WO2008117046A1 - Pyrazolo [4, 3-d] pyrimidines as antibacterial compounds - Google Patents

Abstract

This invention relates to compounds of Formula I and formula II and their use in the treatment of bacterial infections.

Description

PYRAZOLO [4, 3-D] PYRIMIDINES 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'-OH 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 supported the essentiality of this en∑yme (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 111: 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-dependentDNA 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, Brδtz-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 5-(oxy)-3-

(tetrahydrofuranyl)-lH-pyrazolo[4,3-d]pyrimidin-7-amines, e.g., a compound of formula I

or a pharmaceutically acceptable salt thereof, wherein:

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

R^a is hydrogen, C_1-6alkyl, C_2-6alkenyl, hydroxy(C_1-6alkyl), and cyano, any of which may be optionally substituted on one or more carbon atom by R';

R¹, R^1', R², R^2', R³, and R^3' are each independently selected from hydrogen, hydroxy, cyano, azido, C_1-6alkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C_2-6alkenyl, C_2- ealkynyl, heterocyclyl, -OR⁷, NR⁸R⁹, wherein R¹, R¹', 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(C]_-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; - A -

R⁴ at each occurrence is independently -NR⁸R⁹, Q-ealkyl, C_2-6alkenyl, d-βalkoxy, C_{3- 8}cycloalkyl, 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, C_1-6alkyl, C_2-

₆alkenyl, C_3-8cycloalkyl, C_3-8cycloalkenyl, 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., C_2-6alkenyl, C_2-6alkynyl, C_3-8cycloalkyl, C_3-gcycloalkenyl, 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, C_1-6alkyl, C_2-6alkenyl, C_3- scycloalkyl, C_3-8cycloalkenyl, aryl, S(O)_PR⁴, and heterocyclyl wherein R⁷ may be optionally substituted on one or more carbon by one or more R';

R⁸ and R⁹ are each independently selected from hydrogen, C_halky., C_2-6alkenyl, C_2- βalkynyl, -OR⁷, C_3-8cycloalkyl, C_3-8cycloalkenyl, 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, -NR⁸R⁹, azido, cyano, isocyano, Ci_-6alkyl, C_2-6alkenyl, C_2-6alkynyl, aryl, C_3-8cycloalkyl, C_3-8cycloalkenyl, 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 by one or more R ;

R" at each occurrence is independently halo, azido, cyano, C_1-6alkyl, C_2-6alkenyl, C_2- ealkynyl, 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 as shown in formula II

II or a pharmaceutically acceptable salt thereof, wherein R, R^a, R¹' R¹ , R², R² , R³ and

R³ are as defined in formula I.

The invention further provides compounds of Formula I, in free or salt form, e.g., pharmaceutically acceptable salt form, as follows: 1.1 Compounds of Formula I, wherein R is selected from a group consisting of

Ci-galkyl, C_2-8alkenyl, Ca-salkynyl, C_3-8carbocyclyl, aryl and heterocyclyl wherein R is optionally substituted with one or more R'.

1.2 Compounds of Formula I or 1.1 , wherein R is haloC_1-8alkyl.

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

1.4 Compounds of Formula I or 1.1 , wherein R is C_3-8carbocyclyl (e.g., cyclohexyl, bicyclo[3.1.0]hexanyl or bicyclo[4.1.0]heptanyl) or C_{3- 8}carbocyclylalkyl (e.g., cyclobutylmethyl, cyclopentylmethyl, or spiro[2.2]pentanylmethyl) wherein R is optionally substituted with one or more R'.

1.5 Compounds of Formula I or 1.1 or 1.4, wherein R is selected from a group consisting of cyclobutylmethyl, cyclopentylmethyl, methylcyclohexyl (e.g., #-<ms-4-methylcyclohex- 1 -yl), spiro[2.2]pentan- 1 -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'. 1.6 Compounds of Formula I, 1.1 or any of 1.4- 1.5 , wherein R is cyclobutylmethyl.

1.7 Compounds of Formula I or any of 1.1-1.6, wherein R^a is selected from a group consisting of hydrogen, C^aUcyl, C_2-6alkenyl, hydroxy(Ci-₆alkyl), and cyano, wherein R^a is optionally substituted with R'.

