Field of the invention

The present invention relates to a new anhydrooctonic acid, and salts thereof, which is a powerful inhibitor of bacterial lipopolysaccharide synthesis. The compound has potential use as an antibacterial agent and as an intermediate or starting material in the preparation of antibacterial agents. The invention also relates to methods for the preparation of the anhydrooctonic acid as well as to new intermediates useful for the preparation of anhydrooctulosonic acids. The invention further relates to preparation of pharmaceutical preparations containing the said acid or salts thereof.

Background of the invention

Lipopolysaccharides (LPS) form a unique class of acromolecules which are characteristic of Gram-negative bacteria. They constitute the outer part of the outer membrane of the microorganism and are involved in the interaction of the cell with the environment. LPS thus plays a critical role in determining the antigenicity, toxicity, adhesiveness, invasiveness, and penetrability of the cell. The outer membrane constitutes an important structure of the Gram-negative bacterial cell by protecting it against the environment, e.g. the host defense system and antibacterial agents.

The structure of LPS in Salmonella typhimurium is shown schematically in the Figure below.

(Tetrasaccharide)g Hex-Hex-Hep-Hep- DO-KDO Lipid A

0-Antigen Core

The Lipid A constitutes the innermost part of LPS. Hex = Hexose, Hep = Heptose, KDO = 3-deoxy-D-manno-octulosonic acid.

Due to the importance of LPS for bacterial survival it might be expected that damage to it would affect the viability of the microbial cell. It has thus-been found that mutants defective in 3-deoxy-D-ιπanno- -octulosonic acid (KDO) biosynthesis are no longer viable. Enzymes involved in the biosynthesis of this acid might therefore constitute valuable targets for chemical compounds, which by inhibition of the enzymes involved would act as novel antimicrobial agents.

Prior art

3-Deoxy-D-manno-2-octulusonic acid (KDO) is described in Advances in Carbohydrate Chemistry and Biochemistry, 38, 323-388 (1981).

Some analogues of KDO are reported to have an inhibiting effect upon the enzyme CMP-KDO synthetase (p. 388).

Ray et al (ACS Symposium Series 231, 141-169) describes the synthesis of analogues of 3-deoxy-D-manno-2-octulosonate (KDO) and their use in inhibiting the biosynthesis of the LPS of Escherichia coli. Analogues of KDO have been synthesized and tested as inhibitors or alternative substrates for CMP-KDO synthetase. The substrate analogues were weak inhibitors of the enzymes involved in KDO biosynthesis.

In J. Med. Che ., 27, 717-26 (1984) compounds are described which are reported to inhibit arabinose-5-phosphate iso erase in the biosynthesis of lipopolysaccharide.

Disclosure of the invention

The present invention relates to a new compound, 2,6-anhydro-3-deoxy-D- -glycero-D-talo-octonic acid with the formula I and physiologically acceptable salts thereof.

It has surprisingly been found that the compound of the invention exhibits a strong inhibitory activity on the enzyme cytidine monophosphate- 3-deoxy-D-manno-octulosonate synthetase (CMP-KDO synthetase). This activity suggests a potential value of the compound as an antimicrobially active agent. The invention takes into consideration that compounds which structurally deviate from the formula I, after administration to a living organism, may be transported into the bacteria and there be transformed to the compound of formula I by hydrolysis or other chemical reactions, and in this structural form exhibit its inhibitory activity on the CMP-KDO synthetase. The compound of the invention is thus useful as an intermediate in the preparation of such compounds in order to obtain an antimicrobially or antibacterially active agent.

The compound of the invention is preferably used as a salt, e.g. a metal salt or a salt with an organic base,preferably the ammonium salt of the formula

NH.

Methods of preparation

The compound of the invention may be obtained by one of the following methods constituting a further aspect of the invention.

A. Epimerization (base-catalyzed iso erization) of a compound of the formula

followed by removal of the protecting groups

2 5 wherein R -R are the same or different and selected from hydrogen or among the protective groups which are stable towards base, such as arylalkyl, or cyclic acetal preferably benzyl or acetonide, and R is an alkyl group. The epimerization may be performed in an alcoholic solvent, e.g. methanol or ethanol containing a metal alkoxide corresponding to

R 0, or by treatment with a metal amide such as lithium diisopropylamide in a suitable solvent such as tetrahydrofuran or diethyl ether.

