US RE28647 E
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United States Patent 1 Umezawa et a1.
[ PREPARATION OF 3-4 DIDEOXYKANAMYCIN B ACTIVE AGAINST RESISTANT BACTERIA  Inventors: Sumio Umezawa; Hamao Umezawa,
both of Tokyo; Tsutomu Tsuchiya, Yokohama, all of Japan  Assignee: Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai, Tokyo, Japan  Filed: May 1, 1974  Appl. No.: 465,744
Related US. Patent Documents Reissue of:  Patent No.: 3,753,973
Issued: Aug. 21, 1973 Appl. No.: 149,351
Filed: June 2, 1971  US. Cl. 260/210 K; 260/210 AB; 424/181  Int. Cl. C07I-I 5/06  Field of Search 260/210 K, 210 AB Reissued Dec. 9, 1975  References Cited UNITED STATES PATENTS 2,967,177 1/1961 Johnson et a1. 260/210 K OTHER PUBLICATIONS Hudson, Advances in Carbohydrate Chem.," Vol. 8, 1953, pp. 126-127, Academic Press, New York, NY. Pigman, The Carbohydrates," 1957, Academic Press Inc., New York, N.Y., pp. 162-163.
Primary ExaminerJ0hnnie R. Brown Attorney, Agent, or Firm-Brady, OBoyle & Gates 3 Claims, No Drawings PREPARATION OF 3-4 DIDEOXYKANAMYCIN B The synthetic route is as follows: Starting material ACTIVE AGAINST RESISTANT BACTERIA kanamycin B is transformed to the kanamycin B deriva tive as shown in the General Formula I or I, in whicl 5 five amino groups, 4",6"-hydroxyl groups and all or 2 part of 5,3',4' and 2"-hydroxyl groups are protected.
Matter enclosed in heavy brackets I: 1 appears in the original patent but forms no part of this reissue specification; matter printed in italics indicates the additions made by reissue.
CEINKCOB 4' o This invention relates to the ynthesis of 3'-4'- l Pk dideoxykanamycin B which is antibacterial and especially active against infection of kanamycin-resistant at a' o organisms. This invention is based on the findings disozi covered by a series of researches done by H. Umezawa et al., who cleared the mechanism of the drug-inactivation by the resistant bacteria isolated from patents and who then pursued to find, based on the mechanism, l new antibiotic derivatives active against drug-resistant z m bacteria.
Hamao Umezawa et al. [reported in Science," 157, 1559 (1967)] found that drug-resistant gram-negative cmN=cH1v bacteria carrying R factor of patent-origin, resistant 4 Staphyococci and resistant Pseudomonas aeruginosa o k produced enzymes that inactivate kanamycin by transi ferring phosphate from ATP to 3'-hydroxyl group of M33! l kanamycin.
omo n cmmzi/ a" K dz 1' 1' 1' NB: on
\0 NH: 4 [1 R H, alkyl, aralkyl, aryl, alkyloxy, aralkyloxy or arnomc a" ji 35 yloxy group.
R H, alkyl, aralkyl or aryl group. :1; IV X H, H or presented by a I" R x" on kanamycin R=0H 40 kanamycin BR=NH1 (in which A and B are respectively, H, alkyl or aryl Based on these findings, the inventors firstly atgroup) tempted to block the 3'-hydroxyl group and 3-O- methylkanamycin was synthesized; but the derivative had no antibacterial activity. 3'-deoxykanamycin was, H2) in the next step, synthesized and it was found that the derivative had the activity against the above-described resistant bacteria. Therefore, it was roughly concluded Y represented by that the replacement of the hydroxyl group, which is phosphorylated by resistant bacteria, with hydrogen will give the antibiotic a character to attack the resistant bacteria. The inventors applied the principle to kanamycin B and highly active 3',4-dideoxykanamycin B (the formula is shown below) was prepared. (in which A and B respectively alkyl or aryl group) or I NH:
' aralkylsulfonyl or arylsulfonyl group,
)0 Z H or acyl group. 5 0 The derivative (1) or (V) is then transformed to the derivative as shown in the General Formula II (W H), in K 1-0 I "5 if I Z H, acyl, alkyl, aralkyl, aryl group or alkylsulfonyl,
