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Title:
6,7-DIHYDROXY-8-METHYL-4-OXO-4H-1-BENZOPYRAN-5-CARBOXYLIC ACID WITH ANTIBACTERIAL ACTIVITY
Document Type and Number:
WIPO Patent Application WO/1997/047616
Kind Code:
A1
Abstract:
There are provided compounds of formula (I) wherein R is a hydrogen atom or an acetyl group, and their pharmaceutically acceptable salt, having antibacterial activity. The compound of formula (I) of the invention wherein R is hydrogen is prepared by fermentation of the micro-organism Streptomyces sp. identified in our data bank by the number 17040 and registered on 20.3.1996 with the DMSZ under the number 10611. The compound of formula (I) (R = -COCH3) is obtained by acetylation with acetic anhydride, in pyridine, of the compound (I) (R = H).

Inventors:
FEDELI LORENA LUISA
FRANCHI GIULIANO
SOLARI INVENTI AUGUSTO
PIACENZA LUCA
RIVOLA GIOVANNI
ROSSI ROSARIA
GAROFANO LUISA
Application Number:
PCT/EP1997/002899
Publication Date:
December 18, 1997
Filing Date:
June 04, 1997
Export Citation:
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Assignee:
MINI RICERCA SCIENT TECNOLOG (IT)
International Classes:
A61K31/00; A61K31/35; C12P7/40; A61K31/352; A61P31/00; A61P31/04; C07D311/22; C12R1/465; (IPC1-7): C07D311/22; A61K31/35
Foreign References:
EP0114899A11984-08-08
DE2132260A11972-02-03
Other References:
CHEMICAL ABSTRACTS, vol. 88, no. 17, 24 April 1978, Columbus, Ohio, US; abstract no. 117779u, page 270; XP002039093
CHEMICAL ABSTRACTS, vol. 99, no. 25, 19 December 1983, Columbus, Ohio, US; abstract no. 209832m, page 407; XP002039094
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Claims:
CLAIMS
1. Compounds of the formula (I) wherein R is a hydrogen atom or an acetyl group and their pharmaceutically acceptable salts.
2. The compound of claim 1 which is 6,7dihydroxy8methyl4oxo4Hl benzopyran5carboxylic acid and its pharmaceutically acceptable salts.
3. The compound of claim 1 which is 6,7diacetyloxy8methyl4oxo4Hl benzofuran5carboxylic acid and its pharmaceutically acceptable salts.
4. A fermentative process for preparing a compound of the formula I (R=H), which comprises fermentation, in the presence of a strain of Streptomyces sp., identified with the DSMZ collection No. 10611, or a variant or mutant thereof capable of producing said compound, performed under aerobic conditions in a nutritive liquid medium containing an assimilable source of carbon, nitrogen and mineral salts.
5. A fermentative process for preparing a compound of the formula I (R=H), according to claim 4 wherein the fermentation is carried out at a temperature ranging from 23° to 31 °C and for a period varying from 96 to 200 hours.
6. A fermentative process for preparing a compound of the formula I (R=H), as claimed in each of the previous claims 4 and 5, which comprises the step of purifying said compound and/or preparing its pharmaceutically acceptable salt.
7. A therapeutic composition containing one or more compounds as claimed in the previous claims 1 to 3, together with a pharmaceutically acceptable vehicle.
Description:
6,7-DIHYDROXY-8-METHYL-4-OXO-4H-l-BENZOPYRAN-5- CARBOXYLIC ACID WITH ANTIBACTERIAL ACTIVITY

This invention refers to compounds of formula (I)

wherein R is a hydrogen atom or an acetyl group, and their pharmaceutically acceptable salt, having antibacterial activity.

The compound of the formula (I) of the invention wherein R is hydrogen is prepared by fermentation of the microorganism Streptomyces sp. identified in our strain bank by the number 17040 and registered on 20.3.1996 with the DSMZ (DEUTSCHE

SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH

Mascheroder Weg lb, D-38124 Braunschweig, Germany) under the Budapest Treaty with the number 10611.

MACROSCOPIC CHARACTERISTICS OF THE STRAIN 17040

The macroscopic characteristics of the strain 17040 were recorded after 14 days of incubation at 28°C and are given in table 1. Growth is generally satisfactory both on organic as well as synthetic media.

TABLE 1. Cultural characteristics of the strain 17040.

