Intermediates of the mureidomycin group, their preparation, and their use

Abstract: New intermediates of the mureidomycin group have the formula and wherein R¹ and R² have various definitions.

Description: The present invention relates to certain novel intermediates of the mureidomycin group, and also provides methods for preparing them and the use of the compounds, and their salts and esters, as synthetic intermediates in the preparation of other mureidomycins. As resistance to conventional antibiotics becomes increasingly established in common strains of pathogenic bacteria, the need for a wider variety of antibiotics for use in the fight against such bacteria becomes ever more crucial. Although this need can be, and sometimes is, met by chemical modification of existing classes of antibiotic, the discovery of a wholly new class of antibiotic leads to exciting possibilities in the treatment of diseases caused by pathogenic bacteria. Such a new class of antibiotics, named the "mureidomycins", is disclosed in our previous U.S. Patent No. 5 213 974. In this we have disclosed the isolation of the first four members of this class, mureidomycins A, B, C and D, from the fermentation broth produced by a then newly discovered microorganism, Streptomyces flavidovirens SANK 60486 (BIKOKEN JOHKI 1347; FERM BP-1347). We have now discovered two new members of this class of antibiotics, herein named "mureidomycins E and F", together with their salts and esters and certain other derivatives, which are particularly effective against Gram-negative bacteria, most especially strains of the genus Pseudomonas . Mureidomycins E and F can also be isolated from the fermentation broth of Streptomyces strain SANK 60486. Alternatively, these two new mureidomycins as well as certain of their derivatives can be prepared by subjecting mureidomycin A to chemical reaction. These compounds are claimed in European Publication No. 317,292. Mureidomycins A to F referred to herein can be represented by the following planar structural formula: wherein:- for Mureidomycin A , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; for Mureidomycin B , R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; for Mureidomycin C , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; for Mureidomycin D , R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; for Mureidomycin E , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 8-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and for Mureidomycin F , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 6-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl. The invention provides, as new compunds, mureidomycins AP to FP which can be represented by the following planar structural formula: wherein R¹ and R² have the same meanings as before. The invention also provides salts of compounds of formula (II). The present invention also provides a process for producing mureidomycins AP to FP. Mureidomycins AP, BP, CP, DP, EP and FP of the above formula (II) and their salts can be prepared by hydrolysis of the corresponding mureidomycin A, B, C, D, E or F of formula (A) above, or a salt thereof, optionally followed by salification or desalification of the product thus obtained, when appropriate. The hydrolysis can be effected by treating the starting material with a suitable acid, base or protease, optionally in the presence of a solvent. Acids, bases and proteases conventionally employed in previously known similar hydrolysis reactions may be used for this preparation, without particular limitation. For example, there can be used a mineral acid such as hydrochloric, sulphuric or hydrobromic acid; or an alkali or alkaline earth metal hydroxide or carbonate such as sodium hydroxide, potassium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate or calcium carbonate; but it is preferred to use a protease such as pepsin, cathepsin, pronase or chymotrypsin, and particularly pepsin. It is also preferred to carry out the hydrolysis in the presence of a solvent, so that it will proceed more smoothly. There is no particular limitation on the choice of the solvent, provided that it has no adverse effect on the reaction, and suitable examples include water, aqueous methanol, and mixtures of an alcohol such as methanol or ethanol with an ether such as tetrahydrofuran or dioxane or a sulphoxide such as dimethyl sulphoxide. If the hydrolysis is effected with an acid or a base, the reaction temperature is not particularly critical, and for instance the reaction may be performed at room temperature or at the reflux temperature of the solvent (if one is employed). On the other hand, if the hydrolysis is effected with a protease, then conventional enzymatic reaction conditions should be employed; for example, in the case of pepsin the reaction may be carried out in a reaction solvent adjusted to pH 2.5 with dilute hydrochloric acid, at a temperature from 10 to 60°C, and more preferably around 37°C, for a period of from 10 hours to 3 days. After completion of the reaction, the desired compound can be isolated and purified by conventional techniques, utilizing its physico-chemical properties. (The physico-chemical properties for mureidomycins AP, BP, CP, DP, EP and FP are set out below, in Examples 9 to 14, respectively). For example, suitable methods include: extraction with solvents; ion-exchange through resins, for example, anion exchange resins such as Dowex SBR-P (Dow Chemical Co.) or cation exchange resins such as Dowex 50 W (Dow Chemical Co.) or