JP2003512452A - New indole compounds - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of compounds as inhibitors of fatty acid synthase FabH.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the use of compounds as inhibitors of the fatty acid synthase FabH.

[0002] The biosynthetic pathway of background saturated fatty acids invention are very similar in prokaryotes and eukaryotes. However, the chemical reactions may be different and the biosynthetic device configurations are very different. Vertebrate and yeast are type I fatty acid synthases (FASs)
, In which all enzymatic activities are each encoded on one or two polypeptide chains. Acyl carrier protein (ACP) is an essential part of the complex. In contrast, in most bacterial and plant FASs (type II), each reaction is catalyzed by a separate monofunctional enzyme, and ACP is a distinct protein.
Mycobacteria contain FASs of both type I and type II
Are unique in that they are involved in the biosynthesis of basic fatty acids, while the latter are involved in the synthesis of complex cell envelope lipids such as mycolic acid. Therefore, it appears to have considerable efficacy in the selective inhibition of bacterial systems by broad spectrum antibacterial agents (Jackowski, S. 1992. Emerging Targets in
Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgo
papadakou. Chapman & Hall, New York; and Jackowski, S. et al. (19
89). J. Biol. Chem. 264, 7624-7629.).

The first step in the biosynthesis cycle is malonyl-ACP and acetyl-C by FabH.
Condensation with oA. In a subsequent round, malonyl-ACP condensed with acyl-ACP during chain extension (FabB and FabF, synthase I, respectively).
And II). The second step in the chain extension cycle is the ketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequent dehydration with β-hydroxyacyl-ACP dehydrase (FabA or FabZ) gives rise to trans-2-enoyl-ACP, which is then NADH-.
It is converted to acyl-ACP by the dependent enoyl-ACP reductase (FabI). A further round of this cycle adds two carbon atoms per cycle, ultimately giving rise to palmitoyl-ACP, followed mostly by feedback inhibition of FabH and I by palmitoyl-ACP to halt the cycle (Heath . et al. (1996). J. Biol. Chem. 271, 1833-1836). Therefore, FabH is a major biosynthetic enzyme that is also an important regulatory point in the overall synthetic pathway (Heath, RJ and Rock, CO 1996. J. Biol. Chem. 271, 1833-18.
36; Heath, RJ 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 FASs in mammals (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 Fab
It is a potent inhibitor of B and F, bactericidal but toxic to eukaryotes. Because it competes for a fatty acid-acyl binding site common to both FAS types (D'Agnolo, G. et al., 1973. Biochim. Biophys.
Acta. 326, 155-166). Further studies with these inhibitors have demonstrated that these enzymes are essential for viability. There are few studies on Gram-positive bacteria.

There is an unprecedented need for the development of new classes of antibiotic compounds that do not succumb to existing resistance mechanisms. No commercial antibiotics target fatty acid biosynthesis and therefore no new antibiotics of this type can be inactivated by known antibiotic resistance mechanisms. Moreover, this is a potentially broad spectrum target. Therefore, FabH inhibitors will meet such an unprecedented need.

SUMMARY OF THE INVENTION The present invention includes indole derivatives and pharmaceutical compositions containing these compounds, and their use as FabH inhibitors, which are antibiotics for treating Gram positive and Gram negative bacterial infections. It is useful as a substance. The invention further includes a method of treating Gram-negative or Gram-positive bacterial infections in animals, including humans, which method is characterized in that an effective amount of a compound of the invention is administered to the animal in need thereof.

[0007] Compounds of the Detailed Description of the Invention The present invention of formula (I):

[Chemical 2] (I) [wherein R is OH; Q is O, NH or CH ₂ ], or a pharmaceutically acceptable salt thereof.

Also included in the invention is a complex of pharmaceutically acceptable salts. The compounds of the present 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 included within the scope of the invention. Some of the compounds of this invention may be crystallized or recrystallised from solvents such as organic solvents. In such cases, solvates may be obtained. The present invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water obtainable by processes such as lyophilization.

