US3923791A

US3923791A - Preparation of thioethers of rifamycin S and rifamycin SV

Info

Publication number: US3923791A
Application number: US456721A
Authority: US
Inventors: Walter D Celmer
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 1974-04-01
Filing date: 1974-04-01
Publication date: 1975-12-02
Anticipated expiration: 1992-12-02

Abstract

New 3-thioethers of rifamycin SV are prepared by the reaction of rifamycin S with appropriate mercaptans. These thioethers of rifamycin SV are oxidized to yield the corresponding thioethers of rifamycin S. All these compounds exhibit significant antibiotic activity.

Description

Celmer Dec. 2, 1975 PREPARATION OF THIOETHERS OF RIFAMYCIN S AND RIFAMYCIN SV [75] Inventor: Walter D. Celmer, New London,

Conn.

[73] Assignee: Pfizer Inc., New York, NY.

[22] Filed: Apr. 1, 1974 [21] Appi. No.: 456,721

[52] U.S. Cl 260/239.3; 424/244 [51] Int. Cl. C07D 498/02 [58] Field of Search 260/2393 P [56] References Cited UNITED STATES PATENTS 3,625,960 12/1971 Maggi 260/2393 P Primary Examiner-Henry R. Jiles Assistant Examiner-Robert T. Bond Attorney, Agent, or FirmConnolly and Hutz [57] ABSTRACT New 3-thioethers of rifamycin SV are prepared by the reaction of rifamycin S with appropriate mercaptans. These thioethers of rifamycin SV are oxidizedto yield the corresponding thioethers of rifamycin S. All these compounds exhibit significant antibiotic activity.

6 Claims, No Drawings BACKGROUND OF THE INVENTION The rifamycins, a group of closely related hydroquinone-quinone antibiotics, are described in II Farmaco, Ed. Sci. 14, 146 (1959); Antibiotics Ann. 1959/1960, 262 (1960a); Experientia 16, 412 (1960); II Farmaco, Ed. Sci. 16, 165 (1961); Res. Progr. Biol. med. Chem. 1, 337 (1964); and Antibiotics 1, 256-264 (1967), Pergamon Press, 1st edition.

US. Pat. No. 3,625,960and II Farmaco, Ed. Sci. 22, No. 5, 307 (1967) disclose 3-thioethers of rifamycin SV in which the sulfur atom links the rifamycin nucleus to complex radicals of various nature.

SUMMARY OF THE INVENTION This invention is concerned with 3-thioethers of rifamycin having the formula selected from the group consisting of Me Me B K Mo mcoo 0 m on 0 no I and wherein R is alkyl containing from 2 to carbon atoms, allyl, phenyl and cyclohexyl.

This invention is also concerned with a process for preparing 3-thioethers of rifamycin S and rifamycin SV by a sequence of steps comprising a. contacting rifamycin S with a mercaptan of the formula R-SH wherein R is alkyl containing from 1 to 5 carbon atoms, allyl, phenyl and cyclohexyl;

b. oxidizing the product of step (a) comprising rifamycin SV and the thioether of rifamycin SV to fonn rifamycin S and the thioether of rifamycin S;

c. contacting the rifamycin S and the thioether of rifamycin S from step (b) with additional mercaptan of the formula R-SH;

d. repeating process steps (b) and (c) until substantial amounts of rifamycin S starting material have been converted to the 3-thioether of rifamycin SV;

e. oxidizing the mixture obtained from step (d) comprising a major amount of the 3-thioether of rifamycin SV and a minor amount of rifamycin SV and separating the 3-thioether of rifamycin S from the resulting oxidized mixture.

DETAILED DESCRIPTION OF THE INVENTION US. Pat. No. 3,625,960 and II Farmaco, Ed. Sci. 22, No. 5, 307 (1967) describe the preparation of a number of 3-thioethers of rifamycin SV by the process of contacting rifamycin S with an appropriate complex mercapto-compound. The failure to obtain thioethers of rifamycin SV by reacting rifamycin S with simple mercaptans (e.g. ethyl mercaptan, benzyl mercaptan, thiophenol, etc.) was explained in II Farmaco, Ed. Sci. 22, No. 5, 307 1967) as being due to the strong reducing power of these mercaptans on the starting quinone (rifamycin S). In these instances only rifamycin SV, the hydroquinone form of rifamycin S, could be recovered from the reaction mixture.

The present invention is concerned with process conditions for successfully accomplishing the synthesis of the simple 3-thioethers of rifamycin SV. These thioethers can be oxidized to the corresponding thioethers of rifamycin S.