IDENTIFICATION AND CLASSIFICATION OF THE STRAIN 17040

All the characteristics shown by the strain 17040 clearly correspond to those reported for the genus Streptomyces by Waksman and Henrici (BERGEY'S MANUAL OF DETERMINATIVE BACTERIOLOGY 6 th Ed, THE WILLIAMS AND WILKINS BALTLMORA - 1948).

The biochemical and physiological characteristics of the strain 17040 are given in tables 2 and 3.

+ = growth

- = non growth

The medium used for this test was Yeast nitrogen base (Difco) with the addition of

K 2 HPO 4 1%.

TABLE 3. Biochemical characteristics of the strain 17040.

+ - positive reaction - = negative reaction

The medium used for growth of the strain 17040 was ISP3 (Difco). An aqueous suspension of the mycelium was used to determine the enzymatic activity. FERMENTATION

The fermentation in liquid phase can be carried out in two phases: vegetative and productive, at a temperature ranging from 23 to 31°C. The first phase can be carried out for periods varying from 28 to 72 hours; the second for periods varying from 96 to 200 hours. The culture media consist of sources of carbon, nitrogen and mineral salts. The sources of carbon can be starch, dextrine and sucrose; the sources of nitrogen can be cornsteep liquor, soy flour, corn flour, caseine and yeast extract; the sources of mineral salts can be calcium carbonate, potassium phosphate, iron sulphate, zinc sulphate, manganese sulphate, copper sulphate and sodium chloride. The fermentation can be carried out either in a flask or in fermenters of different volumes.

PURIFICATION PROCESS

Upon completion of the fermentation, the compound according to the invention is present in the liquid culture broth, which is separated from the mycelium by centrifuging at neutral pH. The supernatant liquor, with the addition of sodium chloride can be loaded onto LRA 450 Amberlite® resin, from which the new compound is eluted with solutions of phosphate buffer of increasing molarity. The fractions selected by HPLC analysis are loaded onto a XAD-4 Amberlite® resin column from which the active principle is eluted with a mixture of water and miscible organic solvent such as for example acetone, methanol, acetonitrile. Acetonitrile is preferably used. The fractions with a high concentration of active compound (HPLC analysis) are combined and concentrated at reduced pressure. The aqueous

concentrate is acidified to pH=3. After standing at +4°C for 18 hours, the solution separates into a solid which is collected by filtering and dried at reduced pressure. The raw compound can be treated with an absolute ethanol-methanol mixture and the suspension brought to boil. After removal of the insoluble residue by filtering, the solution is concentrated at reduced pressure to eliminate the more volatile solvent. After the residual ethanolic solution is left to stand at +4°C for 24 hours, the compound according to the invention crystallizes and is filtered off and dried at reduced pressure at +45°C. The compound of the formula (I) wherein R is hydrogen can be converted into a compound of the formula (I) wherein R is acetyl by acetylation. The acetylation is preferably carried out by dissolving the starting compound wherein R is H in pyridine, and treating it with acetic anhydride. The resulting acetylated compound is then isolated and characterized by known methods. BIOLOGICAL ACTIVITY Antibacterial activity

The minimum inhibiting concentration (MIC) of the 6,7-dihydroxy-8-methyl-4-oxo- 4H-l-benzopyran-5-carboxylic acid compound (formula I, R=H) was determined on micro-organisms belonging to various Gram positive, Gram negative and acid- resistant genera and species using the standard method of serial dilutions in agar. The results obtained are given in table 4.

TABLE 4 Antibacterial activity of the 6,7-dihydroxy-8-methyl-4-oxo-4H- benzo ran-5-carboxylic acid.

The MIC of the 6,7-dihydroxy-8-methyl-4-oxo-4H-l-benzopyran-5-carboxylic acid compound was also investigated on several clinical strains of Helicobacter pylori and is given in table 5.

TABLE 5. Antibacterial activity of the 6,7-dihydroxy-8-methyl-4-oxo-4H-l- benzo ran-5-carbox lic acid com ound on clinical strains of Helicobacter lori.

The above microbiological data show that the compounds of formula (1) are suitable for use as anti bacterial s, in particular, for combatting infections due to clinical strain of the microorganism Helicobacter pylori.

For the use as antibacterials the compounds of formula (I) and their pharmaceutically acceptable salts can be incorporated into dosage forms by procedures and methods commonly known in the art.

The following examples offer a non-restrictive illustration of the invention.