Since the antibiotic compounds of the invention are intended for use in pharmaceutical compositions, they are each in a substantially pure form, eg at least 60% pure, more suitably at least 75% pure. It will be readily appreciated that, preferably at least 85% pure, especially at least 95% pure, and more particularly at least 98% pure (% by weight). Impure preparations of the compounds may be used to produce the purer forms used in pharmaceutical compositions. Formulations of these less pure compounds should contain at least 1%, more suitably at least 5%, preferably 10 to 49% of a compound of formula (I) or salt thereof.

[0010]   Preferred compounds have Q which is O or NH.

A preferred compound is 1- (6-chloro-3,4-methylenedioxybenzyl)-
5- (2,6-dichlorobenzyloxy) indole-2-carboxylic acid and 1
It is-(6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-dichlorobenzylamino) indole-2-carboxylic acid.

[0012]   The method of Scheme 1 produces compounds of formula I where Q is O.

Scheme 1 Scheme 1 a) 6-chloropiperonyl chloride, NaH, DMF; b) 10% Pd / C, H ₂ , EtOAc; c) 2,6-dichlorobenzyl bromide, NaH, DMF; d) KOH, EtOH / THF, refluxing indole ester 1-Scheme-1 (Aldrich) and base (such as sodium hydride) are treated with solvent (such as DMF), then 6-chloropiperonyl chloride is added and stirred (6 hours to 30 hours, preferably 20 hours),
2-Scheme-1 is obtained. 2-Scheme-1 was dissolved in a solvent (such as ethyl acetate), treated with 10% palladium on carbon and the resulting mixture was shaken (6 to 30 hours, preferably 20 hours), 3-Scheme Get -1. Solvent (DMF
Alkylation of 3-Scheme-1 with 2,6-dichlorobenzyl bromide using a base (such as sodium hydride) to give 4-Scheme-1. Saponification of 4-Scheme-1 with a base (such as potassium hydroxide) in a solvent (such as ethanol and tetrahydrofuran) gives 5-Scheme-1.

[0013]   The method described in Scheme 2 produces compounds of formula I where Q is NH.

Scheme 2 Scheme 2 a) 6-chloropiperonyl chloride, NaH, DMF; b) SnCl ₂ , EtOH, 70 ° C; c) 2,6-di
chlorobenzyl chloride, NaH, DMF; d) 1) NaOH, THF, MeOH. 2) 10% HCl Commercially available 5-nitroindole 6-Scheme-2 using a base such as NaH in a polar aprotic solvent such as DMF. Alkylated with 6-chloropiperonyl chloride. This alkylation method provided Introindole 7-Scheme-2. Tin (II) chloride was used as a reducing agent to reduce the nitro group to give the corresponding amine 8-Scheme-2, although other suitable reagents could be used. N-Alkylation gave 9-Scheme-2 using NaH as base and 2,6-dichlorobenzyl chloride as alkylating agent. The synthesis of 10-Scheme-2 was performed by standard hydrolysis of the ethyl ester of 9-Scheme-2.

Biological assay: FabH inhibition Streptococcus pneumoniae β-ketoacyl-ACP synthase III (Fa
The assay is designed to measure the IC50 for bH). Substrate malonyl-
ACP, [14C] -acetyl-CoA, is mixed with FabH to produce [14C] -acetoacetyl-ACP. Assay conditions: sodium phosphate pH 7.0 100 mM β-mercaptoethanol 1 mM malonyl acyl carrier protein 20 μM acetyl coenzyme A 70 μM [14 C] acetyl coenzyme A 5 μM FabH 1-16 nM Total reaction volume 50 μl 1) Mix all reagents except enzyme Then, dispense into a 96-well plate containing an inhibitor in advance. 2) FabH is diluted with an assay buffer containing 1 mM β-mercaptoethanol, added to the plate to start the reaction, and incubated at 37 ° C. for 40 minutes. 3) Stop the reaction with 150 μl of 10% TCA. 4) Pre-wet GF / C filter mat with 10% TCA, filter the quenched reaction, rinse wells twice with 150 μl 10% TCA, then filter. 5) Oven dry the filter mat at 60 ° C, seal the filter mat in a clear plastic bag, add Betaplate scintillation cocktail, and add Walla
c Count with a Microbeta liquid scintillation counter.

