR with a base such as sodium hydride in a solvent such as TΗF at 50⁰C followed by the addition of the (tetrahydrofuranyl)-3- rnethoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-fi(]- pyrimidin-4-amine. The 3-methoxy of the resulting product may then be reduced to a carbonyl by using an agent such as lithium triethylborohydride in a solvent such as TΗF to afford the compounds of the present invention.

Scheme 8

Oxone

lithium triethylborohydride

Examples:

The syntheses of the compounds of the present invention are illustrated, but not limited to the following Examples. The common intermediates l,2,3-tri-O-acetyl-5- deoxy-β-D-ribofuranose and 3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-ύ(]- pyrimidin-4-amine, used in the Examples are prepared as follows: Intermediate 1: l,2,3-tri-0-acetyl-5-deoxy-β-D-ribofuranose

Methyl 2,3-O-isopropylidine-5-O-(p-tolylsulfonyl)-beta-D- ribofuranoside (100 g) in THF (650 mL) is cooled to -5 to 0 ⁰C. Super hydride (lithium triethyl- borohydride, 1.0 M solution in THF) (650 mL) is added slowly (over 15 min). The reaction mixture is allowed to warm up to room temperature where it is further stirred for 2 h. When TLC (30 % EtOAc / Hexane) confirms the completion of reaction, the reaction mixture is cooled to -10 ⁰C, diluted with EtOAc, and quenched with water. The reaction mixture is then concentrated on a rotary evaporator. The aluminum salts are filtered through a plug of silica gel and the filtrate is concentrated on a rotary evaporator. The resulting oil is diluted in EtOAc and washed with H₂O and brine, dried over sodium sulfate and concentrated on a rotary evaporator to give methyl 5-deoxy-2,3-0-(l-methyl- ethylidene)-D-ribofuranoside as a faint yellow-colored oil (50 g). The product is used directly in the next step. ¹H NMR (300 MHz, CDCl₃) δ 4.92 (s, 1 H), 4.62 (d, 1 H), 4.5 (d, 1 H), 4.32 (dd, 1 H), 3.3 (s, 3 H), 1.46 (s, 3 H), 1.3 (s, 3 H), 1.26 (d, 3 H).

The solution of crude methyl 5-deoxy-2,3-O-(l-methylethylidene)-D- ribofuranoside (25 g, 0.13 mol) in 70 % aq. acetic acid (100 mL) is heated to 100 ⁰C and then to reflux for a minimum of 6 h, or until the ¹H NMR confirms the exhaustive hydrolysis. Upon complete, the reaction mixture is concentrated on a rotary evaporator and further dried under high vacuum to give 5-deoxy-D-ribofuranose. The crude product is used directly in the next step.

To a stirred solution of crude 5-deoxy-D-ribofuranose (10 g) from the above reaction in pyridine (150 mL) is added at room temperature imidazole (15g). The resulting mixture is stirred for 10 minutes at room temperature. Then acetic anhydride (42 ml) is added drop-wise and the reaction mixture is stirred for 6 h at room temperature. When TLC (30 % EtOAc/Hexane) confirms the completion of the reaction, the reaction mixture is diluted with EtOAc, washed with aqueous saturated NaHCO₃, H₂O and brine, and then dried over anhydrous sodium sulfate. The concentrated crude product is purified by silica column chromatography (30% EtOAc in Hexanes) to give the C-I beta isomer (l,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose) as a white solid (5 g). ¹H NMR (300 MHz, CDCl₃) δ 6.1 (s, 1 H), 5.33 (d, 1 H), 5.1 (m, 1 H), 4.26 (m, 1 H), 2.12 (s, 3 H), 2.1 (s, 3 H), 2.08 (s, 3 H), 1.37 (d, 3 H).

Intermediate 2: (25,3/?,4/?)-tetrahydrofuran-2,3,4-triyl triacetate

/⁰N^OAc

OAc OAc

(2.S',3i?,4i?)-tetrahydrofuran-2,3,4-triyl triacetate (0.428 g) may be prepared by methods disclosed in Carbohydrate Research, 1978, 61, 501-509.