HPLC Analysis.

The culture, the fractions deriving from the chromatographic processes and the final product, were analysed by means of a Beckman Gold System HPLC with a photodiode detector on an E. Merck Hibar LiChrosper® 100 RP18 chromatographic column 4x250 mm, H 3 PO 4 mobile phase with gradient from 0 to 100% of CH 3 CN in

15 minutes and 100% continuation of CH 3 CN for 5 minutes, flow rate: 2 ml/min.

The HPLC analyses were conducted at room temperature. The volumes injected were 20 ml and under the aforesaid conditions, the 6,7- dihydroxy-8-methyl-4-oxo-4H-l-benzopyran-5-carboxylic acid showed a RT of 8.1 minutes.

EXAMPLE 1

The Streptomyces sp. 17040 culture was allowed to grow for 14 days at 28°C on a CZY agarized culture medium (sucrose 30 g/1; NaNO 3 3 g/1; K 2 HPO 4 1 g/1;

MgS0 4 .7H 2 O 0.5 g/1; KC1 0.5 g/1; yeast extract 4 g/1; FeSO 4 .7H 2 O 0.01 g/1; agar 20 g/1; deionized H 2 O to 1000 ml; pH uncorrected; sterilization 1 15°C x 20 minutes).

One part of the mycelial growth on the agarized culture medium was used as inoculum for the vegetative phase which occurred in the following culture medium: casein 8 g/1; cornsteep liquor 8 g/1; dextrine 16 g/1; (NH 4 ) 2 S0 4 0.8 g/1; K 2 HP0 4 0.2 g/1; CaC0 3 4 g/1; deionized H 2 O to 1000 ml; pH uncorrected; sterilization 120°C x 20

minutes. The flask containing 30 ml of the aforementioned medium was inoculated with 1/5 of the mycelial growth of the strain on agarized culture medium. It was then left to incubate at 28°C on a rotary agitator at 250 rpm for 54 hours. An amount of 2.5 ml of the vegetative culture broth was used to inoculate a flask containing 30 ml of the following culture medium: pen starch 60 g/1; soy flour 10 g/1; cornsteep liquor 18 g/1; CaCO 3 4 g/1; yeast extract 1 g/1; MgSO 4 .7H 2 O 0.02 g/1; FeSO 4 .7H 2 O 0.02 g/1; deionized H 2 O to 1000 ml; pH uncorrected; sterilization 120°C x 20 minutes. The flask was left to incubate at 28°C on a rotary agitator at 250 rpm for 144 hours. EXAMPLE 2 The culture broth (4.7 litres) was centrifuged (9000 x g/20 minutes) to separate the mycelium from the supernatant liquor. The latter (3.5 litres), with pH=7.63 and concentration in desired active compound of 600 mcg/ml, was brought to pH 6.5-7 by adding 1 M hydrochloric acid then loaded onto a 6x33 cm column (approx. 1 litre) of IRA 458 (OH) Amberlite® resin conditioned with 0.3 M phosphate buffer to pH=7. The elution was carried out by using phosphate buffer solutions with pH=7 and with molarity gradient from 0.05 to 0.3. The fractions in HPLC with a peak with a retention time of 8.1 minutes, were combined, sodium chloride (1% w/v) was added and the resulting solution was loaded on top of a 8x20 cm column (IL approx.) of XAD-4 Amberlite® resin. The column was eluted with gradient from 0 to 50% of acetonitrile in water. The fractions containing the active compound were concentrated under vacuum until the organic solvent was completely eliminated. The residual aqueous solution (approx. 2.5 litres) was acidified to pll=3 by addition of 1M hydrochloric acid, then held at -t 14°C for 18 hours. A solid crystallized and was filtered off, washed on the filter with cold demineralized water (+4°C) acidified to ρH=3 with 0.1 M hydrochloric acid, then dried under vacuum at +45°C for 18 hours. The raw solid thus obtained was dissolved in a mixture of absolute

ethanohmethanol 1 :1 v/v on boiling. The solution was concentrated under vacuum until the methanol was completely eliminated, then held at +4°C for 24 hours. A solid crystallized and was filtered off and dried under vacuum at +45°C for 18 hours to obtain the 6,7-dihydroxy-8-methyl-4-oxo-4H-l-benzopyran-5-carboxylic acid (380 mg), identified with the laboratory code FCE 22325.