Assay for Antimicrobial Activity Document M7-A4, "Methods for Dilution Susceptibili, which is a procedure recommended by the National Committee for Clinical Laboratory Standards (NCCLS).
ty Tests for Bacteria that Grow Aerobically "(incorporated herein by reference) was used to examine whole-cell antimicrobial activity by broth microdilution. 2-fold serial dilutions ranging from 0.06 to 64 mcg / ml. A panel of 12 strains was evaluated in the assay, which panel consisted of the following laboratory strains: Staphylococcus aureus Oxford, Streptococcus pneumoniae.
R6, Streptococcus pyogenes CN10, Enterococcus faecalis I, Haemophilus i
nfluenzae Q1, Escherichia coli DC0, E. coli ESS, E. coli 7623 (AcrAB +) E
.coli 120 (AcrAB-) Klebsiella pneumoniae E70, Pseudomonas aeruginosa K7
99 wt and Candida albicans GRI 681. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of compound that inhibited visible growth.

The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof or an in vivo hydrolysable ester thereof and a pharmaceutically acceptable carrier. The compositions of the present invention, including those in a form suitable for oral, topical or parenteral use, can be used to treat bacterial infections in mammals, including humans. By analogy with other antibiotics, the antibiotic compounds of the invention may be formulated for administration in any conventional manner for use in human or veterinary medicine. The composition may be formulated for administration by any route, such as oral, topical or parenteral, especially oral. The composition can be tablets, capsules, powders, granules, lozenges,
It may be in the form of liquid preparations such as creams or sterile parenteral solutions or suspensions. The topical formulations of the present invention may be provided as, for example, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated bandages and aerosols,
Preservatives, solvents that enhance drug penetration and suitable and conventional additives such as emollients in ointments or creams may be included.

The formulations may contain compatible conventional carriers, such as ethanol or oleyl alcohol for cream or ointment bases or lotions. Such carriers may make up from about 1% to about 98% of the formulation. More usually they will comprise up to about 80% of the formulation. Tablets and capsules for oral administration include unit dose presentati
on form) and binders such as molasses, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; molding lubricants, Conventional excipients such as, for example, magnesium stearate, talc, polyethylene glycol or silica; disintegrants such as potato starch; or acceptable wetting agents such as sodium lauryl sulfate may be included. The tablets may be coated by methods well known in normal pharmaceutical practice. Oral liquid formulations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or provided as a dry product for constitution with water or other suitable carrier before use. May be. Such liquid formulations include suspending agents such as sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible oils; emulsifiers such as lecithin, sorbitan monooleate or arabic. Gums; non-aqueous carriers (which may include edible oils) such as almond oil, oily esters such as glycerin, propylene glycol or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired It may contain conventional additives such as conventional flavors or colors.

Suppositories will contain conventional suppository bases, eg cocoa-butter or other glyceride. For parenteral administration, a fluid unit dosage form is prepared using the compound and a sterile carrier, water being preferred. The compound, depending on the carrier and concentration used, can be either suspended or dissolved in the carrier. In preparing solutions, the compound can be dissolved in water for injection, filter sterilized, then filled into vials or ampoules and sealed. Preferably, the solution has a pH of about 3.5 to 7
Buffer (such as phosphate) to maintain the range. Formula (I)
DMSO or an alcoholic solvent may be present (concentrations such as 0.01 to 10 mL / liter) to enhance the solubility and penetration of the compounds of. Advantageously, agents such as local anesthetics, preservatives and buffers can be dissolved in the carrier. To increase stability, the composition can be filled into vials and then frozen to remove water under reduced pressure. The dried lyophilized powder may then be sealed in a vial and water for injection may be provided with the vial to reconstitute the liquid before use. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the carrier, undissolved, and sterilized by filtration. The compound can be sterilized by exposure to ethylene oxide and then suspended in a sterile carrier. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The composition may contain from 0.1% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration. When the composition comprises a dosage unit,
Preferably, each unit will contain 50-500 mg of active ingredient. The dose used for adult treatment will depend on the route and frequency of administration, but will preferably be between 1 and 140 mg / kg body weight. No unacceptable toxicological effects are expected when the compounds of formula (I) or their pharmaceutically acceptable salts or their in vivo hydrolysable esters are administered in the above dosage range.