Intermediate 3: 3-Methoxy-4,6-bis(methylthio)pyrazolo[3,4-d]pyrimidine

3-Methoxy-4,6-bis(methylthio)pyrazolo[3,4-d]pyrimidine may be prepared as disclosed in Journal of Heterocyclic Chemistry, 1986, 23(6), 1869-78.

Intermediate 4: l-(2,3-di-0-acetyl-5-deoxy-β-D-ribofuranosyl)-3-methoxy-6-(methylsulfonyl)-l//- pyrazolo [3,4-</] pyrimidin-4-amine

3-Methoxy-4,6-bis(methylthio)pyrazolo- [3,4-d]pyrimidine (0.8 g) is dissolved in 1,4-dioxane (40 mL) with heating. The resulting clear solution is allowed to cool down to room temperature and then to 0 ⁰C where a suspension of Oxone^® (12.2 g) in H₂O (12 mL) is introduced dropwise. The resulting suspension is stirred for 24h. The reaction mixture is filtered. The filtrate is diluted with EtOAc, washed with water and brine and dried over anhydrous sodium sulfate. The reaction mixture is concentrated on a rotary evaporator to give

3-methoxy-4,6-bis(methylsulfonyl)-lH-pyrazolo[3,4-ύ0pyrimidine as a light yellow-colored solid (600 mg), which is used directly in the next step. MS (electrospray): 307 (M+l) for C₈Hi ₁N₄OsS₂. ¹H NMR (300 MHz, DMSO) δ 4.2 (s, 3 H), 3.6 (s, 3 H), 3.5 (s, 3 H).

3-Methoxy-4,6-bis(methylsulfonyl)pyrazolo[3,4-d]pyrimidine ( 1.3 g,) is added in 7 N ammonia solution in MeOH (25 mL) at room temperature and a bright orange-colored solution is formed. The color immediately starts to fade away as a yellow-colored precipitate is formed. The progress of the reaction is monitored by LCMS. The reaction mixture is stirred for 2 h. When LCMS confirms the completion of reaction, the precipitate formed is collected by filtration, washed with MeOH and dried under high vacuum to give crude

3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-<^-pyrimidin-4-amine. This crude product (1 g) is used directly in the next step without any further purification. MS (electrospray): 244 (M+l) for C₇Hi₀NsO₃S. ¹H NMR (300 MHz, DMSO) δ 12.2 (bs, 1 H), 8.4 (bs, 1 H), 7.35 (bs, 1 H), 3.95 (s, 3 H), 3.25 (s, 3 H).

To a stirred suspension of 3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-ύfl- pyrimidin-4-amine (775 mg) and the l,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose (1 g), which is prepared as disclosed above, in acetonitrile (25 mL) is added at room temperature SnCl₄ (5 ml of 1 M solution in CH₂Cl₂). The resulting clear solution is stirred at room temperature for 3 h and monitored by LCMS. When LCMS confirms the completion of the reaction, the reaction mixture is diluted with EtOAc, washed with H₂O, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is then purified on a silica column (70% EtOAc in Hexanes) to give l-(2,3-di-0-acetyl-5- deoxy-β-D-ribofuranosyl)-3-methoxy-6-(methylsulfonyl)- IH- pyrazolo[3,4-</]pyrimidin- 4-amine as white solid product (450 mg). MS (electrospray): 444 (M+l) for Ci₆H₂₂N₅O₈S. ¹H NMR (300 MHz, DMSO) δ 8.65 (bs, 1 H), 7.6 (bs, 1 H), 6.2 (d, 1 H), 5.8 (t, 1 H), 5.35 (t, 1 H), 4.2 (m, 1 H), 4.0 (t, 1 H), 4.0 (s, 3 H), 3.3 (s, 3 H), 2.12 (s, 3 H), 2.10 (s, 3 H), 1.3 (d, 3 H). Intermediate 5: (2i?,3/?,4/?)-2-[4-amino-3-methoxy-6-(methylsulfonyl)-lH-pyrazolo- [3,4-</]pyrimidin-l-yl]tetrahydrofuran-3,4-diyl diacetate