i on which 3' and 4'-hydroxyl groups are liberated, and the derivative is then transformed to the derivative W H, alkylsulfonyl, aralkylsulfonyl or arylsulfonyl group. R, R, Y, Z and Z are the same with that described in the General Formula 1 or I. shown in the General Formula 11 (W SO Q. 01a]- 1, aralkyl or aryl group, in which 3' and 4-hydroxyl 'oups are sulfonylated. The derivative (11) (W D Q) is then treated by the procedure described by itson et a1. (Carbohydrate Research, volume 1, pages 58 to 340, 1965), i.e., treated with iodide ion and a etal powder having reducing property such as zinc 1st in an appropriate solvent giving the derivative hav g a 3',4'-unsaturation group. The protecting groups the resulting derivative, after hydrogenation of the isaturation group, are removed by an usual manner to ve the final product, 3',4'-dideoxykanamycin B. The synthesized 3',4'-dideoxykanamycin B inhibits .e growth of many kinds of bacterial strains in the sim- 11' order with that of kanamycin B as shown in Table and moreover inhibits the growth of strains of kanaycin and kanamycin B resistant Staphylococci, resis- .nt Echerichia coli and resistant Pseudomonas aerugi- )sa. 3',4'-dideoxykanamycin B shows the therapeutic feet against the infections caused by Staphylococci, lebsiella pneumoniae, Salmonella typhosa dn Pseudoonas aeruginosa in mice. 3,4'-dideoxykanamycin B as low toxicity (LD 180 mg./kg. mouse, -iv.) and lOWS high blood level after the injection, thus proving 1e derivative to be a valuable chemotherapeutic agent Ir treatment of many kinds of infections including lose cause by gram-negative and gram-positive resisnt bacteria. It shows the strong effect in inhibiting microorganms including pathogenic organism as shown below.
TABLE 1 in the next, examples of the preparation of 3',4'- dideoxykanamycin B are shown. Since 3',4'-dideoxykanamycin B synthesized was proved effective for the remedy of a wide variety of infections, this invention is not limited to the preparations of 3',4'-dideoxykanamycin B as shown in examples, but includes all modified procedures, which are recognized as usual.
EXAMPLE l The synthesis of penta-N-ethoxycarbonylkanamycin B To a stirred mixture of kanamycin B free base (1.0 g.) and anhydrous sodium carbonate (0.9 g.) in aqueous acetone (1:1, 20 ml.), ethoxycarbonyl chloride (1.05 ml.) was added and the stirring was continued for 5 hours at room temperature. Resulting precipitates were filtered, washed with water and dried to give a solid (1.46 g.), M.P. 305 (decomp), [a],, +88 (c. 0.5, dimethylformamide).
EXAMPLE 2 The synthesis of penta-N-ethoxycarbonyl-3',4'; 4",6"-di-O-isopropylidenekanamycin B [in general Formula 1, R=oc.,H,, X=Y CH(CH Z'=H] The penta-N-ethoxycarbonylkanamycin B (13.7 g.) prepared by the procedure described in Example 1 was dissolved in dimethylformamide (70 ml.) and the solution, after addition of 2,2-dimethoxypropane (18.8 g.) and anhydrous p-toluenesulfonic acid (0.35 g.), was heated at 65 C. for 1 hour. The resulting solution was concentrated to approximately m1. and, after addition of 2,2-dimethoxypropane (30 g.), the solution was again heated at C. for 1 hour. Triethylamine (6 ml.) was added and the solution was poured into a stirred mixture of benzene (50 m1.) and water (500 ml.), whereupon a solid precipitated (Solid A). After removal of the Solid A by filtration, the benzene layer was allowed to stand. The precipitates appeared were filtered, washed with benzene and water and dried to give a solid, 35 g., M.P. 236237 C. [a],, +87 (c. 1, dimethylformamide).
The Solid A obtained (8.1 g.) was proved to be penta-N -ethoxycarbonyl-4' ',6' -O-isopropylidenekanamycin B and the compound could be converted to penta- ANTIBACTERIAL SPECTRA OF 3',4'-D1DEOXY- KANAMYCIN B AND KANAMYCIN B Minimal inhibitory concentration utrient agar, 3, C., 18 hours N-ethoxycarbonyl-3',4';6"-di-O-isopropylidenekanamycin B by the same treatment as described above.
