IL39872A - Derivatives of kanamycin a and b - Google Patents

Description

39872/2 Derivatives of kanamycin A and B BRISTOL-MYERS COMPANY Abstract of the Invention Derivatives of kanamycin A and B have been prepared which possess substantially improved antibacterial activity. An example of "such an agent is l-[L-(-)-7-amino-e^hydrcxybutyryl]-kanamycin A [IVa3 BB-JS8J .

Background of the Invention 1. ) Field of the Invention: This invention relates to semisynthetic 1-subetituted derivatives of kanamycin A or B, said compounds being prepared by acylating the 1-amino-function of kanamycin A or 3 with a γ-amino-a-hydroxybutyryl moiety. 2. ) Description of the Prior Art: The kanamycins are known antibiotics described in Merck Index, 8th Edition, pp. 597-598. Kanamycin A is a compound having the formula This invention relates to semi-synthetic derivatives of kanamycin A and B, said compounds being known as l-[L-(-)-y- ¾nino-a-hy4roxybaty^yl]-lc»namycin A and Band having the formul NH-R ' IV in which R5 is OH or NHg and R is L-(-)-7-araino-a-hydroxybutyrylj or a nontoxic pharmaceutically acceptable acid addition salt there For the purpose of this disclosure, the term "nontoxic pharma ceutically acceptable acid addition salt" shall mea a mono, di-, tri- or tetrasalt formed by the interaction of 1 molecule of compo IV with 1-4 moles of a nontoxic, pharmaceutically acceptable acid.

Included among *these acids are acetic, hydrochloric, sulfuric, mandelic, maleic, phosphoric,' nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

The compounds of the present invention may be prepared for example by the following preferred reaction sequence: N-Hydroxysucclnimide ester of -(-)-y- benzyloxycarbonylamino-a-hydroxybutyric acid 2. ) Compound II III A preferred embodiment of the present invention is the compound having the formula t other than Hj or a nontoxic pharmaceutically acceptable acid addi- tion salt thereof.

A more preferred embodiment is the compound of formula V in which ^ is L-(-)-7-amino-o-hydroxybutyryl, R is hydrogen and R- is OH or NI^.

A most preferred embodiment is the compound of formula V wherein R1 is H, R2 is L-(-)-7-amino-a-hydroxybutyl and R^ is OHj or a nontoxic pharmaceutically acceptable acid addition salt thereof.

Another most preferred embodiment is the compound of formula V wherein R1 is H, R2 is L- *f-«mtno- ot-hydroxybutyryl and ^ is NH2S °r a nontoxic pharmaceutically acceptable acid additioa salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride, acetate, malete, mandelate, citrate, ascorbate, nitrate or phosphate salts of compound V.

Another most preferred embodiment is the monosulfate salt of compound V.

Still another preferred embodiment is the disulfate salt of compound V.

The objectives of the present invention have been achieved, by the provision according to the present invention of the process for the preparation of the compounds having the formula CHgNHg NH-Re IV in which R is L- ( - ) -γ-amino-a-hydroxybutyryl and R3 is OH or NH2; or a nontoxic pharmaceutically acceptable acid addition salt thereof which process comprises the consecutive steps of A.) acylating kanamycin A or kanamycin B with an acylating agen ing -tine formulas CH 0 -C-O-C-N, I 3 CH X-CHg-C II-OH , carbodilmide thereof 0 II —C-CH2-CH2-CO2H or a carbodilmide thereof , in which and R5 are alike or different and each is H, F, CI, Br, N©2> OH, (lower) alkyl or (lower) alkoxy, is chloro, bromo or iodo, or with its functional equivalent as an acylating agent, in a ratio of 1 mole or less of acylating agent per mole of kanamycin A or B,in a solvent, preferably selected from the group comprised of dimethyl-formamlde, dimethylacetamide, tetrahydrofuran, dioxane, 1,2-dimethox ethane, methanol, .ethanol, water, acetone, pyridine, N-(lower) alkyl piperidine, or mixtures thereof, but preferably dimethylformamide, a a temperature below about 50° C. and prererably below 250 c. to prod the compound having the formula as defined above; 7 8 9 B.) acylating compound II with an acylating agent having the 0 formula OH 0 I II said acylating agent 11 W-NH-CH2-CHg-CH-C-M 2 being derived from L-(-)-Y-aroino-«(-hydroxybutyric acid, in which in which R and are as above^or with its functional equivalent as an acylating agent for a primary amine, in a ratio of at least 0. 5 mole of compound VII per mole of compound II, but preferably in a ratio of about 0.5 to about 1.4, and most preferably in a ratio of about 0.8 to about 1 . 1 , in a solvent, preferably selected from the group comprising a mixture of water and ethyleneglycol dimethyl ether, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran, propyleneglycol dimethyl ethe or the like and most preferably 1 : 1 water-ethylene glycol dimethy ether, to produce a compound of the formula CHg-NH-Y in which Έ?9 Y and W are as above; and C.) removing the blocking groups W and Y from compound III by methods commonly known in the art, and preferably when W and Y are radicals of the formula by hydrogenating compound III with hydrogen in the presence of a metal catalyst, preferably selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but preferably palladium, and most preferably palladium on charcoal, in a water-water miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydro- furan, ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether, or the like, but most preferably 1 : 1 water-dioxane, and preferably in the presence of a catalytic amount of glacial acetic acid to produce the compound of formula IV, and, if desired, sub- sequently converting the product by methods known per se into a nontoxic pharmaceutically acceptable acid addition salt thereof, it should be apparent to those knowledgeable in the art that other agents can be used in the process above to acylate the amine functions of the intermediate compounds of the present invention. This disclosure is meant to include all such acylating agents that produce labile amine blocking groups, said labile blocking groups ; commonly employed in the synthesis of peptides. The labile block- ing groups must be readily removable by methods commonly known in the art. Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, J. Pharm. Sciences pp. I-27 (1970 ) . Functional equivalents as acylating agent for primary amine groups would include corresponding carboxylic chlorides bromides, acid anhydrides, including mixed anhydrides and particularl aliphatic monoesters of carbonic acid, of alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2 , -dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with the kanamycin derivative (II) after first reacting said free acid with Ν,Ν' -dimethylchloroforminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/6, 360 (1965 ) ] or by the use of enzymes or of an Ν,Ν' -carbonyldiimidazole or an Ν,Ν' -carbonylditjriazole [cf . Sheehan and Hess, J. Amer. Chera. Soc. , 77, IO67, (1955 ) ] or of alkynylamine reagent [cf. R.