3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-d]- pyrimidin-4-amine (0.5 g) may be similarly prepared as described above. (2S,3i?,4/?)-tetrahydrofuran-2,3,4-triyl triacetate (0.428 g) may be prepared by methods disclosed in Carbohydrate Research, 1978, 61, 501-509. The two compounds are combined in acetonitrile (12 mL) and SnCL» (3.25 ml of 1 M solution in CH₂Cl₂) is added at room temperature. The resulting clear solution is stirred at room temperature for 3 h and monitored by LCMS. When LCMS confirms the completion of the reaction, the reaction mixture is diluted with EtOAc, washed with H₂O and brine, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is purified on a silica column (70% EtOAc/Hexane) to give

(2R,3Λ,4/?)-2-[4-amino-3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-c/]- pyrimidin-l-yl]tetrahydrofuran-3,4-diyl diacetate as white solid product (600 mg). MS (electrospray): 430 (M+l) for C₁₅H₂₀N₅O₈S. ¹H NMR (300 MHz, DMSO) δ 8.65 (bs, 1 H), 7.6 (bs, 1 H), 6.25 (d, 1 H), 5.95 (t, 1 H), 5.65 (m, 1 H), 4.4 (dd, 1 H), 4.0 (s, 3 H), 3.3 (s, 3 H), 2.12 (s, 3 H), 2.05 (s, 3 H).

Example 1: 6-(cyclobutylmethoxy)-l-(5-deoxy-β-D-ribofuranosyl)-3-methoxy- lH-pyrazolo [3,4-</J pyrimidin-4-amine

To a stirred suspension of sodium hydride (100 mg, 60% dispersion in oil) is added cyclobutylmethanol (3 ml) slowly under nitrogen. The reaction is heated at 40° C for 15 minutes. l-(2,3-di-O-acetyl-5-deoxy-β-D-ribofuranosyl)-3-methoxy-6-(methylsulfonyl)- lH-pyrazolo[3,4-(i]pyrimidin-4-amine (100 mg) is added to the reaction and the resulting mixture is stirred at 40° C for 30 minutes, then at room temperature for 4 hours. The reaction mixture is diluted with ethyl acetate and washed with water. The organic layer is dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is purified by silica column chromatography (100% EtOAc) to give

6-(cyclobutylmethoxy)- 1 -(5-deoxy-β-D-ribofuranosyl)-3-methoxy- lH-pyrazolo[3,4-cfl- pyrimidin-4-amine solid product (14 mg). MS (electrospray): 366 (M+l) for Ci₆Η₂₃N₅O₅. ¹H NMR (300 MHz, DMSO-D6) δ ppm 1.20 (d, 3 H) 1.62 - 2.15 (m, 6 H) 2.58 - 2.78 (m, 1 H) 3.74 - 4.09 (m, 6 H) 4.18 (d, 2 H) 4.43 (t, 1 H) 5.83 (d, 1 H) 6.51 - 6.89 (br s, 1 H) 7.37 - 7.79 (br s, 1 H).

Lithium triethylborohydride (2.5 ml of IM solution in THF) is then added to 6-(cyclobutylmethoxy)-l-(5-deoxy-β-D-ribofuranosyl)-3-methoxy-lH-pyrazolo[3,4-ύ(]- pyrimidin-4-amine (10 mg), and the resulting mixture is heated at 80° C for 4 hours. The reaction mixture is cooled and quenched with slow addition of water. Acetonitrile is added to the quenched reaction mixture and concentrated on a rotary evaporator. The crude product is purified on a silica column (10-20% MeOH in EtOAc) to give