B. Cyclizat on of a partly protected compound of the formula

wherein Y is a leaving group, e.g. a halogen atom such as chloro, bromo or iodo, an aryl- or alkylsulfonyloxy group, or a phosphoniooxy group (R ) ?0- where R is phenyl or alkyl containing 1-4 carbon atoms. R -R are hydrogen, acyl, arylalkyl such as oenzyl, 2-tetrahydropyranyl, cyclic acetal such as acetonide or a protecting group known in the art. R is hydrogen, an alkyl group containing 1-6 carbon atoms, arylalkyl

or a protecting group known in the art. A great variety of protecting groups are known to those skilled in the art by for instance "Protecting Groups in_.0rganic Chemistry" T.W. Greene, Wiley 1981. The cyclization is initiated by adding a base such as potassium carbonate or metal alkoxide to a solution of the compound of formula IIIA in an alcoholic solvent, e.g. ethanol or ethanol.or a mixture including the said alcohols at room temperature to 50°C,or by treatment of the compound IIIA with sodium hydride in an inert solvent such as tetrahydrofuran or toluene.

The protecting groups of the claimed acid may be removed by one or more chemical reactions known to those skilled in the art,e.g. hydrolysis, to obtain the compound of the formula I.

C. Reduction of a KDO derivative of the formulas IVA or IVB, followed by removal of protecting groups

wherein X is a halogen atom, preferably chloro or bromo, or a sulfur- containing group such as alkylthio, arylthio or aryl-SCOO or thiocarbonyl, R -R are as defined under B.

The reduction is preferably carried out using hydrogen in combination with a metal catalyst such as palladium, platina or nickel. Alternatively, the reduction can be carried out under radical-generating conditions involving a hydride reducing agent such as trialkyltin hydride.

The reduction with hydrogen may be performed in a suitable solvent, such as ethyl acetate, toluene, acetone or dimethyl for amide (DMF) at a temperature between 0°C and 50°C in the presence of a tertiary arnine such as pyridine or triethylamine.

Salts

Physiologically acceptable salts of compounds of the invention are prepared by methods known in the art. The salts are novel compounds and comprise a further aspect of the invention. Addition salts can be prepared by treating the compound of the invention with, for example, ammonia. Metal salts can be prepared by treating a metal hydroxide with a compound of the invention. Examples of metal salts which can be prepared in this way are salts containing Li, Na and K. A less soluble metal salt can be precipitated from a solution of a more soluble salt by addition of a suitable metal compound.

Pharmaceutical preparations

Pharmaceutical preparations of the compound of the invention constitute a further aspect of the invention.

The compound of the invention may be administered in a form aimed at facilitating the transport of the compound of the invention across the bacterial cell wall.

The compounds of the invention can be administered in the form of a pharmaceutical preparation comprising the active ingredient in the form of the original compound or optionally in the form of a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable carrier.

Intermediates

Some of the intermediates or starting materials used in the methods A-C above are known. However, certain intermediates or starting materials are novel and constitute a further aspect of the invention. Thus, in one aspect the invention is related to novel compounds of the formula

wherein Y is a leaving group such as a halogen atom, a sulfonate group or a phosphoniooxy group and R -R are same or different and selected from hydrogen or protecting groups as described above; and to the methods of preparing said chemical intermediates. The preparation of the intermediates will be illustrated by specific examples below.

Methods of preparation of intermediates

The intermediate, of the formula

COOR ¹

wherein Y and R -R are as stated above, may be obtained by one of the following methods.

D. Formation and halogenation of a metal enolate

wherein R ,6-RlO are as stated in Method B, R 12 is a protecting group, which is stable towards base but is removable under conditions where in

R -R are stable, and M is a metal cation. This method has been

1? described by Rathke et al. Tetrah. Lett. 1971, 3995. R is generally selected from the substituted silyl groups such as trimethylsilyl, substituted benzyl groups such as 2,4-dimethoxybenzyl or alkylthioalkyl groups such as methylthiomethyl . M is a metal or mono cation such as lithium or sodium or a metal having additional ligands such as zinc, tin, boron or titanium which confer suitable reactivity on the enolate.

The starting material in this synthesis can be prepared from a suitably protected D-manno-2-octenoate by catalytic hydrogenation and, for example, silylation according to standard procedures known to those skilled in the art. An example of a synthesis of such an octenoate is given by Collins et al. in J.C.S. Chem. Commn. 1981, 1139 who reported the preparation of ethyl 3,4;7,8-di-0-isopropylidene-D-manno-2- -octenoate.

