GB2068366A - A paromomycin derivative - Google Patents

Abstract

3%,4%-Dideoxyparomomycin, useful as an antibacterial agent, active against gram-positive, gram-negative bacteria and against protozoa, is disclosed and claimed. The process for its preparation comprises sulphonylation at the 4%- position of a 6,3%,2_,5_,3>,4>-hexa- O-acyl-6%-O-benzoyl-penta-N- benzyloxycarbonylparomomycin to give its 4%-O-sulphonyl derivative which, on treatment with a strong base, affords 3%,4%-b-epoxy-penta-N- benzyloxycarbonyl paromomycin. After acylation or benzoylation of the free hydroxy groups, the epoxy derivative is treated with sodium acetate, acetic acid and sodium acetate to give 6,6%,6_,5_,3>,4>- hexa-O-acyl or benzoyl-4%,deoxy-4%- iodo-penta-N- benzyloxycarbonylparomomycin from which, by reaction with methane sulphonyl chloride in pyridine, the corresponding 6,6%,2_,5_,3>,4>- hexa-O-acyl or benzoyl-3%,4%-dideoxy- 3%-ene-penta-N- benzoyloxycarbonylparomomycin is obtained. De-O-acylation or de-O- benzoylation of this affords 3%,4%- dideoxy-3%-ene-penta-N- benzyloxycarbonylparomomycin from which, after hydrogenation of the 3%,4%-carbon-carbon double bond and contemporaneous removal of the N- protecting groups, the desired 3%,4%- dideoxy-paromomycin is obtained.

Description

SPECIFICATION A paromomycin derivative The invention relates to a paromomycin derivative, to a process for its preparation, and to a pharmaceutical composition containing it.

The invention provides 3',4'-dideoxyparomomycin or a pharmaceutically acceptable salt thereof.

3',4'-Dideoxyparomomycin has the formula (8) hereinbelow.

3',4'-Dideoxyparomomycin may be prepared from 6'-0-benzoyl-6,3',2",5",3"',4"'-hexa-0- (C2-C5 acyl or benzoyl)-penta-N-benzyloxycarbonyl-pa romomycins. These starting materials may in their turn be prepared from paromomycin, a natural aminoglycoside antibiotic (United States Patent Specification Nos 2916485 and 3065147), by the method described in our British Patent Application No. 8018158, or from the same precursor by using benzoyl-imidazole in chloroform with a shorter reaction time and a comparable yield.The preparation, which is within the scope of the invention, is illustrated by the following reaction scheme, in which A represents an acyl or benzoyl group, Bz represents a benzyl group, Ph represents a phenyl group and R represents an alkyl, aryl or aralkyl residue, optionally halogen substituted.

H CH200CPh 0 A NHCOOBz BZOOCNH NHCCHz AO--' 1; / NHCOOBZ BzOOC BzCCCNH 0 4-os ulphonylation \ CH200CPh t1) \ RSo20 < Mw oMDZ AO WYCA | CA OA BzOOCNH (2) strung cE2CH o he he NHCOOBZ NHCOOBz He H NHCOOBZ HO o0 BzOOCilI OH 2 OH BZOOCNH -Ac3rlatian (3) X 20A. 0 > NHCOOBz BzOO( NHCOOBz AC C1H NHcOOB z AO tgO > OA NHCOOBz BZoOCHNCH2\ X ( 4 ) B zOOCNH O

Hal cHCCCHCCHa 20A Nal \CH3COOH 31 I (5) HO COOBz AC | AT NO ThzCCCHil0 AC nzoo CH o0 CA NHcCCsz BZOOCHNCH2| < A o NHCOOBz < NHCOOBZ BZOOCNH t > AC CH2 COCBz AC oO CA BzOOO(NcH2 00A (6) BzOOCNH / throng (7) < nHCOOBZ HO BzOOCNH < -NHCOOBz H | / H Catalytic BzOOCti H oZ Vydrogenation o 0 > NH2 OH 2 HO o0 H HO 0 CH2 OH 2 H2NCH2 OH (8) H2N O