FCE 22325 was obtained as fine yellowish crystals; it is soluble in dimethylsulfoxide, dimethylformamide and alkaline water, poorly soluble in alcohols, and insoluble in water. Moreover, it is unstable in alkaline water with a pH higher than 8. The pharmaceutically acceptable salt, e.g. the alkali metals salts, of FCE 22325 can be prepared by methods per se known in the art.

CHEMICAL PHYSICAL CHARACTERISTICS

Melting point 240°C

U.V. spectrum ( MeOH) max 224, 252, 340

E 1215, 539, 584 I.R. spectrum (KBr) cm " ' 3500-1400, 1620 570, 1535, 1505, 1490, 1460,

1400, 1 150, 1310, 1 155, 1 10, 1065, 1050, 995, 960, 920, 860, 810, 800, 755, 720 Molecular weight m/z determined for 236 (26, [M]\); 218 (100,[M-H 2 O]\); 190 (14,[M-H 2 O-CO] + .); 189 EI-MS (19,[M-H 2 O-CHO] + ); 173 (10, [M-H 2 O-COOH]'); 162 (12, [M-H 2 O-2CO] + .).

Elemental analysis found C=55.60% H=3.45% calculated C=55.94% H=3.41%

Empirical formula C i ] H 8 O 6

Spectrum Η-NMR (200 MHz; DMSO -d 6 ;) 2.26 (s, 3H, CH 3 -10); 6.33 (d, J-5.8 Hz, I H, H-3); 8.31 (d, J=5.8, I H, H-2); 10.30, 1 1 , 20, 14.60 (extended signals, 3H, OH).

Spectrum , 3 C-NMR ( 100 MHz, DMSO, -d 6 ) 9.4 CH 3 -10; 1 1 1.6 CH 3 ; * 113.7 C5; * 114.1 C4a; * 1 14.4 C8; #145.6 C8a; #151.1 C6; #151.1 #151.5 C7; 156.5 CH-2; 170.0 COOH; 176.8 C-4. * interchangeable # interchangeable

EXAMPLE 3

6.7 diacetyloxy-8-methyl-4-oxo-4H-l-benzofuran-5-carboxylic acid FCE 22325 (240 mg) was dissolved in pyridine (3 ml) and acetic anhydride (2 ml) was added. The reaction mixture was left under magnetical stirring overnight. It was poured into water and ice and shaken after the addition of methylene chloride. After separation of the organic phase, it was washed with saturated solutions of KHSO 4 ,

NaHCO 3 and water, then dried with anhydrous Na 2 SO 4 and filtered on a porous membrane. The filtrate was concentrated under vacuum and by the addition of n- hexane a white crystalline precipitate was obtained which was filtered off and dried to obtain 270 mg of the title compound.

U.V. spectrum (MeOH) max. 224.308

I.R. (KBr) peaks cm. "1 3100-2900, 2550, 1790, 780, 1735, 1625, 1275, 1260, 605, 1590, 1570, 1480, 1445, 400, 1370, 1330, 1205,

1 180, 150, 1090, 1050, 1010, 940, 890, 860, 840. 780. 750, 720

Molecular weight m/z determined 321 [M+Hf ; 279[M+ll-CH2CO] f ; 261 [M M1-

CH 3 COOH] + ; by FAB(+)MS) Elemental analysis found C=56.29% H-3.80% calculated C=56.26% H=3.78%

Empirical formula C 15 H 12 O 8

Spectrum Η-MNR (200 MHz; DMSO- d 6 )

2.24 (s, 3H, CH 3 - 10); 2.27, 2.37 ( 2Xs, 6H, 2 X CH 3 -COOH); 6.39 (d, J=5.9 Hz, IH, H-3); 8.38 (d, J=5.9 Hz, IH, H-2); 13.42 (extended signal, IH, OH). Spectrum 13 C- NMR ( 50 Mz; DMSO-d 6 )

9.5 CH 3 -10; * 19.7 CH 3 -14; *19.8 CH 3 -12; 1 12.0 CH-3; "118.7 C5; # 123.0 C4a; # 125.1 C8; ®136.6 C8a; @ 145.0 C6; @ 152.4; C7; 156.9 CH-2; 165.4 COOH; Λ 167.4 CH3-14; Λ 167.6 CH3-12; 174.8 C-4. * interchangeable # interchangeable ^ interchangeable A interchangeable