The novelty of the process resides in the repeated reworking of the reaction mixture of rifamycin S with mercapto-compound by oxidizing co-produced rifamycin SV to rifamycin S which along with rifamycin S thioether is again contacted with mercapto-compound. Appreciable remaining amounts of co-produced rifamycin SV are again oxidized to rifamycin S, and the process is repeated until all the starting material has been substantially converted to the desired rifamycin SV thioether. Trace amounts of remaining rifamycin SV are oxidized to rifamycin S which is readily separated from co-produced rifamycin S thioether by column chromatography. The eluted rifamycin S thioether is reduced to the corresponding rifamycin SV thioether.

The reaction procedure is generally applicable to a range of simple mercaptans except for minor modifications in reaction temperature. Rifamycin S is dissolved in tetrahydrofuran (2% w/v) and the solution is diluted with stirring with an equal volume of water. A simple mercaptan is added to the solution cooled to 05C. at a level of 2-5 moles of mercaptan per mole of rifamycin S. The reaction of benzenethiol or 2-methyl-2-propanethiol with rifamycin S may be conducted at 0C. to room temperature; the reactions with allyl mercaptan and cyclohexyl mercaptan are preferably run at room temperature. The solution is stirred for approximately 15 minutes. Additional mercaptan is added if unreacted rifamycin S is present as determined by thin layer chromatography employing silica gel and a developing system of acetone-chloroform (1:1).

In addition to the 3-thioether of rifamycin SV, a considerable amount of rifamycin SV (approximately 50%) is obtained in the reaction mixture. The material is re-worked by extracting the reaction mixture with a water-immiscible organic solvent, preferably ethyl acetate. The solvent extract is dried over anhydrous sodium sulfate and the hydroquinone antibiotics (rifamycin SV and the 3-thioether of rifamycin SV) are oxidized to the corresponding quinone antibiotics. The rifamycin quinones (rifamycin S and rifamycin S thioether),are again contacted with mercapto-compound as previously described. The process is repeated until the conversion to the 3-thioether of rifamycin SV is substantially complete as demonstrated by thin layer chromatography.

There is considerable difficulty in effecting the separation of the 3-thioethers of rifamycin SV from small or trace amounts of rifamycin SV by column chromatography. The problem may be obviated by oxidizing all the material to the quinone form in which state the various components are readily separated by column chromatography. The ethyl acetate extract of the final reaction mixture is oxidized and chromatographed on a silica gel column using ethyl acetate-hexane (3:1) as the developing solvent. The column cuts are followed by silica gel thin layer chromatography with a developing system of acetonechloroform (1:1). The chromatograms are examined visually. The rifamycins are highly colored with various shades of orange, yellow and pink.

The column cuts containing the separated 3- thioether of rifamycin S are combined, the solvent removed under vacuum and the residue taken up in methanol. The thioether of rifamycin S in methanol solution is reduced to the rifamycin SV thioether, the methanol removed under vacuum, residue taken up in chloroformwater and the pH adjusted to about 9 with sodium hydroxide solution. The chloroform layer is separated, dried over anhydrous sodium sulfate and taken to dryness under vacuum. The 3-thioether of rifamycin SV may be crystallized from ethyl acetate-hexane.

The quinone-hydroquinone system of rifamycin S and rifamycin SV is described in Antibiotics 1, 256-264 (1967), pergamon Press, 1st edition. This relationship also applies for their 3-thioethers. The quinones are readily reduced to the hydroquinones and the hydroquinones are easily oxidized to the quinones by means well known to those skilled in the art. The preferred oxidizing agent is activated manganese dioxide which is prepared by azeotropic drying of manganese dioxide as described in J. Org. Chem. 34, No. 6, 1979 (1969). A slurry of activated manganese dioxide is added to a methanol or ethyl acetate solution of rifamycin hydroquinone (gram/gram of antibiotic) and stirred for about 30 minutes at room termperature. The reaction mixture is clarified by filtration or centrifugation and the solvent removed under vacuum. The preferred reducing agent is ascorbic acid which is added to a methanol or ethyl acetate solution of the rifamycin quinone (2 grams/gram of antibiotic) and stirred for about 30 minutes at room temperature.

It is to be understood that the present invention includes within its scope a number of simple 3-thioethers of rifamycin S and rifamycin SV which can be readily converted to one form or the other as desired. All of these novel compounds are useful in combatting microorganisms, especially Mycobacterium tuberculosis, Diplococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus, including strains which are resistant to other known antibiotics. In addition, they are useful as disinfectants against such microorganisms and as an aid in the purification of mixed cultures for medical diagnostic and biological research purposes.

Table I illustrates the in vitro and in vivo activity of some of these compounds against an antibiotic resistant strain of Staphylococcus aureus. The in vitro tests were run by preparing tubes of nutrient broth or petri dishes with nutrient agar with gradually increasing concentrations of the pure compound, and then inoculating the media with the specified strain of S. aureus. The minimal inhibitory concentration (M.I.C.) indicated is the minimal concentration of the compound (in micrograms/milliliter) at which the microorganism 4 failed to grow. The in vivo activity was determined in experimentally infected mice.