The preparation comprises sulphonylation of the only free hydroxy group (at position 4') of the starting material (1) to give the 4'-0-suíphonyl derivative (2). The reaction is performed using, as sulphonylating agent, an alkylsulphonyl halide, an aralkylsulphonyl halide or an arylsulphonyl halide, any of which may be halogen substituted (preferably sulphonylating agents are methylsulphonyl chloride, p.bromobenzenesulphonyl chloride, p.toluenesulphonyl chloride, benzylsulphonyl chloride and trifluoromethylsulphonyl chloride), in the presence of a base, preferably pyridine. Reaction of compound (2) with a strong base, suitably sodium methoxide, in methanol or chloroform, affords the key intermediate (3), having deprotected hydroxy groups and position 3' and 4' involved in a oxirane ring.

This provides the possibility of selective modifications at such positions. In order to convert the epoxy group into a methylene group, the free hydroxy groups of compound (3) are protected again, by acylation or benzoylation, and the resultant epoxy derivative (4) is treated with sodium iodide, acetic acid and its sodium salt. This treatment causes opening of the oxirane ring to give the iodohydrin moiety in compound (5). The 3',4'unsaturated derivative (6) is prepared from compound (5) by reaction with methanesulphonyl chloride in pyridine and subsequent heating, suitably at reflux. De-O-acylation of compound (6) affords 3 ',4'dideoxy-3'-ene-penta-N-benzyloxycarbonyl-paromomycin (7).

Hydrogenation of carbon-carbon double bond and removal of the N-protecting groups are achieved by catalytical hydrogenation, suitably by transfer catalytic hydrogenolysis in the presence of 10% palladium-on-charcoal and cyclohexane, to obtain 3 ',4'-dideoxyparomomyci n (8). This may be purified by ion exchange column chromatography and converted into sulphate form. Mass spectrometry data (field desorption) indicate by fragmentation both deoxygenation sites to be in ring A.

3',4'-Dideoxyparomomycin is useful as an antibacterial agent, active against gram-positive and gram-negative bacteria and against protozoa. The most straightforward approach to improving the spectrum of antibacterial activity of natural aminoglycoside antibiotics has been to remove or sterically hinder sites of enzymatic inactivation.It is believed that the hydroxy group at 3'-position in the am inoglycoside antibiotics is susceptible of enzymatic inactivation by phosphotransferase enzymes produced by resistant bacterial strains and aiso 4'-hydroxy group suffers of enzymatic inactivation by 0 nucleotidylation (Kirk-Othmer: Encyclopedia of Chemical Technology, Vol. 2, 3rd Ed., 1978 by John Wiley and Sons, Inc.); removal of these hydroxy groups is expected to a broader spectrum of activity, although we do not wish to be bound by these theoretical considerations.

The invention further provides a pharmaceutical composition comprising 3',4'dideoxyparomomycin or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.

Blological Activity The in vitro antibacterial activity, tested by serial di!ution method in solid medium, is reported in the Table. The potency of 3',4'-dideoxyparomomycin is lower than that of paromomycin, but its spectrum included E. coli K12--R148, APH(3'111 producer, and S. epidermis PS109, AAD(4') producer, both highly paromomycin resistant.

The therapeutic activity was tested subcutaneously versus paromomycin in mice experimentally infected with S. aureus Smith and Shigella flexneri. 3',4'-Dideoxyparomomycin shows (Table 2) slightly lower activity than paromomycin against staphylococcal infection; on the contrary, against Shigella infection, the efficacy is almost 3 times higher.

3',4'-Dideoxyparomomycin is very interesting because of its strong resistance to two aminoglycoside inactivating enzymes widespread in bacterial population, and because of its excellent bioavailability.

TABLE 1 In vitro activity of 3 ",4 O-dideoxyparomomycin versus paromomycin

MIC ( g/ml) 3' A'-Dideoxy Strain paromomycin Paromomycin E. coli K12: 12.5 6.S " " K12--R112(1) > 200 > 200 " 55 K12-R148(2) 25 > 200.

