WO2002055661A2

WO2002055661A2 - Fatty acid synthase inhibitors

Info

Publication number: WO2002055661A2
Application number: PCT/US2001/051330
Authority: WO
Inventors: Robert A. Daines; Jeffrey M. Axten; Israil Pendrak; Wei Peng
Original assignee: Smithkline Beecham Corporation
Priority date: 2000-11-17
Filing date: 2001-11-16
Publication date: 2002-07-18
Also published as: WO2002055661A3; AU2002243439A1

Abstract

This invention relates to the novel use of urea beta-lactam compounds as inhibitors of the fatty acid synthase FabH.

Description

FATTY ACID S YNTHASE INHIBITORS

FIELD OF THE INVENTION This invention relates to the use of urea beta-lactam compounds as inhibitors of the fatty acid synthase FabH.

BACKGROUND OF THE INVENTION The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, although the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeasts possess type I fatty acid synthases (FASs) in which all of the enzymatic activities are encoded on one or two polypeptide chains, respectively. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as my colic acids. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadakou. Chapman & Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264, 7624-7629.)

The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and JJ respectively). The second step in the elongation cycle is ketoester reduction by NADPH-dependent β- ketoacyl-ACP reductase (FabG). Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (Fabl). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), IBioLChem. 271, 1833-1836). Fab H is therefore a major biosynthetic enzyme, which is also a key regulatory point in the overall synthetic pathway (Heath, RJ. and Rock, CO. 1996. J.Biol.Chem. 271, 1833-1836; Heath, R . and Rock, CO. 1996. J.Biol.Chem. 271, 10996- 11000).

The antibiotic thiolactomycin has broad-spectrum antibacterial activity both in vivo and in vitro and has been shown to specifically inhibit all three condensing enzymes. It is non-toxic and does not inhibit mammalian FASs (Hayashi, T. et al.,1984. J. Antibiotics 37, 1456-1461; Miyakawa, S. et al., 1982. J. Antibiotics 35, 411-419; Nawata, Y et al., 1989. Acta Cryst. C45, 978-979; Noto, T. et al., 1982. J. Antibiotics 35, 401-410; Oishi, H. et al., 1982. J. Antibiotics 35, 391-396. Similarly, cerulenin is a potent inhibitor of FabB & F and is bactericidal but is toxic to eukaryotes because it competes for the fatty-acyl binding site common to both FAS types (DAgnolo, G. et al.,1973. Biochim. Biophys. Acta. 326, 155-166). Extensive work with these inhibitors has proved that these enzymes are essential for viability. Little work has been carried out in Gram-positive bacteria.

There is an unmet need for developing new classes of antibiotic compounds that are not subject to existing resistance mechanisms. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics of this type would be rendered inactive by known antibiotic resistance mechanisms. Moreover, this is a potentially broad-spectrum target. Therefore, FabH inhibitors would serve to meet this unmet need.

SUMMARY OF THE INVENTION

This invention comprises novel compounds and pharmaceutical compositions containing these compounds and their use as FabH inhibitors that are useful as antibiotics for the treatment of Gram positive and Gram negative bacterial infections.

This invention further constitutes a method for treatment of a Gram negative or Gram positive bacterial infection in. an animal, including humans, which comprises administering to an animal in need thereof, an effective amount of a compound of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are represented by Formula (I):

wherein:

Ar represents optionally substituted aryl or heteroaryl; and

X represents NRj or O, such that Rj is H or C __[ alkyl; provided that if X is NH or O, Ar cannot be phenyl.

Also included in the invention are pharmaceutically acceptable salt complexes.

Preferred substituents of aryl and heteroaryl groups include halo, cyano, Cl - C4 alkyl, methoxy, nitro, and trifluomethyl.

As used herein, "alkyl" means both straight and branched chains of 1 to 6 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, π-propyl, iso -propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl and the like. The alkyl may cany substituents such as hydroxy, carboxy, alkoxy, and the like.

The compounds of this invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds and diastereomers are contemplated to be within the scope of the present invention.

Some of the compounds of this invention may be crystallised or recrystallised from solvents such as organic solvents. In such cases solvates may be formed. This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation. Since the antibiotic compounds of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 95% pure, particularly at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or salt thereof.