4-amino-6-(cyclobutylmethoxy)- 1 -(5-deoxy-β-D-ribofuranosyl)- 1 ,2-dihydro-3H- pyrazolo[3,4-i/]pyrimidin-3-one solid product (5 mg). MS (electrospray): 352 (M+l) for Ci₅H₂₁N₅O₅. ¹H NMR (300 MHz, DMSO-D6) δ ppm 1.16 (d3 H) 1.65 - 2.16 (m, 6 H) 2.56 - 2.82 (m, 1 H) 3.68 - 3.93 (Br, 2 H) 4.18 (d, 2 H) 4.26 - 4.46 (m, 1 H) 5.01 (d, 1 H) 5.26 (t, 1 H) 5.58 5.90 (m, 1 H) 6.70 (s, 1 H) 7.53 (s, 1 H) 11.10 (s, 1 H).

Example 2: 4-amino-l-(5-deoxy-β-D-ribofuranosyl)-6-[(/rαns-4-methylcyc-ohexyl)- oxy]-l,2-dihydro-3H-pyrazolo[3,4-rf]pyrimidin-3-one

To a stirred suspension of sodium hydride (200 mg, 60% dispersion in oil) in TΗF (3 ml) is added frøΗs-4-methylcyclohexanol (3 ml) slowly under nitrogen. The reaction is heated at 50° C for 30 minutes. l-(2,3-di-0-acetyl-5-deoxy-β-D-ribofuranosyl)-3- methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-i/]pyrimidin-4-amine (200 mg) is then added to the reaction and the resulting mixture is stirred at 50° C for 2 hours. The reaction is cooled, diluted with ethyl acetate and washed with water. The organic layer is dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The crude product is purified on a silica column (100% EtOAc) to give l-(5-deoxy-β-D-ribofuranosyl)-3- methoxy-6- [(t nms-4-methylcyclohexy l)oxy] - 1 H-pyrazolo[3 ,4-c/]pyrimidin-4-amine as solid product (25 mg). MS (electrospray): 394 (M+l) for Ci₈H₂₇N₅O₅.

Lithium triethylborohydride (1 ml of IM solution in THF) is then added to a solution of l-(5-deoxy-β-D-ribofuranosyl)-3-methoxy-6-[(/ra«5-4-methylcyclohexyl)- oxy]-lH-pyrazolo[3,4-</]pyrimidin-4-amine (25 mg) in TΗF (1.5 ml), and the resulting mixture is heated at 80° C for 2 days. The reaction is cooled and quenched with slow addition of water (0.5 ml). Methanol is added to the quenched reaction and concentrated on a rotary evaporator. The crude product is then purified on a silica column (20-30% MeOH in EtOAc) to give 4-amino- 1 -(5-deoxy-β-D-ribofuranosyl)-6-[(frøM.ϊ-4- methylcyclohexyl)oxy]-l,2-dihydro-3H-pyrazolo[3,4-</]pyrimidin-3-one solid product (17 mg). MS (electrospray): 380 (M+ 1) for C_nH₂₅N₅O₅. ¹H NMR (300 MHz, DMS0-D6) δ ppm 0.77 - 1.47 (m, 11 H) 1.60 - 1.79 (m, 2 H) 1.90 - 2.12 (m, 2 H) 3.67 - 3.97 (m, 2 H) 4.35 (d, 2 H) 5.00 (d, 1 H) 5.27 (d, 1 H) 5.70 (d, 1 H) 6.63 (s, 1 H) 7.51 (s, 1 H) 11.10 (s, 1 H).