Buijile and H. G. Viehe, Angew, Chem., International Edition 3, 582 (1964 ) ] , or of a ketenimine reagent [cf. C. L. Stevens and M. E. Monk, J. Amer. Chem. Soc . 80, k06$ (1958 ) ] or of an isoxazolium salt reagent' [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc.,.83 , 1010 (1961 ) ] . Another equi-valent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, Ν,Ν' -carbonyldiimidazole is reacted with a carboxylic acid in equi olar proportions at room temperature in tetra-hydrofuran, chloroform, dimethylformamide or a similar inert solvent to form "the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides.

The by-product, imidazole, precipitates and may be separated These reactions are well-known in the art (cf. U. S. Patent Nos. 3.079* 31^* 3* 117* 126 and 3* 129*224 and British Patent Nos. 932,644, 957, 570 and 959* 054) .

In preparing the 6 '-blocked-amlno kanamycin in Step A it is preferred to regulate the molar proportions of reagents so that a ratio of 1 mole or less of acylating agent is used per mole of 6 '-blocked-amino kanamycin. Increasing the proportion of acylating agent will result in lower yields of the desired intermediate because of the increase of competing side reactions which produce polyacyl derivative(s) impurities.

As with most chemical reactions, temperatures higher or lower than those specifically described herein may be used. Temper- atures substantially higher than those described, i.e. 50 ° C, however, tend to result in reduced yield due to a greater extent of side reactions .

Compound IVa, l-(L- ( -) -Y-amino-a-hydroxybutyryl) -kanamycin A, possesses excellent antibacterial activity that appears superior to kanamycin A itself. Illustrated below are two tables showing the minimal inhibitory concentrations (MIC^s) of kanamycin A and compound IVa (BB-K8) against a variety of gram-positive and gram^- negative bacteria as obtained by the Steers agar-dilution method (Table I) and the two-fold dilution method (Table 2). Mueller-Hinton, Agar Medium was used in the study of Table 1 and Heart Infusion Broth was used in the study of Table 2.