" 55 k12-.R55(3) 12.5 6.2 S. aureus 209 P 1.55 0:7 S. epidermis PS 109(4) 1.55- room (1) APH (3') I producer (2) APH (3') II (3) AAD(2'') (4) AAD (4') ,, TABLE 2 Therapeutic activity in mouse experimentally infected

ED50 (mg/kg) (base) 3 '54'-D ideoxy- Strain paromomycin Paromomycin S. aureus Smith OA' 0;26 S. flexneri 9.9 28.8 The followiny Examples, in which all temperatures are in degrees Centigrade, illustrate the invention.

EXAMPLE 1.

Hexa-O-acetyl-6'-O-benzoyl-4'-O-p-bromobenzenesulphonyl-penta-N-benzyloxycarbonylparomomycin 2 g of 6,3',2",5",3"',4"'-hexa-O-acetyl-6'-benzoy-penta-N-benzyloxycarbonylparomomycin prepared as described in our British Patent Application No. 7919778 were dissolved in 20 ml of methylene dichloride and 4 ml of triethylamine and 0.1 g of 4-dimethylaminopyridine were added. 1 g of p-bromobenzenesulphonyl chloride in 3 ml of methylene dichloride was added to the ice-cold solution. After 2 hours at 00, the reaction mixture was stirred for 4 days at ambient temperature. The mixture was evaporated under vacuum and the residue dissolved in chloroform, washed with 0.5 N hydrochloric acid, water, sodium bicarbonate solution and water, in that order. The organic solution was dried and evaporated giving 2.5 g of residue.Purification by preparative TLC on silica gel plates afforded 1.5 g of the title compound (65% yieid) in pure form. The compound had Rf 0.35 on TLC in the solvent system toluene:ethyl acetate (1:1 by volume) starting product; Rf 0.20).

EXAMPLE 2 3 ' ,4'-p-epoxy-penta-N-benzyloxyca rbonylparomomyci n To a solutionof 1.1 g of the compound prepared in Example 1 in 11 ml of methanol were added 11 ml of 0.5 N methanolic sodium methoxide, the mixture was stirred at room temperature for 12 hours and water was added. After one extraction with ethyl acetate, the aqueous phase was evaporated to dryness. The residue was redissolved in water and extracted with ethyl acetate. The organic extracts were washed with water and the solvent was evaporated off. The residue was reprecipitated from acetone:diethyl ether giving 0.72 g of crude product.Purification by silica gel column chromatography eluting with chloroform:ethyl acetate:methanol (40:25:9 by volume), gave 0.45 g of the title compound (60% yield) in pure form, having Rf 0.26 on TLC in the solvent system chloroform:ethyl acetate:methanol (40:24:9 by volume). M.p. 126--1280, [aiD25 + 30.6 (c 1.044 CHCl3), NMR spectrum (60 MHz, CD3 C0CD3) 8 : 3.,1 5 (2 H, s, oxirane ring protons).

Elemental Analysis: Calculated for C63H73NsO23: C% 59.66, H% 5.80, N% 5.52 Found: 59.02, 5.75, 5.44.

EXAMPLE 3 Hexa-O-acetyl-6'-0-benzoyl-4'-0-methanesulphonyl-penta-N-benzyloxywarbonylparomomycin To a solution of 13 g of 6,3',2",5",3"',4"'-hexa-0-acetyi-6'-0-benzoyl-penta-N- benzoyloxycarbonylparomomycin in 260 ml of anhydrous pyridine were added 4.6 mi of methanesulphonyl chloride. The reaction mixture was stirred at 4050 for 2 hours. At ambient temperature, 30 ml of water were added and the solution evaporated. Water was added to the residue and, after extraction with chloroform, the organic extracts were washed with water, dried and evaporated under vacuum. The residue was reprecipitated from chloroform:diethyl ether:n.hexane, to obtain 13.5 g of the title compound (99% yield) in a form sufficiently pure for further transformations.