Example 3: 4-amino-l-(5-deoxy-β-D-ribofuranosyl)-6-[(l-methylcyclopropyl)- methoxy]-l,2-dihydro-3H-pyrazolo[3,4-</]pyrimidin-3-one

To a stirred suspension of sodium hydride (250 mg, 60% dispersion in oil) in THF (2.5 ml) is added (l-methylcyclopropyl)methanol (2.5 ml) slowly under nitrogen. The reaction is heated at 50° C for 30 minutes. l-(2,3-di-O-acetyl-5-deoxy-β-D- ribofuranosyl)-3-methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-c(]pyrimidin-4-amine (300 mg) is then added to the reaction and the resulting mixture is stirred at 50° C for 2 hours. The reaction is then cooled, quenched with water and 5% aqeous citric acid, and extracted with ethyl acetate. The organic layer is washed with water, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is purified on a silica column (100% EtOAc) to give l-(5-deoxy-β-D-ribofuranosyl)-3-methoxy-6-

[(l-methylcyclopropyl)methoxy]-lH-pyrazolo[3,4-<^pyrimidin-4-amine solid product (52 mg). MS (electrospray): 366 (M+l) for Ci₆H₂₃N₅O₅.

Lithium triethylborohydride (2 ml of IM solution in THF) is then added to l-(5-deoxy-β-D-ribofuranosyl)-3-methoxy-6-[(l-methylcyclopropyl)methoxy]-lH-pyrazolo[3,4- c/Jpyrimidin-4-amine (52 mg) and the resulting mixture is heated at 80° C for 12 hours. The reaction is cooled and quenched with slow addition of water (1 ml). Methanol (20 ml) is added to the quenched reaction and concentrated on a rotary evaporator. The crude product is purified on a silica column (20-30% MeOH in EtOAc) to give 4-amino- 1 -(5-deoxy-β-D-ribofuranosyl)-6-[( 1 -methylcyclopropytymethoxy]- 1 ,2-dihydro-3H-pyra zolo[3,4-J]pyrimidin-3-one solid product (48 mg). MS (electrospray): 350 (M-I) for C₁5Η₂1N5O5. ¹H NMR (400 MHz, DMSO-D6) δ ppm 0.30 - 0.38 (m, 2 H) 0.47 - 0.54 (m, 2 H) 1.13 (s, 3 H) 1.16 (d, J=6.06 Hz, 3 H) 3.74 - 3.90 (m, 2 H) 3.93 - 4.08 (s, 2 H) 4.33 (s, 1 H) 5.00 (s, 1 H) 5.24 (s, 1 H) 5.74 (d, J=3.54 Hz, 1 H) 6.68 (br s, 1 H) 7.54 (br s, 1 H) 10.73 - 11.53 (br s, 1 H).

Example 4: 4-amino-l-(5-deoxy-β-D-ribofuranosyl)-6-(2,2-dimethylpropoxy)-l,2- dihydro-3H-pyrazolo [3,4-rf] pyrimidin-3-one

To a stirred suspension of sodium hydride (250 mg, 60% dispersion in oil) is added 2,2-dimethylpropan-l-ol (2.5 ml) in THF (2.5 ml) slowly under nitrogen. The reaction is heated at 50° C for 30 minutes. l-(2,3-di-O-acetyl-5-deoxy-β-D-ribofuranosyl)-3- methoxy-6-(methylsulfonyl)-lH-pyrazolo[3,4-c/]pyrimidin-4-amine (300 mg) is then added to the reaction and the resulting mixture is stirred at 50° C for 2 hours. The reaction is cooled, quenched with water and 5% aqueous citric acid, and extracted with ethyl acetate. The organic layer is washed with water, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is purified on a silica column (100% EtOAc) to give l-(5-deoxy-β-D-ribofuranosyl)-6-(2,2-dimethylpropoxy)-3- methoxy-lH-pyrazolo [3,4-</]pyrimidin-4-amine solid product (47 mg). MS (electrospray): 368 (M+l) for C₁6Η₂5N5O5.