The compound of formula (I) may be the sole therapeutic agent in the composition of the invention, or may be combined with other antibiotics or compounds which enhance the antibacterial activity of the compound of formula (I). A combination may be used. The antibiotic compounds of the present invention include Gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae, as well as Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus fa, including isolates resistant to existing antibiotics.
It is active against a wide range of organisms, including both Gram-positive organisms such as ecalis and Enterococcus faecium. The following examples illustrate the preparation of compounds of this invention and intermediates thereof.

Example 1 Preparation of 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-dichlorobenzyloxy) indole-2-carboxylic acid a) 1- (6-chloro Ethyl -3,4-methylenedioxybenzyl) -5- (benzyloxy) indolecarboxylate NaH (6.1 g, 0.15) rinsed with hexane in DMF (500 mL).
mol; 60% dispersion in hexane) was added 5-benzyloxy-2-indolecarboxylic acid ethyl ester (30 g, 0.1 mol). The resulting mixture was stirred at room temperature for 1 hour. Chloropiperonyl chloride (20.85 g, 0.1
mol) was added. The resulting mixture was stirred at room temperature for 24 hours. The resulting mixture was poured into water and extracted with EtOAc (2x). The combined organic layers are H ₂ O,
Wash with brine, dry (MgSO ₄ ), filter, then concentrate under reduced pressure to give a solid. Recrystallization of the solid from ethanol gave the title compound as a white solid (30.68g, 66%). MS (ES +) m / e 464.5 [M + H] ⁺ b) 1- (6-Chloro-3,4-methylenedioxybenzyl) -5- (hydroxy) indolecarboxylate Ethyl 1- in EtOAc (800 mL). Ethyl (6-chloro-3,4-methylenedioxybenzyl) -5- (benzyloxy) indolecarboxylate (22.67
g, 0.046 mol) in a solution of 10% Pd on carbon (11.34 g, 50% w)
/ W) was added. The resulting mixture was hydrogenated in a Parr shaker at 60 psi for 18 hours. The resulting mixture was filtered through diatomaceous earth and concentrated under reduced pressure to give the title compound as a white solid (14.54%, 80%). MS (ES +) m / e 374.4 [M + H] ⁺ c) 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-
Ethyl dichlorobenzyloxy) indolecarboxylate NaH (2.34 g, 0.0) rinsed with hexane in DMF (390 mL).
6 mol; 60% dispersion in hexane), to a solution of ethyl 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (hydroxy) indolecarboxylate (
14.54 g, 0.04 mol) was added. The resulting mixture was stirred at room temperature for 1 hour. 2,6-Dichlorobenzyl bromide (9.36 g, 0.045 mol) was added. The resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was poured into water and extracted with EtOAc (2x). The combined organic layers were washed with H _{2 O,} brine, dried (MgSO _4), filtered, and then to obtain a solid and concentrated under reduced pressure. Et
Recrystallization of the solid from OAc gave the title compound as a white solid (17.07g).
, 82%). MS (ES +) m / e 515.2 [M + H] ⁺ d) 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-
Dichlorobenzyloxy) indole-2-carboxylic acid 1- (6-chloro-in THF (210 mL) and EtOH (210 mL).
A solution of ethyl 3,4-methylenedioxybenzyl) -5- (2,6-dichlorobenzyloxy) indolecarboxylate (5 g, 9.7 mmol) in KOH (21
0 mL, was added in _{H 2} O 3N solution). The resulting mixture was refluxed for 2 hours.
The resulting mixture was partitioned between EtOAc and 3N HCl. The combined organic layers were washed with H ₂ O, brine, dried (MgSO ₄ ), filtered and then
Concentration under reduced pressure gave a solid. Recrystallisation of the solid from EtOAc gave the title compound as a white solid (4.7g, 97%). MS (ES +) m / e 505.2 [M + H] ^＋