EXAMPLE 3 The synthesis of 2"-O-benzoyl-penta-N-ethoxycarbonyl-3',4;4",6"-di- O-isopropylidenekanamycin B [in general Formula 1, R=OC,H X=Y=CH(Cl-l Z is COC H and Z is H] The kanamycin B derivative (3.24 g.) prepared by the procedure described in Example 2 was dissolved in pyridine (48 ml.) and to the solution, benzoyl chloride (2 g.) was added. After allowing to stand at room temperature for 1 hour, the solution was evaporated and the residue was dissolved in chloroform. The solution was washed with water, dried over sodium sulfate and concentrated to approximately 20 ml. Addition of nhexane gave crystals, 3.35 g., M.P. 205209 C. [al -H 14 (c. 1, dimethylformamide).
EXAMPLE 4 The synthesis of 2"-O-benzoyl-penta-N-ethoxycarbonyl-4",6"-O-isopropylidenekanamycin B The kanamycin B derivative (2.82 g.) prepared by the procedure described in Example 3 was dissolved in aqueous acetic acid (1:3, 40 ml.) and the solution was heated at 95 C. for 30 minutes. The resulting solution was evaporated and the deacetonated product obtained (2.58 g.) was dissolved in dimethylformamide (15 ml.) and, after addition of 2,2-dimethoxypropane (0.65 g.) and anhydrous p-toluenesulfonic acid (55 mg.), the solution was allowed to stand at room temperature for 1 hour. The solution was poured into water and the resulting precipitates were filtered, washed with water and treated with benzene to give benzene-insoluble solid, 2.01 g., M.P. 285-287 C., [a] "+105 (c. 1, dimethylformamide); NMR spectrum (in dimethylsulfoxide-d 1' 8.72 and 8.60 (each 3-proton singlet assignable to isopropylidene protons), r 1.5-2.8 (5-proton multiplet assignable to benzoyl protons).
EXAMPLE 5 The synthesis of 2"-0-benzoyl-penta-N-ethoxycarbonyl-4",6"-O-isopropylidene-3',4'-di-O-mesyl kanamycin B [in General Formula 11, R=OC,l-1 Y=CH(CH W=SO CH Z=COC,,H and Z'=l-l] The kenamycin B derivative (1.18 g.) prepared by the procedure described in Example 4 was dissolved in pyridine (15 ml.) and the solution, after addition of methanesulfonyl chloride (CH,SO,C|, 0.66 g.), was allowed to stand at room temperature for 1.5 hours. The solution was concentrated to the one-third of the original volume and the solution was poured into water. Resulting precipitates were filtered and washed with water to give a solid, 1.32 g., M.P. 198 C., ,," +107 (c. 1.5, dimethylformamide); NMR spectrum (in pyridined,): r 6.60 and 6.45 (each 3-proton singlet assignable to mesyl protons).
EXAMPLE 6 The synthesis of 2"-O-benzoyl-3',4'-dideoxy-3,4'-didehydro-penta-N- ethoxycarbonyl-4",6"-O-isopropylidenekanamycin B To a solution of the kanamycin B derivative (1.0 g. prepared by the procedure described in Example 5, in
dimethylformamide (20 ml.), sodium iodide (11 g.) and zinc dust (5 g.) were added and the mixture was heated at C. for 1 hour under vigorous stirring. The mixture was poured into a hot mixture of chloroform ml.) and water (100 ml.) under stirring and the chloroform layer isolated was washed with water, dried over sodium sulfate and evaporated to give a solid (940 mg.). The solid was treated with hot ethyl acetate and the solvent-insoluble solid (310 mg.) was recrystallized from a mixture of methanol-chloroform-ethyl acetate, M.P. 282-284 C., ,,1-36" (c. 0.4, dimethylformamide); NMR spectrum (in pyridine-d r 4.06 (2-proton a little broadened singlet, H-3' and 4').
EXAMPLE 7 The synthesis of 2"-O-benzoyl-3',4'-dideoxy-penta-N-ethoxycarbonyl- 4",6"-O-isopropylidenekanamycin B A solution of the kanamycin B derivative (1.15 g), prepared by the procedure described in Example 6, in a mixture of p-dioxane (45 ml.), methanol (25 m1.) and water (25 ml.) was hydrogenated with hydrogen under 2 atm. pressure in the presence of platinum oxide (0.3 g.), which was preactivated before use, at room temperature for 5 hours. The resulting solution was filtered and evaporated to give a colorless powder, 1.10 g., M.P. 254-256 C., ,, +87 (c. 0.2, dimethylformamide); NMR spectrum (in pyridine-d r 7.8-8.5 (4- proton broad signal, H-3',4').