The enolate is formed and allowed to react in an inert organic solvent such as tetrahydrofuran, diethyl ether or tetrarπethylurea at a temperature of between -120-0 ^C C.

Suitable reagents by which to form the enolate may be chosen among the metal amides, such as lithium diisopropylamide, or metal triflates, such as tin(II) triflate, in the presence of a tertiary amine. A suitable modifying metal salt such as zinc chloride may also be added to the

solution of a lithium enolate. The addition of a modifying salt in a halogenation of an enolate is new and constitutes a further valuable aspect of the invention.

After reaction and work-up the product is separated from its C-2 epimer, for example by chromatography on silica gel.

12 The group R may be removed by methods known to those skilled in the art and the claimed compound isolated thereafter. We have found, however, that it is more convenient to postpone this step until the intermediate will be cyclized and then instead to perform the deprotection and the cyclization as an one-pot synthesis.

E. Hydrosilylation of a D-manno-2-octenoate and reacting the formed silyl ketene acetal with a halogen or pseudohalogen.

R ,6- _n R12 and Y are as described above and R ,13 is a lower alkyl group such as ethyl .

The starting octenoate can be prepared by standard techniques, such as for example trimethylsilylation, from known compounds such as the one used as starting material in Method D.

The hydrosilylation is performed in excess trialkylsilane as the solvent or in an inert solvent such as toluene or tetrahydrofuran at a tempera- ture of between 15-100°C using a trialkyl- or diarylsilane such as triethylsilane in the presence of a noble metal catalyst such as tris-triphenylphosphine rhodium chloride.

10

After reaction is complete pyridine or another suitable base is added and the mixture is treated with a halogen such as bromine or, prefer¬ ably, with a pseudohalogen such as iodine chloride. The product is purified and treated as described above.

F. Hydrosilylation of an octenoate and reacting the formed silyl ketene acetal with a peracid

R ,6- _n R13 and Y are as described above and R ,14 is an aryl- or alkylsulfonyloxy group as described above. The starting material can be obtained as described in Methods D and E. The hydrosilylation is carried out as described above. The silyl ketene acetal is then oxidized by using a peracid such as 3-chloroperbenzoic acid. After purification by chromatography the resulting silyl ether is treated with a fluoride salt in a suitable solvent such as methanol. The resulting alcohol is then converted into a sulfonate using methods known to those skilled in the

12 art. R is then removed as described above.

Working examples

Preparation of intermediates or starting material for use in Method B

Example 1 Ethyl 2,3-dideoxy-4,5;7,8-di-0-isopropylidene-6-0-(trimethyl- silyl)-D-manno-octonate

Ethyl 2,3-dideoxy-4,5;7,8-di-0-isopropylidene-D-manno-octenoate, which is a known compound (Collins et al. Chem. Soc. Chem. Comm. 1981 , 1139) is hydrogenated over 10% palladium-on-charcoal and then silylated using chlorotrimethylsilane in pyridine-ether to give after silica gel chromatography the title compound as a syrup in 90% yield. [tt} _D +38.7 (£ 1.04, CHC1 ₃ ). ¹³ C-NMR (CHC1 ₃ , TMS-0 in product as zero ppm) : 6172.6, 108.6, 106.9, 76.2, 75.8, 74.9, 71.0, 66.2, 59.4, 29.9, 27.3, 25.3, 25.1, 24.5, 13.5.

Example 2 Ethyl 4,5;7,8-di-0-isopropylidene-2,3-dideoxy-2-iodo-6-0- -(trimethylsilyl)-D-glycero-D-galacto-octonate

The compound from Example 1 (1.0 g;2.5 mmol) in 10 ml of tetrahydrofuran (THF) was added during 5 min to a stirred solution of lithium diisopropylamide (2.8 mmol) in 70 ml of THF, which was kept at -78°C under nitrogen. After 15 min dry ZnCl ₂ (0.34 g; 2.5 mmol) was added in one portion and the mixture was stirred for 1 hour. While keeping the temperature of the reaction mixture at -70°C a solution of iodine (0.76 g; 3 mmol) in 10 ml of THF was added. The temperature was allowed to rise to about 0°C and solvents were evaporated under vacuum while keeping the temperature below 20°C. The residue was dissolved in 100 ml of ether - light petrol (1:4) and treated with 5 ml of a saturated solution of sodium thiosulfate and sodium hydrogen carbonate in water for 5 min. The mixture was dried over Na _^ SO _* .