M.p. 125--1300, [aio25 + 27.8 (c 1.024 CHCt3), NMR spectrum (60 MHz, CDCI3) S: 2.8 (3 H, s, CH3S03-).

Elemental Analysis: Calculated for Ca3Hg3NsO33S: C% 57.93, H% 5.44, N% 4.07, S%1.86 Found 57.47, 5.40, 4.GO, 1.55.

EXAMPLE4 3',4'-,(-epoxy-penta-N-benzyloxycarbonylparnmomycin 13 g of the compund prepared in Example 3 were dissolved in 130 ml of chloroform. To the ice cold solution 25 ml of 1.45 N methanolic-sodium methoxide were added dropwise. The solution was stirred for 13 hours at 0 and then brine was added. The mixture was extracted with ethyl acetate. The organic extracts were washed with salted water, water, dried and evaporated. The residue was reprecipitated as white amorphous powder (9 g) from acetone:diethyl ether. Purification by preparative HPLC on silica gel column, eluting with chloroform:ethyl acetate:methanol (40:25:9 by volume) afforded 5 g of pure 3',4'-epoxy-penta-N-benzyloxycarbonylparomomycin (52% yield).

EXAMPLE 5 3',4'-X3-epoxy-hexa-0-benzoyl-penta-N-benzyloxyCarbonylparomomycin To an ice cold solution of 770 ng of the compound prepared in Examples 2 and 4 in 10 ml of anhydrous pyridine was added benzoyl chloride (1.5 ml). After 1 hour at 00, the mixture was stirred for 24 hours at ambient temperature. 3 ml of water were added and solvent was removed under reduced pressure. The residue was dissolved in chloroform, washed with water, dried and evaporated.

Reprecipitation from chloroform:diethyl ether:n.hexane TLC on silica gel yielded 765 mg of the title compound (66% yield) in pure form, having Rf 0.43 on TLC (silica gel) in the solvent system toluene: ethyl acetate (60:40 by volume).

EXAMPLE 6 6,6',2", 5",3''',4'''-hexa-O-benzoyl-4'-deoxy-4'-iodo-penta-N-benzyloxycarbonylparomomycin 760 mg of the compound prepared in Example 5 were dissolved in 14 ml of acetone. To the solution were added 300 mg of sodium iodide, 25 mg of sodium acetate and 0.4 ml of acetic acid. The mixture was refluxed for 7 hours, then the solvent was removed under reduced pressure. The residue was dissolved in chloroform, washed with water and dried. Evaporation of the solvent left 705 mg of the title compound (86% yield) in a form sufficiently pure for subsequent reaction.

EXAMPLE 7 6,6',2" ,5",3"',4" '-hexa-0-benzoyl-3' ,4'-dideoxy-3'-ene-penta-N-benzyloxycarbonylparomomycln Methanesulphonyl chloride (0.25 ml) was added to an ice cold solution of 700 mg of the compound prepared in Example 6 for 7.5 ml of dry pyridine. The reaction mixture was refluxed for 45 minutes, then concentrated under vacuum, and after dilution with water, extracted with chloroform. The extracts were washed with sodium thiosulphate solution and with water, dried and evaporated. The residue was purified by preparative TLC (silica gel) obtaining 375 mg of the title compound (58% yield) in preform. The compound had Rf 0.34 on TLC (silica gel) in the solvent system toluene:ethyl acetate (70:30 by volume): the spot immediately yellowed after spraying with cold 1% potassium permanganate solution. NMR spectrum (60 MHz, CDCl3) S :5.8 (2 H, m, olefinic protons).

EXAMPLE 8 3',4'-dideoxy-3'-ene-penta-N-benzyloxycarbonylparnmomy & n 370 mg of the compound prepared in Example 7 were dissolved in 14 ml of 0.05 N methanolic sodium methoxide and stirred for 10 hours at ambient temperature. The reaction was quenched by adding solid carbon dioxide and water and the solution was evaporated to dryness. Water was added to the residue and the mixture was extracted with ethyl acetate. The extracts were washed with water and evaporated to give 245 mg of crude product. Purification by preparative TLC (silica gel), using chloroform:ethyl acetate:methanol (40:25:9 by volume) as eluant, yielded 135 mg of the title compound (55% yield) in pure form, having Rf 0.24 on TLC (silica gel) in the same solvent system.