1.8 Compounds of Formula I or any of 1.1 - 1.7, wherein R^a is hydrogen.

1.9 Compounds of Formula I or any of 1.1-1.8, wherein R¹ and R^1' are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., cyanomethyl), azido, Ci-βalkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C_2-6alkenyl, C_2-6alkynyl, heterocyclyl,

-OR⁷ and NR⁸R⁹, wherein R¹ and R¹ are optionally substituted with one or more R'.

1.10 Compounds of Formula I or any of 1.1 - 1.9, wherein one of R¹ or R^{1 '} is hydroxy. 1.11 Compounds of Formula I or any of 1.1 - 1.10, wherein one of R¹ or R¹ is hydroxy and the other is hydroxy.

1.12 Compounds of Formula I or any of 1.1-1.10, wherein one of R¹ or R¹ is hydroxy and said hydroxy group is cis to R^a.

1.13 Compounds of Formula I or any of 1.1 - 1.12, wherein R² and R^2' are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., cyanomethyl), azido, Ci_-6alkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C_2-6alkenyl, C_2-6alkynyl, heterocyclyl,

-OR and NR R wherein R and R are optionally substituted with one or more R'. 1.14 Compounds of Formula I or any of 1.1 - 1.13 , wherein R² and R² are independently selected from a group consisting of halo (e.g., flouro or chloro), hydroxy, cyano, cyanoalkyl and azide. 1.15 Compounds of Formula I or any of 1.1-1.141.12, wherein one of R² or R² is hydroxy. 1.16 Compounds of Formula I or any of 1.1-1.151.12, wherein one of R² or R² is hydroxy and the other is hydrogen. 1.17 Compounds of Formula I or any of 1.1-1.15, wherein one of R¹ or R¹ and another of R² or R² are cώ-diol and said ciy-diol is cis to R^a. 1.18 Compounds of Formula I or any of 1.1-1.17, wherein R³ and R^3' are independently selected from a group consisting of hydrogen, hydroxy, cyano, cyanoalkyl (e.g., cyanomethyl), azido, C^alkyl, C₃.₈carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C₂.₆alkenyl, C_2-6alkynyl, heterocyclyl, -OR⁷ and NR⁸R⁹ wherein R³ and R³ are optionally substituted with one or more R'.

1.19 Compounds of Formula I or any of 1.1 - 1.18, wherein R³ and R³ are independently hydrogen, methyl or hydroxymethyl.

1.20 Compounds of Formula I or any of 1.1 - 1.19, wherein one of R³ or R^3' is hydroxymethyl.

1.21 Compounds of Formula I or any of 1.1 - 1.19, wherein one of R³ or R³ is hydroxymethyl and the other is hydrogen.

1.22 Compounds of Formula I or any of 1.1-1.19, wherein one of R³ and R³ is hydroxymethyl and said hydroxymethyl group is trans to R^a. 1.23 Compounds of Formula I or any of 1.1 - 1.19, wherein one of R³ or R³ is methyl.

1.24 Compounds of Formula I or any of 1.1 - 1.19 or 1.22, wherein one of R³ or R³ is methyl and the other is hydrogen.

1.25 Compounds of Formula I or any of 1.1 - 1.19 or 1.22, wherein either R³ or R^3' is methyl and said methyl group is trans to R^a.

1.26 Compound of Formula formula I or formula II selected from

and

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 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3-d]pyrimidin- 7-amine, e.g., compound of formula I, formula II or any of 1.1 - 1.26, 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 5-(oxy)-3-(tetrahydrofuranyl)-lH- pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26 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 5-(oxy)-3-(tetrahydrofuranyl)-lH- pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1 - 1.26, in free or pharmaceutically acceptable salt form.

The invention also provides a 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3- d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26 in free or pharmaceutically acceptable salt form, for use as a medicament. The invention also provides the use of 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3- d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26 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 5-(oxy)-3-(tetrahydrofuranyl)-lH- pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-

1.26, 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 5-(oxy)-3-(tetrahydrofuranyl)-lH- pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1- 1.26, 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 compound of formula I, formula II or a pharmaceutically acceptable salt thereof 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 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3- d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1 - 1.26, 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 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3- d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26, in free or pharmaceutically acceptable salt form, 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 5-(oxy)-3- (tetrahydrofuranyl)-lH-pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26 in free or pharmaceutically acceptable salt form, and a pharmaceutically acceptable diluent or carrier.