Chromatography on silica gel (light petrol - ether, 3:1) gave the title compound as a syrup and its C-2 epimer having talo configuration (0.60 and 0.38 g, respectively; 74% total yield). R _f values in the same solvent system were 0.55 and 0.44, respectively.

Physical data: |pO _D 62 ^G (cl.10, CHC1 ₃ ). ¹³ C-NMR: <_.171.0, 109.6, 107.6, 80.2, 77.0, 76.3, 72.0, 67.7, 61.3, 35.7, 28.0, 26.2, 25,8, 25.1, 19.9 (CHI), 13.5 ppm.

Example 3 Ethyl 2,6-anhydro-2,3-dideoxy-2-iodo-4,5;7,8-diiso- propylidene-D-glycero-D-talo-octonate

13

The galacto iodide from example 2 was allowed to react with 0.4 equivalents of tetrabutylammonium fluoride at room temperature in a 9:1 mixture of ethyl acetate and ethanol until all starting material had disappeared (1-5 min). This treatment removed the trimethylsilyl group to give the title compound in 90% yield after purification on silica gel (pentane ether, 1:1) ^13C -NMR (CHCI3): άl71.1, 109.3, 108.2, 76.5, 76.2, 76.2, 70.6, 67.3, 61.6, 35.4, 27.0, 26.7, 25.2, 24.8, 20.5, 13.6 IR: 3400 cm ^'1 (strong).

Preparation of Partially Protected Final Products

Example 4 Methyl 4,5,7,8-tetra-0-acetyl-2,6-anhydro-3-deoxy-D-glycero- -D-talo-octonate (Method C)

cOOMe

A mixture of 2.0 g (4.6 mmol) of methyl 4,5,7,8,tetra-0-acetyl-2-chloro- -2,3-dideoxy- -D-manno-2-octulosonate (Bhattacharjee et al., Biochemistry 1978, J_7, 645) in 25 ml of toluene containing 1 ml of pyridine and 1 g of 10% palladium-on-charcoal was hydrogenated in a Parr apparatus at 40 psi for 8 h. After filtration and evaporation of solvents the residue was chromatographed on silica gel with ether-pentane 2:1 as eluent to give 1.40 g (68%) of the title compound. M.p. 102-104 ^C C. [x] _D + 108.8° (£ 2.17, CHClg). ¹³ C-NMR (CHC1 ₃ ); ^170.8-169.6 (Cl + acetyl), 72.3 (C2), 70.6 (C6), 68.1, 66.8, 65.0, 62.6 (C8), 52.5 (MeO), 26.5 (C3). Anal. Calc. for ^H^O^: C, 50.50; H, 5.98. Found: C, 50.5; H, 6.2. From the reaction mixture was also isolated 135 mg (6%) of the C-2 epimer with galacto configuration which was briefly described by Unger et al. (Communication at the 9th International Symposium on Carbohydrate Cheπn ^' stry, London 1978. Abstract No B49).

Deprotection

Example 5_ Methyl 2,6-anhydro-3-deoxy-D-gIycero-D-talo-octonate

The tetraacetate-methyl ester from Example 4 was treated with 0.1 equivalents of sodium methoxide in dry methanol for 1 h at room temperature. Methanol-washed H ion-exchange resin was added, the mixture was filtered and solvents were evaporated to give crystalline title compound (100%). M.p. 74-5°C; £0 _D + 76° (£ 2.40, MeOH). ¹³ C-NMR (D ₂ 0, dioxan ref. 67.4) ; <_.174.4, 75.6, 73.4, 70.2, 67.2, 67.0, 64.3, 53.6, 28.4.

Example 6 Ethyl 2,6-anhydro-3-deoxy-4,5;7,8-di-0-isopropylidene-D- -g ycero-D-talo-octonate (Method B).

COOEt

The compound from Example 3 was dissolved in a 9:1 mixture of ethyl acetate and ethanol and an excess of dry K ₂ C0 ₃ (about 3 equivalents) was then added and the stirred mixture was then left at room temperature for 30 h. Evaporation of solvents and silica gel chromatography (ether-pentane 1:1) gave a 95% yield of the title compound.