Lithium triethylborohydride (2 ml of IM solution in THF) is then added to l-(5-deoxy-β-D-ribofuranosyl)-6-(2,2-dimethylpropoxy)-3-methoxy-lH-pyrazolo[3,4-(i]pyrimidi n-4-amine (45 mg), and the resulting mixture is heated at 80° C for 12 hours. The reaction is cooled and quenched with slow addition of water (1 ml). Methanol (20 ml) is added to the quenched reaction and concentrated on a rotary evaporator. The crude product is purified on a silica column (20-30% MeOH in EtOAc) to give 4-amino-l-(5-deoxy-β-

D-ribofuranosyl)-6-(2,2-dimethylpropoxy)-l,2-dihydro-3H-pyrazolo[3,4-^pyrimidin- 3-one solid product (19 mg). MS (electrospray): 354 (M+l) for C₅H₂₃N₅O₅. ¹H NMR (400 MHz, DMS0-D6) δ ppm 0.96 (s, 9 H) 1.16 (d, 3 H) 3.78 - 3.87 (m, 2 H) 3.89 (s, 2 H) 4.35 (d, 1 H) 5.00 (s, 1 H) 5.25 (d, 1 H) 5.77 (d, 1 H) 6.68 (br s, 1 H) 7.59 (br s, 1 H) 11.16 (br s, 1 H).

Example 5: 4-amino-l-[(2/?,3/?,4/?)-3,4-dihydroxytetrahydrofuran-2-yl]-6- [(frα/is^-methylcyclohexylJoxyl-l^-dihydro-SH-pyrazoloP^w/lpyrimidin-S-one

To a stirred suspension of sodium hydride (200 mg, 60% dispersion in oil) is added frvmϊ-4-methylcyclohexanol (2.5 g) in THF (2.5 ml) slowly under nitrogen. Reaction is heated at 50° C for 30 minutes. (2/?,3i?,4i?)-2-[4-amino-3-methoxy-6-

(methylsulfonyl)-lH-pyrazolo[3,4-(/|pyrimidin-l-yl]tetrahydrofuran-3,4-diyl diacetate (200 mg) is then added to the reaction and the resulting mixture is stirred at 50° C for 3 hours. The reaction cooled, quenched with water and 5% aqueous citric acid, and extracted with ethyl acetate. The organic layer is washed with water, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product is purified on a silica column (70-100% EtOAc in hexanes) to give (2/?,3/?,4/?)-2-{4-amino-3-methoxy-6-

[(/rα«^-4-methylcyclohexyl)oxy]-lH-pyrazolo[3,4-c(]pyrimidin-l-yl}tetrahydrofuran-3,4-diol solid product (16 mg). MS (electrospray): 380 (M+l) for Ci₇H₂₅N₅O₅.

Lithium triethylborohydride (1 ml of IM solution in THF) is then added to (2i?,3/?,4/?)-2-{4-amino-3-methoxy-6-[(/rαλ?5-4-methylcyclohexyl)oxy]-lH-pyrazolo[3,4-<f]pyrim idin-l-yl}tetrahydrofuran-3,4-diol (16 mg), and the resulting mixture is heated at 80° C for 8 hours. The reaction is cooled and quenched with slow addition of water (1 ml). Methanol (20 ml) is added to the quenched reaction and concentrated on a rotary evaporator. The crude product is then purified on a silica column (20-30% MeOH in EtOAc) to give 4-amino-l-[(2i?,3/?,4i?)-3,4-dihydroxytetrahydronjran-2-yl]-6-[(/ra«5-

4-methylcyclohexyl)oxy]-l,2-dihydro-3H-pyrazolo[3,4-<flpyrimidin-3-one solid product (10 mg). MS (electrospray): 366 (M+ 1) for C₆H₂₃N₅O₅. ¹H NMR (300 MHz, DMSO-D6) δ ppm 0.88 (d, 3 H) 0.92 - 1.47 (m, 5 H) 1.61 - 1.82 (m, 2 H) 1.92 - 2.11 (m, 2 H) 3.57 - 3.74 (m, 1 H) 3.91 - 4.11 (m, 1 H) 4.22 (s, 1 H) 4.56 (s, 1 H) 4.68 - 4.90 (m, 1 H) 5.06 (br s, 1 H) 5.28 (br s, 1 H) 5.72 (d, 1 H) 6.75 (br s, 1 H) 7.38 (br s, 1 H) 11.1 (br s, IH).