The invention also provides a pharmaceutical composition comprising a 5-(oxy)-3- (tetrahydrofuranyl)-lH-pyrazolo[4,3-d]pyrimidin-7-amine, e.g., compound of formula I, formula II or any of 1.1-1.26, 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 a further embodiment, the invention also provides a process (Process I) for preparing 5-(oxy)-3-(tetrahydrofuranyl)- lH-pyrazolo[4,3-d]pyrimidin-7-amines or compounds of formula I, formula II or any of 1.1-1.26, in free or pharmaceutically acceptable salt form, which process comprises the step of treating 5-chloro-3-(tetrahydrofuran-2-yl)-lH- pyrazolo[4,3-d]pyrimidin-7-amines with:

(i) a compound of the general formula of H-O-R as hereinbefore described; and

(ii) a base, e.g., sodium hydroxide, sodium hydride, and potassium hydride.

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. C_3-12carbocyclyl, 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-C_1O cycloalkyl rings, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclooctyl radicals, adamantanyl, norbomyl, decahydronapthyl, octahydro-lH-indenyl, spiro [2.2] pentanyl, andbicyclo [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, morpholinyl, 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 Q-βalkyl, C_2- βalkenyl, C_2-6alkynyl, aryl, Cs-gcycloalkyl, Ca^cycloalkenyl, heterocyclyl, C(O)R⁵, S(O)_PC_1- βalkyl, -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, R^{1 '}, R², R² , R³ and R^3' 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, 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 on the b-carbon is on the same side of the plane of the ring as R^a that is on the a-carbon. Similarly, "wherein one of R¹ or R^1' and another of R² or R² are cis-diol" refers to compounds wherein the two hydroxy groups (i.e., diol) that are on the b-carbon and c-carbon are on the same side of the plane of the ring. Therefore, cw-diol may be illustrated below as examples 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 that is on the a-carbon as illustrated below by way of examples only:

wherein R³ or R³' is methyl or hydroxymethyl

"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-methyipiperidine, iV-ethylpiperidine, procaine, dibenzylamine, iV.N-dibenzylethylamine, trø-(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 DNA 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 II, 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 DNA 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 DNA 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 p-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, polyvinylpyrrolidone, 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 rj-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 formulas I 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 niM) reactions as 100% inhibition controls. Compound potency will be based on IC5₀ measurements determined from reactions performed in the presence often different compound concentrations.

The compounds are predicted to have an IC_5O 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 are 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 l-in-10 dilution is made into the same liquid medium to prepare the final organism suspension, and 100 μL of this suspension is 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 t-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 t-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 /ra(trifluoroacetate). 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:

Synthesis from the commercially available starting materials formycin A and formycin B (available from Berry & Associates), using procedures such as those shown in Schemes 1 - 3.

Scheme 1

heat

1. J. Heterocyclic Chem (1984) 21 , 1865-70

2. Nucleic Acid Res. (1986) 14, 1747-64

3. Nucleosides & Nucleotides (1991) 10, 1417-27

Scheme 2

3 Steps

Scheme 3

Compounds of formula I, formula II or any of 1.1-1.26 can be prepared by converting a particular compound of formula I to a different compound of formula I (or compounds of formula II can be prepared 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 4, and one non-limiting example of how the 2'- and 3'-positions of the ribose can be modified is shown in Scheme 5. 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 4

Y = F, H, R'

The alcohols used in the displacement reaction on the 2-halo formycins 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 6.

Scheme 6

PhCOCl, (CH₃CH_j)₃N, CH₂CI

KOH (5%), water, THF

6 : 4

Examples

The invention is now illustrated but not limited by the following Examples in which unless otherwise stated:

(i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration;

(ii) temperatures are quoted as ⁰C; operations were carried out at room temperature, that is typically in the range 18-26 ⁰C and without exclusion of air unless otherwise stated, or unless the skilled person would otherwise work under an inert atmosphere;

(iii) column chromatography (by the flash procedure) was used to purify compounds and was performed on Merck Kieselgel silica (Art. 9385) unless otherwise stated; Jones Flashmaster and Biotage refers to automated normal phase chromatography instruments using silica cartridges for the stationary phase; the instruments were used according to the manufacturers instructions;