The D-talo-iodide from Example 2 was also converted in part to the title compound by first treating it in acetonitrile with a catalytic amount of tetrabutylammonium iodide to give an equilibrium mixture of the iodides, then separating the epimers and cyclizing the isolated galacto iodide as described in the present Example.

Deprotection

Example 7 Ethyl 2,6-anhydro-3-deoxy-D-glycero-D-talo-octonate.

The bisacetonide (1.08 g) from example 6 was dissolved in 20 nil of methanol and 5 ml of trifluoroacetic acid were added. The mixture was left for 0.5 h and volatiles were then removed on a vacuum evaporator. The residue was dissolved in methanol which was removed under vacuum.

This procedure was repeated three times. The residue crystallized to give 0.65 g of the title compound. M.p. 126-128=C. ¹³ C-NMR (MeOH): .173.4, 76.3, 73.8, 71.0, 68.0, 68.0, 65.6, 62.4, 29.6, 14.5.

Preparation of salts

Example 8 Ammonium 2,6-anhydro-3-deoxy-D-glycero-D-talo-octonate.

A solution of either of the compounds from Examples 5 or 7 in 2M NaOH was kept until all material was hydrolyzed. The solution was passed through an ammonium-saturated cation exchange resin followed by

evaporation of water. The residue crystallized on standing in the refrigerator to give a monohydrate of the title compound (90%). M.p. 175-182° (decomposition). jδr] _D + 69° (£ 1.68, H ₂ 0). ¹³ C-NMR (DgO, t-BuOH ref £= 30.6): 179.2, 75.0, 74.6, 70.1, 67.7, 67.3, 64.8, 29.4 ppm.

Anal. Calcd. for CgH _1g N0g: C, 37.35; H, 7.44; N, 5.45. Found: C, 37.4; H, 7.28; N, 5.44.

Biological Tests

The inhibitory effect of the compound of the invention on Gram-negative bacteria was tested using the method described below.

In the following the compound ammonium 2,6-anhydro-3-deoxy-D-glycero- -D-talo-octonate is denoted compound I.

Enzyme inhibition studies

Potential inhibitors were tested for inhibitory action against two key enzymes involved in the biosynthesis of bacterial LPS.

1. CMP-KDO synthetase (CKS) which catalyses the reaction

KDO + CTP CKS CMP-KDO + PPi

CMP-KDO lipid A transferase (KLT) which transfers CMP-KDO to an intermediate in the biosynthesis of LPS known as lipid A precursor

CMP-KDO + lipid A ^■ !=____ KDO-lipid A + CMP precursor precursor

CKS Assay

The CKS enzyme was prepared essentially as described by Osborne [J.Biol.Chem. 242 (1978) 4904-4903} and the enzyme assayed by the method of Ghala bor and Heath (J.Biol.Chem. 241 (1966) 3216-3221).

KLT Assay

The KLT enzyme was purified essentially by the method of Osborne (J.Biol.Chem. 252 (1977) 4895-4903). KLT activity was determined by monitoring the incorporation of 14-C]-labelled CMP-KDO into acid- insoluble product. The radiolabelled CMP-KDO was generated in situ from CTP and p4-C]-KD0 by the action of added CKS enzyme. The incubation mixture contained CKS enzyme, KLT enzyme, 110 mM Tris buffer pH 8.0, 10.6 mM NaCl, 0.5 mM CTP, 0.4%, Alfonic 1012-6 (Trade name), 0.16 M [14-c KDO and lipid A precursor. The final reaction volume was 150 μl. The mixture was incubated at 25 ^C C for 15 min., when 50 μl was transferred to a filter paper circle (Munktell No. 5,24 mm.) and then washed copiously with 10% TCA. The radioactivity adsorbed on the paper circle, which represents radiolabelled KDO bound to precursor was determined using liquid scintillation counting.

Results

Inhibition of CKS and KLT enzymes by compound I is given in Table 1. The inhibitory action is expressed in percent of a control without inhibitor run simultaneously.

Table 1 Percentage of inhibition of CKS and KLT enzymes by compound I.

Compound Konc. I ( M) 10.16 2.54 0.64 0.16 0.04 0.01 0.0025 0.000625

CKS 98% 99% 98% 90% 68% 36% 8% KLT 98% 89% 81% 57% 24% 5%