Claims

We claim:

1. A compound which is a 6-oxy-4-amino- 1 -(tetrahydrofuranyl)-7H- pyrazolo[3,4-d]pyrimidin-3(2H)-one, in free or salt form.

2. A compound of formula I

or a pharmaceutically acceptable salt thereof, wherein:

R is selected from Ci_₈alkyl, C_2-8alkenyl, C₂-salkynyl, C_3-8carbocyclyl, aryl, and heterocyclyl, any of which may be optionally substituted on one or more carbon atoms by R';

R^a is hydrogen,

C₂-₆alkenyl, hydroxy(Ci_₆alkyl), and cyano, any of which may be optionally substituted on one or more carbon atoms by R';

R¹, R^1', R², R^2', R³, and R³ are each independently selected from hydrogen, hydroxy, cyano, azido, Ci.₆alkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C₂-₆alkenyl, C₂-₆alkynyl, heterocyclyl, -OR⁷, NR⁸R⁹, wherein R¹, R^1', R², R^2', R³, and R³' may be optionally substituted on one or more carbon atoms by one or more R ; or alternatively,

R¹, and R¹ , R² and R^2', or R³ and R³ , taken together with the carbon to which they are attached, form C=O or C=N- 0-R⁶, or an optionally substituted 3, 4, 5, 6, or 7-membered ring containing O, 1, or 2 heteroatoms selected from O, S, NH, or N(Ci-₆alkyl); or alternatively, R¹ and R² or R² and R³ taken together with the carbons to which they are attached, form an optionally substituted 3, 4, 5, or 6-membered ring containing 0, 1 , or 2 heteroatoms selected from O, S, NH, or N(C,.6alkyl);

p is independently at each occurrence 0, 1 or 2;

R⁴ at each occurrence is independently -NR⁸R⁹, Ci_₆alkyl, C₂_₆alkenyl, d-βalkoxy, C₃.₈cycloalkyl, heterocyclyl, and aryl wherein R⁴ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁵ at each occurrence is independently hydrogen, -NR⁸R⁹, -OR⁷, Ci-βalkyl, C₂.₆alkenyl, C₃.₈cycloalkyl, C₃_₈cycloalkenyl, heterocyclyl, and aryl wherein each R⁵ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁶ at each occurrence is independently hydrogen, Ci-βalkyl, C₂-βalkenyl, C₂-₆alkynyl,

C₃_₈cyc llooaallkkyyll,, CCss--ββccyyccllooaallkkeennyyll,, hheetteerrooccyyccllyyll aanndd aarryyll,, wwhheerrein each R⁶ may be optionally substituted on one or more carbon atoms by one or more R ;

R⁷ at each occurrence is independently hydrogen, Ci-βalkyl, C₂-₆alkenyl, C₃.₈cycloalkyl, C₃.gcycloalkenyl, aryl, S(O)_PR⁴, and heterocyclyl wherein R⁷ may be optionally substituted on one or more carbon atoms by one or more R';

R⁸ and R⁹ are each independently selected from hydrogen,

C₂-₆alkenyl, C₂-₆alkynyl, -OR⁷, C_3-8cycloalkyl, C₃.scycloalkenyl, heterocyclyl, and aryl, wherein each R⁸ or R⁹ may be optionally substituted on one or more carbon atoms by one or more R ;

R at each occurrence is independently halo, hydroxy, nitro, -NR⁸R⁹, azido, cyano, isocyano, Cuβalkyl, C₂-₆alkenyl, C₂-6alkynyl, aryl, C3.₈cycloalkyl, Cs-scycloalkenyl, heterocyclyl, keto(=O), -OR⁷, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴; =N-O-R⁶, -NHC(O)NR⁸R⁹, -N(C,.₆alkyl)C(O)NR⁸R⁹, -NHC(O)R⁷, -NHCO₂R⁷, -NHSO₂(R⁴), -amidino i.e.-NHC(NH)NH₂, wherein each R may be optionally substituted on one or more carbon atoms by one or more R ; R at each occurrence is independently halo, azido, cyano, Ci-βalkyl, C₂-6alkenyl, C₂-6alkynyl, aryl, cycloalkyl, cycloalkenyl, heterocyclyl, hydroxy, -OR⁷, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, -NR⁸R⁹, -amidino i.e.-NHC(NH)NH₂.