(iv) in general, the course of reactions was followed by TLC, HPLC, or LC/MS and reaction times are given for illustration only; yields are given for illustration only and are not necessarily the maximum attainable; (v) the structure of the end-products of the invention were generally confirmed by NMR and mass spectral techniques. Proton magnetic resonance spectra were generally determined in DMSOd₆ unless otherwise stated, using a Bruker DRX-300 spectrometer operating at a field strength of 300 MHz. In cases where the NMR spectrum is complex, only diagnostic signals are reported. Chemical shifts are reported in parts per million downfield from tetramethysilane as an internal standard (δ scale) and peak multiplicities are shown thus: s, singlet; d, doublet; dd, doublet of doublets; dt, doublet of triplets; dm, doublet of multiplets; t, triplet, m, multiplet; br, broad. Fast-atom bombardment (FAB) mass spectral data were generally obtained using a Platform spectrometer (supplied by Micromass) run in electrospray and, where appropriate, either positive ion data or negative ion data were collected or using Agilent 1 lOOseries LC/MSD equipped with Sedex 75ELSD, and where appropriate, either positive ion data or negative ion data were collected. The lowest mass major ion is reported for molecules where isotope splitting results in multiple mass spectral peaks (for example when chlorine is present). Reverse Phase HPLC was carried out using YMC Pack ODS-AQ (100x20 mmID, S-5μ particle size, 12 nm pore size) on Agilent instruments; (vi) each intermediate was purified to the standard required for the subsequent stage and was characterized in sufficient detail to confirm that the assigned structure was correct; purity was assessed by HPLC, TLC, or NMR and identity was determined by infra-red spectroscopy (IR), mass spectroscopy or NMR spectroscopy as appropriate; (vii) the following abbreviations may be used:

TLC is thin layer chromatography; HPLC is high pressure liquid chromatography; MPLC is medium pressure liquid chromatography; NMR is nuclear magnetic resonance spectroscopy; DMSO is dimethylsulfoxide; CDCl₃ is deuterated chloroform; MeOD is deuterated methanol, i.e. D₃COD; MS is mass spectroscopy; ESP (or ES) is electrospray; EI is electron impact; APCI is atmospheric pressure chemical ionization; THF is tetrahydrofuran; DCM is dichloromethane; MeOH is methanol; DMF is dimethylformamide; EtOAc is ethyl acetate; LC/MS is liquid chromatography/mass spectrometry; h is hour(s); min is minute(s); d is day(s); TFA is trifluoroacetic acid; v/v is ratio of volume/volume; Boc denotes t- butoxycarbonyl; Cbz denotes benzyloxycarbonyl; Bz denotes benzoyl; arm denotes atmospheric pressure; rt denotes room temperature; mg denotes milligram; g denotes gram; μL denotes microliter; mL denotes milliliter; L denotes liter; μM denotes micromolar; mM denotes millimolar; M denotes molar; N denotes normal; nm denotes nanometer; (viii) microwave reactor refers to a Smith Microwave Synthesizer, equipment that uses microwave energy to heat organic reactions in a short period of time; it was used according to the manufacturers instructions and was obtained from Personal Chemistry Uppsala AB; and Example l; (lSl-l-fT-amino-S-fcvcIobtttylmethoxyVljH-PyrazoIo^^-cπpyrimidin-S-vn- 1,4-anhvdro-D-ribitol