IRC-50, CG-50 (Rohm & Haas Co.); chromatography through active carbon as the absorbent or through non-ionic absorption resins such as Amberlite XAD-2, XAD-4 or XAD-7 (Rohm and Hass Co.) or Diaion HP 10, HP 20, CHP 20P or HP 50 (Mitsubishi Chemical Industries, Ltd.); and chromatography through silica gel or alumina. Furthermore, separation, collection and purification of the metabolites may be performed by using any one or more of the following operations, which may be combined in any order or repeated, if desired: partition column chromatography over cellulose such as Avicel (Asahi Chemical Industry Co., Ltd.) or Sephadex LH-20 (Pharmacia Co.); gel filtration using Sephadex G-10, G-25, G-50 or G-100 (Pharmacia Co.) or Toyopearl HW-40 (Toyo Soda Manufacturing Co., Ltd.); crystallization; and recrystallization. ("Dowex", "Amberlite", "Diaion", "Avicel", "Sephadex" and "Toyopearl" are all trade marks.) Where the mureidomycin AP, BP, CP, DP, EP or FP is isolated in the form of a salt, it may be converted to the free unsalified compound by conventional means, such as the use of ion-exchange resins or of adsorbents for reverse phase chromatography. Equally, the free unsalified compound may be salified by conventional means, for instance by treatment with an appropiate acid, such as one of those listed above, or with an appropiate base (e.g. a metal hydroxide or carbonate, such as sodium or potassium hydroxide, or sodium or calcium carbonate). The production, isolation, purification and properties of mureidomycins A to D, used as starting materials for the preparation of mureidomycins AP to DP in the above reaction are described in our aforementioned U.S. patent application serial number 51,665 filed on May 18, 1987. The production isolation, purification and properties of mureidomycins E and F, used as starting materials for the preparation of mureidomycins EP and FP in the above reaction, are described in our co-pending European publication No 317,292, from which this application is derived. Mureidomycins AP to FP can be used as starting materials for the chemical synthesis of other mureidomycins, using per se conventional reaction steps. By way of example, the procedure described below and shown schematically in Reaction Scheme 1 can be used to synthesise mureidomycin C from mureidomycin AP. Dicyclohexylcarbodiimide (DCC) is added with stirring and under ice-cooling to a solution of N -carbobenzoxyglycine (CBZ-glycine), and a solution of mureidomycin AP is added dropwise to the resulting mixture. The reaction is allowed to proceed under ice-cooling for about 3 to 4 hours. After filtration, the solvent is removed by distillation and the residue is purified by a conventional method, e.g. liquid chromatography or TLC to give a glycine derivative having the formula (V) in Reaction Scheme 1. A ureido derivative having the formula (VI) is prepared from methionine and m -tyrosine by a conventional method, and dissolved in a suitable organic solvent. DCC is added to this solution with stirring and under ice-cooling, and then a solution of the glycine derivative (V) is added to the resulting mixture. The reaction is allowed to proceed under ice-cooling for about 3 to 4 hours. After filtration, the solvent is removed by distillation. (In all of the preceding steps, any suitable organic solvent, such as ethyl acetate or acetonitrile, may be used for making up the solutions). The resultant residue is dissolved in a suitable organic solvent (e.g. 80% aqueous methanol) and subjected to catalytic reduction with a suitable catalyst (e.g. 10% palladium on carbon) for about 2 to 3 hours. After completion of the reduction, the catalyst is removed by filtration and the organic solvent is distilled off. The aqueous layer is freeze-dried to afford mureidomycin C in the form of a powder. In the reaction scheme, "CBZ" stands for N -carbobenzoxy. The invention is further illustrated by the following Examples. Example 1 Mureidomycin AP from mureidomycin A A solution of 100mg of mureidomycin A in 50 ml of deionized water was adjusted to pH 2.5 with 1N aqueous hydrochloric acid, 200gm of pepsin (Boehringer Mannheim Co.) were then added, and the mixture was kept stirred overnight at 37°C to complete the reation. The reaction mixture was then adjusted to pH 5.0 with 1N aqueuos sodium hydroxide and adsorbed onto a column of 70 ml of Diaion CHP 20P resin (Mitsubishi chemical Industries, Ltd.). The column was eluted successively with 350 ml of deionized water and 210 ml of 0.1% aqueous trifluoroacetic acid. The eluate was concentrated and lyophylized to afford 34 mg of mureidomycin AP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 502; 3) Molecular formula: C₂₃H₃₀N₆O₇; 4) Thin-layer chromatography: Rf value: 0.13; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 8.3 minutes. Example 2 Mureidomycin BP from mureidomycin B The procedure described in Example 1 was repeated, but reacting 100 mg of mureidomycin B and 200 mg of pepsin for 24 hours, to give 10 mg of mureidomycin BP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 504; 3) Molecular formula: C₂₃H₃₂N₆O₇; 4) Thin-layer chromatography: Rf value: 0.13; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 8.1 minutes. Example 3 Mureidomycin CP from mureidomycin C The procedure described in Example 1 was repeated, but reacting 100 mg of mureidomycin C and 200 mg of pepsin for 48 hours, to give 28 mg of mureidomycin CP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 559; 3) Molecular formula: C₂₅H₃₃N₇O₈; 4) Thin-layer