3. A compound according to claim 2 of formula II

or a pharmaceutically acceptable salt thereof, wherein R, R^a, R¹, R¹', R², R^2', R³, and R³' are as defined in claim 1.

4. The compound according to claim 1, 2 or 3, which compound is selected from any one of the following:

in free or pharmaceutically acceptable salt form.

5. The compound according to any of the preceding claims, wherein the compound is

in free or pharmaceutically acceptable salt form.

6. A method for: a) producing an antibacterial effect, b) inhibition of bacterial DNA ligase, or c) treating a bacterial infection in a warm blooded animal in need of such treatment, comprising administering to said animal an effective amount of a compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form.

7. A compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form for use as a medicament.

8. The use of a compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form in the manufacture of a medicament for use in a method according to claim 6.

9. A compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form for use in a method according to claim 6.

10. A pharmaceutical formulation comprising a compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form and a pharmaceutically acceptable diluent or carrier.

11. A pharmaceutical composition comprising a compound according to any of claims 1-5, in free or pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable excipient or carrier for use in the production of an anti-bacterial effect in a warm-blooded animal, such as a human being.

12. The method of claim 6, wherein said warm-blooded animal is a human being.

13. A process for preparing compounds according to claim 1 in free or pharmaceutically acceptable salt form, which process comprising the step of treating

6-oxy- 1 -(tetrahydrofuranyl)-3 -methoxy- /H-pyrazolo [3 ,4-d]pyrimidin-4-amines with a hydride.

14. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises the step of treating a 6-oxy-3-methoxy- 7H-pyrazolo[3,4-d]pyrimidin-4-amine with a hydride.

15. The process of claim 13 or 14, wherein said hydride is lithium triethylborohydride.

16. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises the step of:

(i) treating a compound of the general formula of Η-O-R with base selected from a group consisting of sodium hydride, lithium hydride and potassium hydride; and

(ii) adding 3-methoxy-6-(methylsulfonyl)-l-(tetrahydrofuranyl)- /H-pyrazolo

[3,4-d]pyrimidin-4-amine to step (i).

17. The process of claim 16, wherein said hydride is sodium hydride.

18. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises the step of treating 3-methoxy-6- (methylsulfonyl)- lH-pyrazolo[3,4-d]pyrimidin-4-amine with:

(i) a tetrahydrofuranyl electrophile of formula (2):

Formula (2) wherein L is a leaving group (e.g., acetate, methoxy, benzoyl or halo); and (ii) a reagent selected from a group consisting of (a) a base, (b) a Lewis Acid (e.g., tin (VI) chloride, aluminum chloride), (c) palladium and (d) triphenylphosphine and diethylazodicarboxylate.

19. The process of claim 16, wherein said reagent is tin (VI) chloride.

20. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises the step of treating a 3-methoxy-4,6-bis(methylsulfonyl)-pyrazolo[3,4-d]pyrimidine with ammonia.

21. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises the step of treating 3-methoxy-4,6-bis(methylthio)- pyrazolo[3,4-d]pyrimidine with persulfuric acid.

22. A process for preparing compounds according to any of claims 2-5, in free or pharmaceutically acceptable salt form, which process comprises performing the steps according to:

(i) Claim 20;

(ii) Claim 19;

(iii) Claim 17 or 18;

(iv) Claim 15 or 16; and

(v) Claim 13 or 14.