Acetic anhydride (7 ml) was added slowly to a solution of (lS)-l-(7-ammo-5-hydroxy-lH- pyrazolo[4,3-cf]pyrimidin-3-yl)-l,4-anhydro-D-ribitol (1.5 g, 5.3 mmol) (prepared as in J. Heterocyclic Chem. 1984, 21, 1865 from formycin A) and 4-dimethylaminopyridine (100 mg) in dry pyridine (40 ml) at rt. The solution was stirred for 48 h then concentrated to dryness. The residue was dissolved in dichloromethane (250 ml), washed with 5% aqueous sodium bicarbonate and brine, dried over sodium sulfate, filtered and concentrated to dryness to give 2.1 g of (l»S)-2,3,5-tri-O-acetyl-l-[l-acetyl-7-(diacetylamino)-5-hydroxy-lH-pyrazolo[4,3- _<flpyrimidin-3-yl]-l,4-anhydro-D-ribitol. To a solution of crude (15)-2,3,5-tri-O-acetyl-l-[l- acetyl-7-(diacetylamino)-5-hydroxy-lH-pyrazolo[4,3-cT]pyrimidin-3-yl]-l,4-anhydro-D-ribitol (0.51 g) in dry dimethylformamide (7 ml) was added successively cesium carbonate (1.2 g, 3.8 mmol) and (bromomethyl)cyclobutane (320 μL). The mixture was stirred at rt for 48 h, diluted with dichloromethane (18 ml) and methanol (2 ml), and filtered through Celite. The filtrate was concentrated to dryness and the residue was taken up in 7N ammonia in methanol (3 ml). The solution was heated in a microwave reactor for 1 h at 70 °C then concentrated to dryness. The residue was purified by RP- ΗPLC using 0.1% trifluoroacetic acid and acetonitrile as the mobile phases with a gradient of 5-35% in 15min. Relevant fractions were combined and concentrated to dryness. The residue was re-purified by RP- ΗPLC with 1OmM ammonium acetate and acetonitrile as the mobile phases with a gradient of 5-25% in 15min. Relevant fractions were combined to give 6.4 mg of the desired product. MS (ESPV. 352 (MΗ⁺)for C₁₅H₂₁N₅O₅ 1H NMR (400 MHz. MeOD) δ ppm 1.77 - 1.89 (m, 4 H) 1.96 - 2.06 (m, 2 H) 2.67 (m, 1 H) 3.61 (dd, 1 H) 3.76 (dd, 1 H) 3.99 (q, 1 H) 4.15 (dd, 2 H) 4.18 (dd, 1 H) 4.49 (dd, 1 H) 5.00 (d, 1 H) Example 2: AZ12594626-001; (lS)-l-f7-amino-5-(cyclobutvlmethoxvVlH-pvrazoIor4,3- rfJpyrimidin-3-yl]-l,4-anhydro-5-deoxy-D-ribitol

A roundbottom flask was charged with (l.S)-l-(7-amino-5-chloro-lH-pyrazolo[4,3- fi0pyrimidin-3-yl)-l,4-anhydro-5-deoxy-2,3-O-(l-methylethylidene)-D-ribitol (50mg, 0.15mmol). Cyclobutane methanol (3ml) was added followed by one sodium hydroxide pellet. The reaction was stirred at 80 ⁰C for 8 days and then concentrated in vacuo. The crude residue was purified by RP-ΗPLC using acetonitrile, water/ammonium hydroxide as the mobile phase. Relevant fractions were pooled and lyophilized resulting in approximately 20mg of (15)- 1 -[7-amino-5-(cyclobutylmethoxy)-lH-pyrazolo[4,3-i/]pyrimidin-3-yl]- 1 ,4- anhydro-5-deoxy-2,3-O-(l-methylethylidene)-D-ribitol as a white solid. (15)-l-[7-amino-5- (cyclobutylmethoxy)-lH-pyrazolo[4,3-c/]ρyrimidin-3-yl]-l,4-anhydro-5-deoxy-2,3-O-(l- methylethylidene)-D-ribitol (20mg, 0.05 mmol) was dissolved in 2ml water. Then 2ml formic acid was added and the reaction was stirred at rt for 15 h. Volatiles were removed in vacuo and the crude material was purified by flash chromatography uisng 5-15% methanol in chloroform as eluent. Relevant fractions were pooled and the material was redissolved in water and lyophilized to give 4.5mg of desired product as a white solid. MS (ESP): 336.25 (MH⁺) for C₁₅H₂]N₅O₄ 1H NMR (300 MHz. MeOD) δ ppm IH NMR (300 MHz, MeOD) δ ppm 0.88 (m, 2 H); 1.37 (d, 3 H); 1.9 - 2.1 (2 m, 4 H); 2.80 (m, 1 H); 3.98 - 4.13 (m, 2 H); 4.30 (d, 2 H) 4.61 (m, 1 H); 5.06 (d, 1 H). The intermediates for this compound were prepared as follows:-

(l-^-l-CV-amino-S-chloro-lH-pyrazolo^_jS-rflpyrimidin-S-y^-l^-anhydro-S-deoxy-l^-O-

(l-methylethylidene)-D-ribitol

A flask was charged with (l^-l-CV-amino-S-chloro-lH-pyrazolo^^-c/Ipyrimidin-S-yl)-!^- anhydro-2,3-C>-(l-methylethylidene)-5-O-[(4-methylphenyl)sulfonyl]-D-ribitol (0.36g, 0.73 mmol) and cooled to 0 ⁰C. Lithium triethylborohydride in tetrahydrofuran (IM, 5ml) was added slowly. After stirring at 0 ⁰C for lOmin, the reaction was allowed to warm to rt and stir an additional 3h. The reaction was quenched by careful addition of ImI water. Volatiles were removed in vacuo and the resulting residue was dissolved in dichloromethane (50ml) and washed with cold water (50ml), saturated sodium bicarbonate (50ml), and dried over sodium sulfate. The aqueous layer was back-extracted with dichloromethane and the organic layers combined. Purification by flash chromatography using 2-6% methanol in dichloromethane as eluent gave the desired product as a clear glass (108mg). MSjΕSP): 326.14 (MH⁺) for C₁₃Hi₆ClN₅O₃