Preferred compounds of the present invention include: 2-Oxo-N-(4-chloro-3-trifluoromethyl)phenyl-l-azetidinecarboxamide; 2-Oxo-N-(3-chloro-2-methyl)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(3-chloro-2-methoxy)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(2-chloro)phenyl- 1 -azetidinecarboxamide ; 2-Oxo-N-(2,5-dichloro)phenyl-l-azetidinecarboxamide; 2-Oxo-N-(3-chloro)phenyl-l-azetidinecarboxamide; and 2-Oxo-N-(2-chloro-5-trifluoromethyl)phenyl- 1 -azetidinecarboxamide.

METHODS OF PREPARATION

Compounds of the formula I were prepared via the method outlined in Scheme 1. For this particular example, X is N, Rl is H and Ar is 4-chloro-3- (trifluoromethyl)phenyl.

Scheme 1

a) 4-chloro-3-(trifluoromethyl)phenyl isocyanate, triethylamine, DMAP cat.,CH₂Cl2, 15 h, it.

Commercially available 2-azetidinone 1, Scheme 1 (Aldrich), was treated with 4-chloro-3-(trifluoromethyl)phenyl isocyanate in the presence of an amine base such as triethylamine in an aprotic solvent such as methylene chloride. This process provides the desired urea-containing compound, in this example, 2-oxo-N-(4-chloro- 3-trifluoromethyl)phenyl-l-azetidinec-irboxamide. Any desired isocyanate may be untilized and the process is not limited to aryl isocyanates or only to commercially available reagents. Following methods well known in the art, a wide range of isocyanates may be generated and used.

SYNTHETIC EXAMPLES The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. All temperatures are given in degrees centigrade, and all solvents are highest available purity unless otherwise indicated.

Example 1 Preparation of 2-oxo~N-(4-chloro-3-trifluoromethyl)phenyM- azetidinecarboxamide

To a solution of 4-chloro-3-(trifluoromethyl)phenyl isocyanate(200 mg, 0.903 mmol) in 5 ml dichloromethane (DCM) in a screwcap vial was added 2- azetidinone (32 mg, 0.451 mmol), triethylamine (0.903 mmol, 126 ul), and DMAP(cat. amount). The reaction was shaken 15 h at room temperature, and the solvent was removed under reduced pressure. Purification by HPLC (30% -100% acetotritrile/water containing 0.1% TFA) yielded the desired compound as a white solid (20 mg). LC/MS (ES+) m/e 293.0 [M+l].

Example 2 Preparation of 2-oxo-N-(3-chloro-2-methyl)phenyl-l- azetidinecarboxamide

The title compound was prepared according to the procedure described for Example 1 substituting 3-chloro-2-methylphenyl isocyanate for 4-chloro-3- (trifluoromethyl)phenyl isocyanate. LC MS (ES+) m/e 239.0 [M+l]. Example 3 Preparation of 2-oxo-N-(3-chloro-2-methoxy)phenyl-l- azetidinecarbcxamide

The title compound was prepared according to the procedure described for Example 1 substituting 3-chloro-2-methoxyρhenyl isocyanate for 4-chloro-3- (trifluoromethyl)phenyl isocyanate. LC/MS (ES+) m/e 255.0 [M+l].

Example 4 Preparation of 2-oxo-N-(2-chloro)phenyl-l-azetidinecarboxa ide

The title compound was prepared according to the procedure described for Example 1 substituting 2-chlorophenyl isocyanate for 4-chloro-3- (trifluoromethyl)phenyl isocyanate. LC/MS (ES+) m/e 225.0 [M+l].

Example 5 Preparation of 2-oxo-N-(2,5-dicMoro)pheϋyI-l-azetidinecarboxamide

The title compound was prepared according to the procedure described for Example 1 substituting 2,6-dichlorophenyl isocyanate for 4-chloro-3- (trifluoromethyl)phenyl isocyanate. LC/MS (ES+) m/e 259.0 [M+l].

Example 6 Preparation of 2-oxo-N-(3-chloro)phenyt-l-azetidinecarboxamide

The title compound was prepared according to the procedure described for Example 1 substituting 3-chlorophenyl isocyanate for 4-chloro-3- (trifluoromethyl)phenyl isocyanate. LC/MS (ES+) m/e 225.0 [M+l]. Exaκιple 7 Preparation of 2-oxo-N-(2-chloro-5-trifluororaethyl)phenyH- azetidinecarboxamide

The title compound was prepared according to the procedure described for Example 1 substituting 2-chloro-5-(trifluoromethyl) phenyl isocyanate for 4-chloro- 3-(trifluoromethyl)phenyl isocyanate. LC/MS (ES+) m/e 293.0 [M+l].

