FI62843C - FOERFARANDE FOER FRAMSTAELLNING AV ETT SYRAADDITIONSSALT AV 1 - ETYLSISOMICIN - Google Patents

Description

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C Patentti myönnetty 10 03 1933 • 255 w Patent undated ^^ (51) K ».lk? /Lnt.CL3 C 07 H 15/22 SUOMI — FINLAND pi) μμιμμμ-ρημοιμι ^ 80271 + 6 (22) H * k «Mi« paiv · - AnaOfcnlnpd ^ 01.09.80 (23) AlkupUvf — CIW | h * t * d «j 01.08.?1+ (41) Tulkit (ulklMkil - Bllvtc oflwwHj 01.09.80 FMttl ·] · rakirtmri halittu * ( 44) NlhtMkripenen „kwlU) wMtitoun taan * · ohe rafistarityralMn AMdkan utltgd odi mtakrlltM puMicarad 30.11.82 (32) (33χ31) pyritty« uoMtm — acid prior »* 06.08.73 19.03.7 ^, 19.03.7 * + USA (US ) 385 751, 1 + 52 600, 1 + 52 586 (71) Oy Kolster Ab, Lönnrotsgatan 19 B, 00120 Helsinki 12, Suomi-Finland (Fl) (72) Marvin Joseph Weinstein, East Brunswick,. New Jersey, Peter J. Lovell Daniels, Cedar Grove, New Jersey, Gerald Howard Wagman,

East Brunswick, New Jersey, Raymond Testa, Verona, New Jersey,

Alan Keith Mallams, West Orange, New Jersey, John Jessen Wright,

Orange, New Jersey, Tattanahalli Laksminarayan Nagabhushan,

East Orange, New Jersey, USA (US) (51+) Process for preparing an acid addition salt of 1-N-ethyl-sisomicin - Menetelmä 1-N-ethyl-isomysinine happoadditiosuolan valmi stami seksi (62) Divided from application 230U / 7I + (patent 62100) -

The present invention relates to a process for preparing an acid addition salt of 1-N-ethylsisomicin of the formula CH_NH_NNH₂Nh5H

v ~ \ I

H3 <Y y-1

OH

2 62843

The salt is prepared by known methods, such as by neutralizing the free base with an acid to about pH 5. Suitable acids for this purpose are hydrochloric, sulfuric, phosphoric, nitric, hydrobromic, acetic, propionic, raleic, ascorbic and citric acids. and similar. Physically, the acid addition salts of the compounds of the invention are characterized as white solids which are soluble in water, soluble in most polar solvents and insoluble in non-polar organic solvents.

The compounds of the process are broad-spectrum antibacterial agents which advantageously exhibit activity against many organisms, especially gram-negative organisms. They can be used separately or in combination with other antibiotic agents to prevent growth or reduce the number of bacteria in different environments. For example, they can used for disinfection of laboratory glass and dental and medical equipment. The activity of the compounds against gram-negative bacteria makes these compounds useful for controlling infections caused by gram-negative bacteria, such as species of Proteus and Pseudomonas. They have veterinary use, especially in the treatment of cattle mastitis and Salmonella-induced diarrhea in pets, such as in dogs and cats.

The starting material for the process of the invention can be prepared by known methods from sisomicin or by fermentation of the strain Micromonospora inyoensis 1550F-1G, a culture of which has been deposited with U.S. Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, under the designation NRRL 5742.

1-N-ethylation of sisomicin is preferably carried out by reaction with acetaldehyde in the presence of a reducing agent.

Cultivation of NRRL 5742 must be carried out in the presence of 1-N-ethyl-2-deoxy-streptamine.

Example 1-N-ethylsisomicin sulfate 1. 1-N-ethylation of sisomicin

A solution of 5 g of sisomicin in 250 ml of water is added with 1-n sulfuric acid until the pH of the solution is adjusted to about 5. The resulting solution of sisomicin sulfuric acid addition salt is added with 2 ml of acetaldehyde stirring for 10 minutes, and 0.85 g of sodium cyanoborohydride is added. . Continue stirring for 15 minutes at room temperature, concentrate the solution in vacuo to a volume of about 100 ml, treat the solution with a basic ion exchange resin / sasora Axnberlite ® IRA 401S (OH) J and lyophilize to an ether residue, comprising N etylsisomi-cin.

Purify by chromatography on 200 g of silica, eluting with the lower phase of the system chloroform / methanol / 7-% ammonium hydroxide (2: 1: 1). Combine equal eluate as determined by thin layer chromatography and concentrate the combined eluate of the main component in vacuo to an ether, comprising 1-N-ethyl isomicin (yield 1.25 g). Purify by chromatography on 100 g of silica gel and elute with the system chloroform / methanol / 3.5% ammonium hydroxide (1: 2: 1). Allow the combined, similar eluates (as determined by thin layer chromatography) to pass through a basic ion exchange resin column and lyophilize the eluate to give 1-N-ethyl iso-chemin (yield 0.54 g); + 164 ° (0.3%, H 2 O); PMR (ppm) (D20): 61.05 (3H, t, J = 7Hz {-CH2CH3); 1/19 (3H, s, -C-CH 3); 2.5 (3H, s, N-CH 3), 4r 85 (1H, m, = CH-) · 4.95 (1H, d, J = 4Hz, H +); 5.33 (1H, d, J = 2.5Hz, H1 ').

Mass spectrum: (M + 1) + m / e 476 also m / e 127, 154, 160 ^ 173, 191, 201, 219, 256, 299, 317, 332, 345, 350, 360, 378, 390, 400.