¹H NMR (300 MHz. DMSO-d6) δ ppm 1.20 (d, 3H); 1.30 (s, 3H); 1.49 (s, 3H); 4.05 (m, IH); 4.57 (m, IH); 5.01 (m, IH); 5.32 (m, IH); 8.15 (br s, 2H); 12.98 (br s, IH).

(l^-l-CV-amino-S-chloro-lH-pyrazoIo^jS-ΛTIpyriniidin-S-yO-l^-anhydro^jS- -^- methylethylidene)-5-0-[(4-methylphenyl)sulfonyl]-D-ribitol

(lS)-l-(7-amino-5-chloro-lH-pyrazolo[4,3-c(]pyrimidin-3-yl)-l,4-anhydro-2,3-O-(l- methylethylidene)-D-ribitol (0.25g, 0.73 mmol) was dissolved in pyridine (8ml) and cooled to -10 ⁰C. A solution of p-toluenesulfonyl chloride (0.28g, 1.46mmol) in pyridine (2ml) was added via addition funnel. The reaction was stirred at -10 ⁰C for 2 h, then the volatiles were removed in vacuo. The resulting residue was dissolved in chloroform (50ml) and washed with saturated sodium bicarbonate (2x50ml), water (50ml), and brine (50ml). The organic layer was dried over sodium sulfate. The crude material was purified by flash chromatography using 1-6% methanol in dichloromethane as the eluent. Relevant fractions were pooled. The product contained a mixture of mono- and di-tosylated products which were used in the subsequent reaction without further purification. MS (ESPV 496.14 (MH⁺) for C₂₀H₂₂ClN₅O₆S (monotosylated)

¹H NMR GOO MHz, DMSO-d6) δ ppm 1.27 (s, 3 H); 1.47 (s, 3 H); 2.34 (s, 3 H); 4.06 - 4.21 (m, 2 H); 4.75 (m, 1 H); 5.09 - 5.34 (series of m, 2 H); 7.28 (d, 2 H); 7.62 (d, 2 H); 12.97 (s, 1 H).

(15)-l-(7-amino-5-chloro-lH-pyrazolo[4,3-ff]pyrimidin-3-yl)-l,4-anhydro-2,3-0-(l- methylethylidene)-D-ribitol

(15)-l-(7-amino-5-chloro-lH-pyrazolo[4,3-c(]ρyrimidin-3-yl)-l,4-anhydro-D-ribitol

(l.lmmol) (prepared as described in Nucleic Acids Res. 1986, 14, \141-64 from oxoformycin B) was suspended in acetone (10ml); dimethoxypropane (0.27ml, 2.2 mmol) and p- toluenesulfonic acid monohydrate (38mg, 0.2 mmol) were added and the reaction was heated to 40 °C. To completely dissolve all solids, N-methylpyrrolidine (6ml) was added. After stirring at 40 ⁰C for 4h, additional dimethoxypropane (0.3ml) and p-toluenesulfonic acid (40mg) were added. The reaction was stirred an additional 3.5 h at 40 °C then at rt overnight. Organics were removed in vacuo and water (40ml) was then added. This solution was extracted with ethyl acetate (3x50ml). The combined organic layers were washed with saturated sodium bicarbonate (50ml) and dried over sodium sulfate. Purification by flash chromatography using 1-7% methanol in dichloromethane as eluent gave the desired product as a white foam (257mg). MS (ESP): 342.15 (MH⁺) for C₁₃Hi₆ClN₅O₄ ¹H NMR (300 MHz. DMSO-d6) δ ppm 1.30 (s, 3 H); 1.51 (s, 3 H;) 3.46 (m, 2 H); 4.06 (m, 1 H); 4.79 (dd, 1 H); 5.04 (m, 1 H); 5.17 (m, 1 H); 8.21 (br s, 1 H) 12.96 (br s, 1 H).