EXAMPLE 8 The synthesis of 2"-O-benzoyl-3,4-dideoxy-penta-N-ethoxycarbonylkanamycin B The kanamycin B derivative (980 mg.) prepared by the procedure described in Example 7 was dissolved in a mixture of acetic acid (8 ml.) and water (5 ml.) and the solution was heated at 100 C. for 5 minutes. Resulting precipitates were filtered and dried, 750 mg., M.P. 247248 C., ,, +89 (c. 2.7, dimethylformamide). Found: C, 52.41; H, 6.54; N, 7.48%. Calcd. for C H N O C, 52.45; H, 6.71; N, 7.65%.
EXAMPLE 9 The synthesis of3',4'-dideoxykanamycin B The kanamycin B derivative (150 mg.), prepared by the procedure described in Example 8, and barium hydroxide octahydrate (2.3 g.) were added to a mixture of water (5 ml.) and P-dioxane (4 ml.), and the resulting mixture was heated in a boiling water bath for 8 hours. Carbon dioxide was introduced and the resulting precipitates were removed by centrifuge. The supernatant layer isolated was evaporated. The residue obtained was dissolved in water and the solution, after filtration, was chromatographed on a column of Amberlite [RC-50 (l-l form) with 0.1-0.4 N ammonium hydroxide. The fraction containing 3',4-dideoxykanamycin B was concentrated. Addition of acetone gave crystals, 47 mg., lab" (c. 0.65, water). Found: C, 47.71; H, 8.38; N, 15.31%. Calcd. for C H MO C, 47.88; H, 8.26;N,15.51%.
EXAMPLE 10 The synthesis of penta-N-salicylidenekanamycin B To a suspension of kanamycin B base (400 mg.) in aqueous methanol (1:8, 20 ml.), salicylaldehyde (520 mg.) was added and the resulting solution was poured into water. Precipitates were filtered and dried yielding 750 mg.
EXAMPLE 1 1 The synthesis of 3',4';4",6"-di-O-cyclohexylidene-penta-Nsalicylidenekanamycin B (in General Formula I, R=C H (OH),X=Y=(CH C,Z,Z'=H) EXAMPLE 12 The synthesis of 2"-O-benzoyl-3',4';4",6"-di-O-cyclohexylidene-penta-N-salicylidenekanamycin B (in General Formula I,
The kanamycin B derivative (590 mv.) prepared by the procedure described in Example ll was dissolved in pyridine ml.) and to the solution, benzoyl chloride (120 mg.) was added. After allowing to stand at room temperature for 1 hour, the solution was poured into water. Precipitates occurred were filtered and dried to yield 6l2 mg.
EXAMPLE 13 The synthesis of 2"-O-benzoyl-4",6"-O-cyclohexylidene-penta-N- ethoxycarbonylkanamycin B A mixture of the kanamycin B derivative (600 mg.), prepared by the procedure described in Example 12, in aqeuous acetic acid (1:3, 20 ml.) was heated at 95 C. for 30 minutes. The reaction mixture was evaporated and the residue was treated with ether. The ether insoluble part was dissolved in aqueous acetone (1:1, 15 ml.) and to the solution, after addition of anhydrous sodium carbonate (400 mg), ethoxycarbonyl chloride (270 mg.) was added and stirring was continued for 5 hours at room temperature. Resulting precipitates were filtered, washed with water and dried to give a solid (515 mg.), [al +98 (c. l.5, dimethyl-formamide). The substance could be transformed to 3',4'-dideoxykanamycin B by a series of similar procedures described in Examples 5-9.
What we claim is:
l. A process for the synthesis of 3'4'-dideoxykanamycin B which comprises protecting the functional groups of kanamycin B by acylating, alkoxycarbonylating, alkylidenating or arylidenating the amino groups; alkylidenating or arylidenating the 4" and 6" hydroxyl groups; acylating, arylmethylating, arylating, alkylsulfonylating or arylsulfonylating the 2" hydroxyl group, and sulfonylating the 3 and 4 hydroxyl groups of kanamycin B to produce a derivative having disulfonic ester groups; removing the 3, 4'-disulfonic ester groups by reaction with iodide ion and a metal dust having reducing properties in a solvent to produce the 3, 4 unsaturated compound; reducing the 3, 4' unsaturated derivative by catalytic hydrogenation and removing the remaining protective groups to product 3',- 4-dideoxykanamycin B.
2. The method of claim 1 including the step of protecting the S-hydroxyl group of kanamycin B by acylation.
3. The compound having the formula