chromatography: Rf value: 0.13; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 13.6 minutes. Example 4 Mureidomycin DP from mureidomycin D The procedure described in Example 1 was repeated, but reacting 100 mg of mureidomycin D and 200 mg of pepsin for 48 hours, to give 11 mg of mureidomycin DP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 561; 3) Molecular formula: C₂₅H₃₅N₇O₈, 4) Thin-layer chromatography: Rf value: 0.13; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 9.2 minutes. Example 5 Mureidomycin EP from mureidomycin E The procedure described in Example 1 was repeated, but reacting 100 mg of mureidomycin E and 200 mg of pepsin for 24 hours, to give 38 mg of mureidomycin EP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 514; 3) Molecular formula: C₂₄H₃₀N₆O₇; 4) Thin-layer chromatography: Rf value: 0.13; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 10.1 minutes. Example 6 Mureidomycin FP from mureidomycin F The procedure described in Example 1 was repeated, but reacting 100 mg of mureidomycin F and 200 mg of pepsin for 24 hours, to give 29 mg of mureidomycin FP having the following physico-chemical properties:- 1) Character and appearance: Amphoteric, colourless powder soluble in water; 2) Molecular weight: 514; 3) Molecular formula: C₂₄H₃₀N₆O₇; 4) Thin-layer chromatography: Rf value: 0.12; Adsorbent: Silica gel plate (Merck, Kieselgel 60 F₂₅₄); Developing solvent: a 4:1:2 by volume mixture of n-butanol, acetic acid and water; 5) High performance liquid chromatography: Column: ODS H2151(Senshu Kagaku Co.); Developing solvent: 5% acetonitrile - 0.1% aqueous trifluoroacetic acid; Flow rate: 1.0 ml/minute; Retention time: 5.3 minutes.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, IT, LI, LU, NL, and SE Compounds having the formula wherein: i) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; ii) R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; iii) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; iv) R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; v) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 8-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and vi) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 6-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and salts and esters thereof. A process for the preparation of a compound of formula (II), as defined in claim 1, which process comprises hydrolysis of the corresponding mureidomycin A, B, C, D, E or F of formula (A): wherein: for Mureidomycin A , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; for Mureidomycin B , R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; for Mureidomycin C , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; for Mureidomycin D , R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; for Mureidomycin E , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 8-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and for Mureidomycin F , R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 6-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; or a salt thereof, optionally followed where appropriate by salification of desalification of the product thus obtained. A process according to Claim 2 wherein hydrolysis is effected by treating the appropriate starting material with a suitable acid, base or protease, optionally in the presence of a solvent. Use of a compound of formula (II), as defined in claim 1, as an intermediate in the synthesis of a mureidomycin. Use of a compound of formula (II), as defined in Claim 1, wherein R¹ is 2,4-dioxopyrimidin-1-yl and R² is α-amino-3-hydroxyphenethyl as an intermediate in the preparation of mureidomycin C. Claims for the following Contracting States : ES, GR A process for preparing a compound of formula wherein: i) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; ii) R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-amino-3-hydroxyphenethyl; iii) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; iv) R¹ represents 2,4-dioxodihydropyrimidin-1-yl and R² represents α-glycylamino-3-hydroxyphenethyl; v) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 8-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and vi) R¹ represents 2,4-dioxopyrimidin-1-yl and R² represents 6-hydroxy-1,2,3,4-tetrahydroisoquinolin-3-yl; and salts and esters thereof; which comprises hydrolysis of a compound of formula (A): wherein R¹ and R² are as defined above. A process according to Claim 1 wherein hydrolysis is effected by treating the appropriate starting material with a suitable acid, base or protease, optionally in the presence of a solvent. Use of a compound of formula (II), as defined in claim 1, as an intermediate in the synthesis of a mureidomycin. Use of a compound of formula (II), as defined in Claim 1, wherein R¹ is 2,4-dioxopyrimidin-1-yl and R² is α-amino-3-hydroxyphenethyl as an intermediate in the preparation of mureidomycin C.

Inventors: HANEISHI TATSUO C O SANKYO COM, INUKAI MASATOSHI C O SANKYO CO, ISONO FUJIO C O SANKYO COMPANY, KINOSHITA TAKESHI C O SANKYO C, SAKAIDA YOSHIHARU C O SANKYO C, SHIMIZU KEIKO C O SANKYO COMPA, HANEISHI, TATSUO, C/O SANKYO COMPANY LIMITED, INUKAI, MASATOSHI, C/O SANKYO COMPANY LIMITED, ISONO, FUJIO, C/O SANKYO COMPANY LIMITED, KINOSHITA, TAKESHI, C/O SANKYO COMPANY LIMITED, SAKAIDA, YOSHIHARU, C/O SANKYO COMPANY LIMITED, SHIMIZU, KEIKO, C/O SANKYO COMPANY LIMITED
Applicants: SANKYO CO, SANKYO COMPANY LIMITED
Application year: 1988
Main classification: C07D405
IPC codes: C07D405, A61K38, C07K5