Claims

We claim:

1. A compound which is a 5-(oxy)-3-(tetrahydrofuranyl)-lH-pyrazolo[4,3- d]pyrimidin-7-amine in free or salt form.

2. A compound of formula I

in free or pharmaceutically acceptable salt form, wherein:

R is selected from d-salkyl, C_2-8alkenyl, C_2-8alkynyl, C_3-8carbocyclyl, aryl, and heterocyclyl, any of which may be optionally substituted on one or more carbon atom by R';

R^a is hydrogen, C_1-6alkyl, C_2-6alkenyl, hydroxy(Ci-₆alkyl), and cyano, any of which may be optionally substituted on one or more carbon atom by R';

R »1 , R r> l , R , R , R , and R are each independently selected from hydrogen, hydroxy, cyano, azido, C_1-6alkyl, C_3-8carbocyclyl, halo, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴, C_2-6alkenyl, C_2- βalkynyl, heterocyclyl, -OR⁷, NR⁸R⁹, wherein R¹, R¹', R², R²', R³, and R³' may be optionally substituted on one or more carbon atoms by one or more R ; or alternatively,

R¹, and R^1', R² and R²', 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 0, 1, or 2 heteroatoms selected from O, S, NH, or N(C].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_1-6alkyl);

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

R⁴ at each occurrence is independently -NR⁸R⁹, Ci_-6alkyl, C_2-6alkenyl, C_1-6alkoxy, C_{3- 8}cycloalkyl, 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, C_1-6alkyl, C_{2- 6}alkenyl, C_3-8cycloalkyl, C_3-8cycloalkenyl, 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_-6alkyl, C_2-6alkenyl, C_2-6alkynyl,

C_3-8cycloalkyl, C₃-₈cycloalkenyl, 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, C^aUcyl, C_2-6alkenyl, C_3- scycloalkyl, C^cycloalkenyl, aryl, S(O)_PR⁴, and heterocyclyl wherein R⁷ may be optionally substituted on one or more carbon by one or more R';

R⁸ and R⁹ are each independently selected from hydrogen, C_halky., C_2-6alkenyl, C_{2- 6}alkynyl, -OR⁷, C_3-8cycloalkyl, C_3-8cycloalkenyl, 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, -NR⁸R⁹, azido, cyano, isocyano, Ci^alkyl, C_2-6alkenyl, C_2-6alkynyl, aryl, C_3-8cycloalkyl, C_3-8cycloalkenyl, heterocyclyl, keto(=O), -OR⁷, -C(O)R⁵, -OC(O)R⁵, S(O)_PR⁴; =N-O-R⁶, -NHC(O)NR⁸R⁹, - N(C_1-6alkyl)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 by one or more R ; R" at each occurrence is independently halo, azido, cyano, d-βalkyl, C_2-6alkenyl, C_2- ealkynyl, 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

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

4. The compound according to claim 1 - 3, wherein said compound is

in free or pharmaceutically acceptable salt form.

5. A method for: i. producing an antibacterial effect, ii. inhibition of bacterial DNA ligase, or iii. 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 1-4, in free or pharmaceutically acceptable salt form.

6. A compound according to any of claims l-4,in free or pharmaceutically acceptable salt form for use as a medicament.

7. A use of a compound according to any of claims 1-4, in free or pharmaceutically acceptable salt form in the manufacture of a medicament for use in a method according to claim 5.

8. A compound according to any of claims 1-4, in free or pharmaceutically acceptable salt form for use in a method according to claim 5.

9. A pharmaceutical formulation comprising a compound according to any of claims 1-4, in free or pharmaceutically acceptable salt form and a pharmaceutically acceptable diluent or carrier.

10. A pharmaceutical composition comprising a compound according to any of claims 1-4, in free or pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable excipient or carrier for use in a method according to claim 5.

11. The method of claim 5 wherein said warm-blooded animal is a human being.

12. A process for preparing compounds according to any of claims 1-4, in free or pharmaceutically acceptable salt form, which process comprises the step of treating 5-chloro-3-(tetrahydrofuran-2-yl)-lH-pyrazolo[4,3-d]pyrimidin-7-amines with:

(i) a compound of the general formula of H-O-R as hereinbefore described; and

(ii) a basel

13. The process according to claim 12, wherein said base is selected from a group consisting of sodium hydroxide, sodium hydride and potassium hydride.

14. The process according to claim 12, wherein said base is sodium hydroxide.