Table 1 Activity vs Staphylococcus aureus 01A0O5 *Dose that protects 50% of mice "US. 3,625,960

The corresponding thioethers of rifamycin S exhibit a comparable range of in vitro and in vivo activities. The more active compounds can be administered via the oral or parenteral routes for the treatment in animals, including humans, of pneumococcal, streptococcal, staphylococcal, tubercular and other antibiotic-sensitive infections. In general, these antibiotics are most desirably administered in daily oral doses of 0.5-1 gram or parenteral injections of to 500 mg., depending on the type and severity of the infection and weight of the subject being treated.

The compounds of this invention may be administered alone of in combination with pharmaceuticallyacceptable carriers, and such administration can be carried out in both single and multiple doses.

For purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and dicalcium phosphate may be employed along with various disintegrants such as starch, alginic acid and certain complex silicates together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and gum acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules; preferred materials include lactose as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes, and if desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerol and various combinations thereof.

For purposes of parenteral administration, solutions of these antibiotics in sesame or peanut oil or in aqueous propylene glycol may be employed as well as sterile aqueous solutions of the corresponding water-soluble alkali metal or alkaline-earth metal salts. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.

The 3-thioethers of rifamycin S and rifamycin SV are slightly soluble in water and petroleum ether, and somewhat more soluble in alkaline solutions. They are readilysoluble in methanol, ethanol, acetone and ethyl acetate.

Useful analytical determinations to characterize the rifamycin thioethers of this invention included elemental analysis, ultraviolet light and infrared absorption spectra, molecular weights by the molecula'rion in the mass spectra and NMR data for thioether isomers.

' EXAMPLE I Rifamycin. S 2:%w]v).is dissolve d in tetrahydrofuran and the solution is diluted with an equal volume of water. Methanethiol is added as a liquid to the solution cooled to 0-5C. at a'level of :2-5 moles of mercaptan per mole of rifamycin S, and stirred for about minutes. Additional methanethiol is added if unreacted rifamyc in S remains as demonstrated by silica gel thin layer chromatography witha developing solvent system of acetone-chloroform (1:1).The reaction mixture is then extracted withethyl acetate. To the solvent extract, dried over sodium sulfate, is added activated manganese dioxide (gram/gram of antibiotic). After stirring for about 30 minuted at room temperature, the reaction mixture is filtered and' the solvent removed under vacuum. The residue is again contacted with methanethiol until the conversion to 3- th iomethylrifamycin SV is substantially complete ,as demonstrated by thin layer chromatography. The final reaction mixture is extracted with ethyl acetate. The solvent extract containing 3-thiomethylrifamycin SV and traoJ amounts of rifamycin SV is oxidized with activated manganese dioxide, and chromatographed on a silica gel column which is developed with ethyl acetate-hexane (3:1). The column cuts are followed by the use of silica gel thin layer chromatograms developed with acetonechloroforrn (1:1), and examined visually. The column cuts containing separated 3-thiomethylrifamycin S are combined, the solvent removed under vacuum and the residue taken up in methanol. Ascorbic acid (2 grams/- gram of antibiotic) is added and stirred for about 30 EXAMPLE III A dry solid pharmaceutical composition is prepared by blending the following materials together in the proportions by weight specified below:

3-Thiomethylrifamycin SV Sodium citrate 25 Alginic acid. l0 Polyvinylpyrrolidone 10 Magnesium stearate 5 EXAMPLE lV Vials are prepared containing weighed amounts of the sterile sodium salt of 3-thiomethylrifamycin SV. These vials are reconstituted for parenteral administration to 100 or 200 mg/ml with sterile water or 5% dextrose solution.

EXAMPLE V To 3-thioethylrifamycin SV dissolved in methanol (2% w/v) is added a slurry of activated manganese dioxide (gram/gram of antibiotic). After stirring for about 30 minutes at room temperature, the reaction mixture is filtered and the methanol removed under minutes at room temperature. The methanol is removed under vacuum, the residue taken up in chloroform-water and the pH adjusted to about 9 with sodium hydroxide solution. The chloroform layer is separated, dried over anhydrous sodium sulfate and taken to dryness under vacuum. 3Thiomethylrifamycin SV is crystallized from ethyl acetate-hexane, yield-80%. Ultraviolet light absorption maxima in 0.01 N l-lCl occur at 225, 305 and 440 mu.