The preparation a of Example 2 1- (6-chloro-3,4-methylenedioxy-benzyl) -5- (2,6-dichlorobenzoyl lolo benzylamino) indole-2-carboxylic acid) 1- (6- Ethyl chloro-3,4-methylenedioxybenzyl) -5-nitroindole-2-carboxylate Ethyl 5-nitroindole-2-carboxylate (0 in anhydrous DMF (4 mL).
． A solution of 5 g, 2.1 mmol) was added to NaH (60% dispersion in mineral oil) (93 mg,
2.3 mmol) and stirred for 5 min, during which time 6-chloro-3,4-chloride
Methylenedioxybenzyl (0.47 g, 2.3 mmol) was added. The resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with EtOAc and washed with water then brine. The organic extract was dried over Na ₂ SO ₄ , filtered and then concentrated under reduced pressure. The resulting residue is 5%, 10%, 15%, then 20%
Chromatography on silica gel eluting with EtOAc / hexanes gave the title compound as a slightly off-white solid (1.32g, 82%). ES
(MS) m / e 403.2 [M + H] ⁺ b) Example 1 in ethyl 1- (6-chloro-3,4-methylenedioxybenzyl) -5-aminoindole-2-carboxylate ethanol (5 mL). Compound of 2a (0.31 g, 0.77 mmo
The solution of l) was treated with tin (II) chloride (0.73 g, 4 mmol) and heated at 70 ° C. for 6 hours. The reaction was poured onto ice, basified with 50% aqueous NaOH, then washed thoroughly with EtOAc. The combined organic extracts were dried over Na ₂ SO ₄ , filtered and then concentrated under reduced pressure. 25%, 30%, 3
Chromatography on silica gel eluting with 5% then 40% EtOAc / hexanes gave the title compound as a brick solid (172 mg, 60%).
ES (MS) m / e 372.9 [M + H] ⁺ c) 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-
Ethyl dichlorobenzylamino) indole-2-carboxylate The compound of Example 2b was used in place of ethyl 5-nitroindole-2-carboxylate and 2 chloride of 2-chloro-3,4-methylenedioxybenzyl chloride was used instead of 6-chloro-3,4-methylenedioxybenzyl chloride.
The title compound was obtained as a pale yellow glassy foam according to the method of Example 2a, except that 6-dichlorobenzyl was used. ES (MS) m / e 530.78 [M] ⁺ d) 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-
Dichlorobenzylamino) indole-2-carboxylic acid A solution of the compound of Example 2c (46 mg, 0.086 mmol) in anhydrous THF (0.4 mL) and methanol (0.2 mL) was treated with 1N NaOH (0.22 m).
L, 0.22 mmol) and stirred for 20 hours. After removing the solvent mixture under reduced pressure, the obtained sodium salt was suspended in water and acidified with 10% HCl (pH = 6.
Up to 5) to give the title compound as a dull yellow solid (35 mg, 80%
). ES (MS) m / e 504.65 [M + 2H] ⁺

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor George Wye-Kin Chan             United States 19096 U, Pennsylvania             Innwood, Wiltshire Road 249             Turn (72) Inventor Kelvin Kin Chong Sham             United States 91362 Sa, California             Thousand Oaks, La Casa Court             No. 3111 F-term (reference) 4C063 AA01 BB03 CC81 DD06 EE01                 4C086 AA01 AA02 BC13 GA02 GA07                       MA01 MA04 NA14 ZB32 ZC20

Claims (3)

[Claims] 1. An effective amount of formula (I): A bacterial infection by administering a compound represented by I [wherein R is OH; Q is O, NH or CH ₂ ] or a pharmaceutically acceptable salt thereof to a patient in need thereof. How to treat. 2. The compound of formula (I) is 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-dichlorobenzyloxy) indole-2-.
The treatment method according to claim 1, which is a carboxylic acid. 3. The compound of formula (I) is 1- (6-chloro-3,4-methylenedioxybenzyl) -5- (2,6-dichlorobenzylamino) indole-2-.
The treatment method according to claim 1, which is a carboxylic acid.