Biological Assay:

FabH was assayed in a coupled format using his-tagged S.aureus FabD, and acyl carrier protein (ACP) purchased from Sigma. Lyophilized ACP was reduced using β-mercaptoethanol in phosphate buffer. Malonyl-CoA, and FabD were added to the reduced ACP, thus generating malonyl-ACP. After the FabD reaction reached equilibrium, [^C] acetyl-CoA and inhibitors were added, and the reaction started by the addition of FabH. TCA precipitation and filtration was used to separate [^C] acetyl-CoA substrate from [^C] acetoacetyl-ACP product.

Secondary and tertiary screens of suitable reproducibility, sensitivity, throughput and analytical power to progress primary screen hits are characterized, validated and in current use. Compounds are evaluated against purified mammalian fatty acid biosynthetic enzymes, E.coli FabH, FabB and a human lung cell cytotoxicity assay.

In addition, whole-cell antibacterial activity is determined against a range of clinically relevant wild type and efflux impaired bacteria using standard and novel fluorescence based technologies. The FabH assay has been thoroughly characterized kinetically and a reaction mechanism proposed. Detailed studies have generated novel data about mechanism of inhibition by tool compounds, including thiolactomycin. Screens in use are of direct relevance to the therapeutic goal - eradication of bacteria from sites of infection ('cure'). Several state-of-the-art animal models of bacterial infection are available, meaningful and in current use in this and numerous other studies at SB. Extensive prior experience with known antibacterials confirm that bacterial kill in vitro and in animal models is an excellent indicator of bacterial kill in vivo and cure of infection.

The present invention also provides a pharmaceutical composition, which comprises a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof, and a pharmaceutically acceptable carrier. The compositions of the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of bacterial infection in mammals including humans.

The antibiotic compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The composition may be formulated for administration by any route, such as oral, topical or parenteral, especially oral. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

The topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for , example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. The solution preferably contains a buffer (such as phosphate) to keep the pH in the range of about 3.5 to 7. DMSO or alcoholic solvents may also be - present (at concentrations such as 0.01 to 10 mL/liter) to aid solubility and penetration of the compound of Formula (I) Advantageously, agents such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The compositions may contain from 0.1% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration. Where the compositions comprise dosage units, each unit will preferably contain from 50-500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 1 to 140 mg/kg of body weight, depending on the route and frequency of administration.. Inhibitors of β-ketoacyl-ACP Synthase (FabH) can be administered by injection in solutions either intravenously, intramuscularly, intraperitoneally, or orally. The solution preferably contains a buffer (such as phosphate) to keep the pH in the range of about 3.5 to 7. DMSO or alcoholic solvents may also be present (at concentrations such as 0.01 to 10 mL/liter) to aid solubility and penetration of the β- ketoacyl-ACP Synthase (FabH) inhibitor.

No unacceptable toxicological effects are expected when a compound of formula (la) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof is administered in the above-mentioned dosage range.

The compound of formula (I) may be the sole therapeutic agent in the compositions of the invention or a combination with other antibiotics or compounds which enhance the antibacterial activity of a compound of formula (I) may be employed.

The antibiotic compounds of the present invention are active against a wide range of organisms including both Gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae and Gram-positive organisms such as Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis and Enterococcus faecium, including isolates resistant to existing antibiotics.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the area can, using the preceding description, utilize the present invention to its fullest extent. Therefore the Examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments, of the invention in which an exclusive property or privilege is claimed are defined as follows.

Claims

What is claimed is:

1. A method of treating bacterial infections by administering to a patient in need thereof an effective amount of a compound of Formula (I) :

wherein,

Ar represents aryl or heteroaryl, such that the aryl or heteroaryl group may be unsubstituted or substituted; and

X represents NR1 or O, such that Rl is H or Cl-4 alkyl; provided that if X is NH or O, Ar cannot be phenyl.

2. A method of treatment according to Claim 1 wherein the compound of Formula (I) is selected from the group consisting of: 2-Oxo-N-(4-chloro-3-trifluoromethyl)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(3-chloro-2-methyl)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(3-chloro-2-methoxy)phenyl-l-azetidinecarboxamide; 2-Oxo-N-(2-chloro)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(2,5-dichloro)phenyl- 1 -azetidinecarboxamide; 2-Oxo-N-(3-chloro)phenyl- 1 -azetidinecarboxamide; and 2-Oxo-N-(2-chloro-5-trifluoromethyl)phenyl-l-azetidinecarboxamide.