EXAMPLE ll vacuum to yield 3-thioethylrifamycin S. Its ultraviolet light absorption maxima in 0.01 N l-lCl in methanol solution occur at 220, 272, 312 (inflection) and 375 my, with E values of 375, 335, I65 and respectively. When pelleted in KBr, characteristic absorption in the infrared region occur at the following wavelengths in microns: 2.90, 3.40, 5.75, 5.95, 6.15, 6.35, 6.80, 7.05, 7.25, 7.60, 7.75, 8.00, 8.35, 8.60, 9.00, 9.40, 10.30, 10.60, 10.85, 11.20, 12.20, 12.40, 12.95, 13.25 and 13.90.

EXAMPLE VI The method of Example V is repeated with 3-n-thiopentylrifamycin SV to yield 3-thiopentylrifamycin S with ultraviolet light absorption maxima in 0.01 N l-lCl in methanol at 220, 272, 312 (inflection) and 375 mg. with E 0,, values at 350, 310, 160 and 80 respectively. When pelleted in KBr, characteristic absorption in the infrared region occur at the following wavelengths in microns: 2.90, 3.40, 5.75, 5.95, 6.15, 6.35, 6.80, 7.05, 7.30, 7.60, 7.75, 8.00, 8.35, 8.60, 9.00, 9.40, 10.30, 10.60, 10.85, 11.20, 12.20, 12.40, 13.00 and 13.90.

EXAMPLE VII The method of Example V is repeated with 3-thio-tbutylrifamycin SV to yield 3-thio-t-butylrifamycin S with ultraviolet light absorption maxima in 0.01 N l-lCl in methanol at 220, 275, 212 (inflection) and 390 mp. with E values at 380, 290, and 60 respectively.

EXAMPLE VIII The method of Example V is repeated oxidizing the appropriate 3thioethers of rifamcyin SV to yield the following:

3-thio-isopropylrifamycin S 7 3-thio-n-butylrifamycin S- 3-thio-3-methyl-l-butylrifamycin S 3-thiophenylrifamycin S 3-thiocyclohexylrifamcyin S 5 3-thioallylrifamycin S What is claimed is:

l. A 3-thioether of rifamycin S and rifamycin SV having the formulae selected from the group consisting of Mo MnH wherein R is alkyl containing from 2 to 5 carbon atoms, allyl, phenyl and cyclohexyl.

2. A process for preparing a 3-thioether of rifamycin S and rifamycin SV as claimed in claim 1, which process comprises a. contactin'g'rifamycin S in aqueous tetrahydrofuran solution at O5C. for 15 minutes with a mercaptan, 2-5 molesof mercaptan per mole of rifamycin S beiplg used, of the formula wherein R is alkyl containing from 1 to 5 carbon atoms, allyl, phenyl or cyclohexyl;

b. oxidizing an ethyl acetate extract of the product of step (a) by stirring for 30 minutes at room temperature with 1 part of activated manganese dioxide per part of said product, by weight;

0. contacting the product of step (b) with additional mercaptan of the formula R-SH by the process of step (a);

i (d). repeating process steps (b) and (c) until substantial amounts of rifamycin S starting material have been converted to a 3-thioether of rifamycin SV;

(e) oxidizing the mixture obtained from step (d) by the process of step (b) and separating a 3-thioether of rifamycin S and of rifamycin SV by absorption on silica gel column developed with ethyl acetatehexane (3:1).

3. 3-Thioethylrifamycin SV.

4. 3-Thio-n-propylrifamycin SV.

5. 3-Thio-isopropylrifamycin SV.

6. 3-Thio-nbutylrifamycin SV.

Claims

1. A 3-THIOETHER OF RIFAMYCIN S AND RIFAMYCIN SV HAVING THE FORMULAE SELECTED FROM THE GROUP CONSISTING OF

2. A process for preparing a 3-thioether of rifamycin S and rifamycin SV as claimed in claim 1, which process comprises a. contacting rifamycin S in aqueous tetrahydrofuran solution at 0*-5*C. for 15 minUtes with a mercaptan, 2-5 moles of mercaptan per mole of rifamycin S being used, of the formula R-SH wherein R is alkyl containing from 1 to 5 carbon atoms, allyl, phenyl or cyclohexyl; b. oxidizing an ethyl acetate extract of the product of step (a) by stirring for 30 minutes at room temperature with 1 part of activated manganese dioxide per part of said product, by weight; c. contacting the product of step (b) with additional mercaptan of the formula R-SH by the process of step (a); (d). repeating process steps (b) and (c) until substantial amounts of rifamycin S starting material have been converted to a 3-thioether of rifamycin SV; (e) oxidizing the mixture obtained from step (d) by the process of step (b) and separating a 3-thioether of rifamycin S and of rifamycin SV by absorption on silica gel column developed with ethyl acetate-hexane (3:1).

3. 3-Thioethylrifamycin SV.

4. 3-Thio-n-propylrifamycin SV.

5. 3-Thio-isopropylrifamycin SV.

6. 3-Thio-n-butylrifamycin SV.