2. Preparation of 1-N-ethylsisomicin by fermentation A. Preparation of graft of Micromonospora inyoensis strain 1550F-1G.

Germination Step 1: Under aseptic conditions, a lyophilized culture precipitates cells obtained from an oblique culture of Micromonospora inyoensis 1550F-1G in a 300 ml shake flask containing 100 ml of the following sterile medium:

Beef extract 3 g

Tryptose 5 g Yeast extract 5 g

Dextrose 1 g

Starch 24 g

Calcium carbonate 2 g

Water tap water 1000 ml

Incubate the flask and its contents for 2-5 days at 35 ° C on a rotary shaker (280 rpm, 5 cm stroke).

Germination step p. (Aseptically transfer 25 ml of fermentation medium from seed stage 1 to a 2-liter shaker flask containing 500 ml of said sterile seed medium. Incubate the piston and its contents for three days at 28 ° C on a rotary shaker (280 rpm, 5 cm stroke).

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Fermentation step: aseptically transfer 500 ml of the inoculum obtained from germination step 2 into a 14-liter fermentation tank containing 9.5 liters of the following sterile medium:

Dextrin 50 g

Dextrose 5 g

Soybean meal 35 g

Calcium carbonate 7 g

Cobalt chloride 10 6 molar

Water tap water 1000 ml

Anti-foaming agent (GE 60) 10 ml B. Adjust to pH 8 before sterilizing the medium described in step A and add an aqueous solution containing 8.0 g of 1-N-methyl-2-deoxy-D-streptamine. Ferment the contents under aerobic conditions under 48 to 240 hours of stirring at a rate of 250 rpm and an air intake of 4.5 liters per minute at 2.5 at. The pH of the fermentation medium changes slightly during the antibiotic product within a range of about 6.8 to 7.3.

Monitor the antibiotic production using the assay method described in J. Antibiotics (Japan) Voi. XXIII, and when peak production is achieved, harvest the product (fermentation is usually complete within about 7 days).

C. Isolation of 1-N-ethylsisomicin Add, while stirring, 7.0 g of oxalic acid to fermentation whole liquid from step B. Acidify the liquid to pH 2.0 using 6-n sulfuric acid. Stir the mixture for about 15 minutes and filter using a suitable filter aid. Neutralize (pH 7.0) the filtrate with 6m of ammonium hydroxide. Pass the filtrate through a 1.0-liter (S) column of ammonium cation exchange resin (such as Amberlite® IRC-50, Rohm and Haas, Philadelphia, Pa.). Discard spent liquid, elute the column with 2-n ammonium hydroxide and collect about 100 ml fractions. monitor the pillar eluate by shell testing each fraction against Staphylococcus aureus ATCC 6538P. Combine the active fractions, evaporate to about 100 ml in vacuo and lyophilize to bind a solid product. Triturate the product several times with warm ethanol, filter and evaporate the felt rate to an ether residue. Chromatograph the product on 25 g of silica gel using the lower phase of the chloroform / methanol / concentrated ammonium hydroxide (1: 1: 1) system as eluent. Combine and evaporate those fractions exhibiting antibiotic activity to form 1-N-ethylsisomicin.

62843 3. 1-N-ethylsisomine sulfate Dissolve 5.0 g of 1-N-ethylsisomicin in 25 ml of water and adjust the pH of the solution to 4.5 with 1-n sulfuric acid. Pour the solution into about 300 ml of methanol with vigorous stirring, continue stirring for about 10-20 minutes and filter. Wash the precipitate with methanol and dry at about 60 ° C in vacuo.

NMR spectrum for 1-N-ethyl isomicine sulfate; 20% (w / v) solution

1 denterium oxide with sodium 2,2-dimethyl-2-sila-pentane-5-sulfonate as internal reference; 20 ° C

Proton Chemical Shift Intensity Multiplicity $ (ppm) 1-NHCH 2 CH 3 1.33 3H tripletU; J = 7Hz 4 "-CH3 1.35 3H singlet 2.3 1.67 - 2.83 4H multiplet 3" -NHCH3 2.93 3H singlet 1-NHCH2CH3 3f 18 2H quartet; J = 7Hz 3 "3 ', 45 1H doublet; J = 11Hz 5" axial 3.47 1H doublet, J = 12Hz OI,, doublet of doublets 2 4'26 1H J = 4Hz, J = 10Hz 1 "5, 1 H doublet; J = 4Hz 4 '5.25 1H multiplet 1' 5.67 1H doublet; J = 1.5Hz HDO 4.78 6 62843 IR spectrum of 1-N-ethylsisomicin sulfate; KBr briquette -1

Frequency (cm) Structural groups and ions 3500 - 2500 (s, br) OH, NH3 +, NH2 +, NH stretch 1690 (w) cyclic C = C stretch 1620 (m) NH3 + NH2 + asymmetric rotation 1530 - 950 (vs , br) CO, SO 2 stretch 610 (s, 440 (w) SO 2 ~ twist (w = weak; m = medium · s = strong; vs = very strong »br = wide)

Claims (2)

7 62843 1. A process for the preparation of a particular drug useful acid addition site of 1-N-ethyl isomicin, characterized in that a compound of the formula I ch2nh2 nh2 γγ ^ ο-γγ NH2 h OH is reacted with an acid and precipitated in the same manner. the acid addition salt is isolated. 2. A process according to claim 1 for the preparation of 1-N-ethyl isomicin sulfate, characterized in that the compound of formula I is reacted with sulfuric acid.