EXAMPLE 9 3',4'-dideoxyparomomycin To a solution of 130 mg of the compound prepared in Example 8 in 14 ml of 80% ethanol, 1.4 ml of cyclohexene and 230 mg of 10% palladium-on-charcoal were added. The mixture was refluxed for 1 hour, filtered and evaporated in vacuo to give 55 mg of residue. The crude product was purified on a column of Amberlite CG 50 (NH4+ form, 1 00-200 mesh), washing with water and eluting with increasing concentrations of ammonia (0.05-0.2 N) to give 30 mug of the title compound (49% yield).

The product (free base) was converted into its sulphate form by adding 0.2 N sulphuric acid at pH 6 and reprecipitated from acetone. The product was homogeneous on TLC (silica gel) in the solvent system chloroform:methanol: concentrated ammonia (1 :3:2) by volume) and had an Rf of 0.35. Mass spectrum (field desorption) (free base) showed a peak at m/e 584 (MH+) and fragments at 424 and 455 indicating both deoxygenation sites to be in ring A.

Claims (9)

CLAIMS:

1. 3',4'-Dideoxyparomomycin or a pharmaceutically acceptable salt thereof.

2. A process for the preparation of 3',4'-dideoxyparomomycin, the process comprising sulphonylating the free hydroxy group of a 6,3',2",5",3"',4"'-hexa-0-acyl-6'-0-benzoyl-penta-N- benzyloxycarbonylparomomycin of the general formula (1) as herein defined is reacted in the presence of a base musing, as sulphonylating agent, an alkyisulphonyl halide, an aralkylsulphonyl halide or an arylsulphonyl halide, any of which may be halogen substituted; reacting the resultant 4'-0-sulphonyl derivative of the general formula (2) as herein defined with a strong base in methanol or chloroform; reprotecting by acylation or benzoylation the deprotected hydroxy groups of the resultant 3',4'-p-epoxy derivative of the general formula (3) as herein defined; opening the epoxy ring of the resultant 3',4'-p- epoxy derivative of the general formula (4) as herein defined by treating it with sodium iodide, acetic acid and sodium acetate; heating the resultant 4'-iodo derivative of the general formula (5) as herein defined with methanesulphonyl chloride in pyridine; removing the O-benzoyl groups or the O-acyl groups from the resultant 3',4'-unsaturated derivative of the general formula (6) as herein defined by treatment at room temperature with dilute methanolic sodium methoxide; and catalytically hydrogenating the resultant compound of the general formula (7) as herein defined.

3. A process according to claim 2 in which the sulphonylating agent is methylsulphonyl chloride, a p.bromobenzenesulphonyl chloride, p.toluenesulphonyi chloride, benzylsulphonyl chloride or trifluoromethyl sulphonyl chloride.

4. A process according to claim 2 or claim 3 in which the base used in the sulphonylation step is pyridine.

5. A process according to any of claims 2 to 4 in which the base used to convert the 4'-sulphonyl derivative (2) to the 3',4'-p-epoxy derivative (3) is sodium methoxide.

6. A process according to any of claims 2 to 5 in which the catalytic hydrogenation is a transfer catalytic hydrogenolysis carried out in the presence of 10% palladium-on-charcoal and cyclohexane.

7. A process according to any of claims 2 to 6 further comprising purifying the 3',4'dideoxyparomomycin by ion exchange chromatography and isolating it as its sulphate.

8. A process for the preparation of 3',4'-dideoxyparomomycin the process being substantially as described herein with reference to the Examples.

9. A pharmaceutical composition comprising 3',4'-dideoxyparomomycin or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable diluent or carrier.