• TABLE I CMIC eg. ml.) Ranamycin A Organism Γ6-δΐ98) 1. Alk. faecalis A-9423 16 8 2. 11 " A-20648 >125 >125 3. Ent. cloacae A-9656 4 4 4. " species A-20364 ' >125 2 . " hafniae 1 A-2067 1 1 6. E. coli A-0636 2 1 7. 11 A-20664 ■■'' ···:'■'■■ " · ' 16· 4 8. " A-20665 >125 1 . " A-20507 32 2 .10. 11 A-20520 > 125 4 11. 11 A-20365 >125 1 12. " A-20684 2 2 13. " A-20682 >125 2 14. " A-20683 >125 8 . " A-20681 >125 2 16. " A-I5II9 4 4 17, . pneumoniae A-9867 4 4 18.. " species A-20328 >125 2 19. " " A-20330 32 32 . " " A-20634 > 125 4 21. " pneumoniae A-20680 r > 125 4 22. " " A-9977 1 1 2 . - 1 Table I (VdC m./ml . ) (Continued) 2 Compound XVa Kanamycln A [BB- 8] Organism (6-8198 ) lot No. 4 . 24. Pr. morganii A-15153 2 2 6 25. " vulgaris A-9555 '■■·■■■ "··;■..'■;'. 1 7 26. " rettgeri A-9636 0.25 Ο.25 8 27. " mirabilis A-20645 4 4 9 28. " mirabilis A-20454 2 2 0 29. Provldencia stuartli A-20615 2 . 1 11 30. " alkalifaciene A-20676 1 1 12 13 31. Ps . aeruginosa A-20229 32 2 ι1» 32 . " 11 A-9843A 125 16 33. " " A-20653 > 125 32 16 34. " species A-20601 125, 63 16 17 35. " " A-20621 > 125 > 125 18 36. " maltophilia A*2062O 32 > 125 37. Sal. enteritidis A-9531 1 0.5 38. " derby A-20087 > 125 1 21 59- Ser. marcescens A-20019 2 4 22 40. " A-9933 4 ■ ; 8 23 41. " " A-20460 > 125 4 2 24 42. " " A-20459 4 16 43 . Shig. flexneri A9684 4 . 4 26 44. Aeromonas sp. A-20670 2 2 27 45. Arizona sp. A-20671 1 28 46. Citrobacter sp. A-20673 . 4 29 47. Edwardsiella sp . A-20678 ?■ ¾·. , 4;'.y■ 4 - Table I (MIC mg./ml.) (Continued) Compound rVa Kannaammyycciinn A Organism [BB-K8] (6-8198 3 lot No. 4 49. Staph, aureus A- 7 9 0. 1 50. " » A-9537 2 1 51. " A-20610 > 125 2 52. " " A-20240 >125 8 53. A-15197 1 2 Mueller-Hinton Medium +# sheep blood 54. Str. faecalis A-9854 63 . 6? 55. " " A-9575 125 >125 56. Str. pyogenes A-20200 32, 16 32 57. " " A-9604 125 125 58. M " A-15040 125 125 59. " " A-20065 125 125 60. D. pneumoniae A-9585 63, 32 63 61. " " A-20159 > 125 > 125 3' ΤΑΚΓίΕ 2 f IC mg./ml. ) Compound TVa ■JEanaraycin A [BB-K8] Organism {6-8196} lot No. 4 2. D- pneumoniae +5 serum A-9585 63 2. Str. pyrogenes ± 5 serum A-9604 225 225 3. staph, aureus Smith A-9537 0.5 0.5 4. Staph, aureus A-9497 0.5 0.5 . Staph, aureus A-20239 >225 4· 6. Staph, aureus A-202 0 >125 4 7. -Enter, cloacae A-0656 2 2 8. Elnter. species A-20364 >225 2 9· K. pneumoniae A-9867 2 4 . E. coli K-12 MI1410 A-20361 2 4 11. E. coli K-12 U630 A-20363 >125 2 12. E. coli K-12 A-963 2 1 13. E. coli A-20664 32 8 14. E. coli A-20665 >125 8 . Pr. mirafcilis A-9900 2 16 16. Pr. morganii A-15153 4 16 17. Pr. vulgaris A-9436 -1 2 18. Ps. aeruginosa A-20227 4 1 19. Ps. species A-20499 4 . Ps. aeruginosa A-20653 >125 4 21. Ps. species A-2c62i >125 >125 22. Ser. marcescens A-20019 2 4 23. Ser. marcescens A-20141 16 16 1 The above MIC data show that compound IVa(BB-K8) is superior to kanamycin A in activity, particularly against kanamycin A re- sistant organisms .

^ The MIC data also correlate well with the in vivo results for . all three organisms against which kanamycin A and compound XVawere 6 tested. 7 Compound I\a and kanamycin A were equally effective in infec- 8 tions of mice caused by kanamycin A-sensitive strains of E. coli 9 AI5II9 and Staph . aureus Α9537· Although the CD^Q values (curra- 0 tive dose in 50$ of mice lethally infected) for Stap . aureus 1 A9537 suggest that compound IVais slightly less active than kanamycin 2 A, this small difference is probably not significant because the dose 3 levels were far apart (5X-dilutions) .

^ Against the kanamycin-resistant strain of E. coll A20520, kana- 5 mycin A as expected was not effective in vivo, whereas compound 6 Ia demonstrated a marked protective action. Compound awas approxi- 7 mately 10 times more active against this E. coli strain when ad ini- 8 stered in a -treatment regimen rather than in a 2-treatment one.

A comparison of In Vitro and In Vivo Act as β test medium.

CD. curative dose, 50 (mg./kg./treated X 50 taneously at 1 and 4 hours post-infect 0, 2, 4, and 6 hours post-infection wh test were carried out as described by = not tasted.

The compounds IV are valuable as antibacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially valuable in the treatment of infectious diseases caused by Gram-positive and Gram-negative bacteria.

The compounds IV when administered orally are useful as an ad- junctive treatment for preoperative sterilization of the bowel.

Both aerobic and anaerobic flora which are suseptible to these drugs are reduced in the large intestine. When accompanied by adequate mechanical cleansing, they are useful in preparing for colonic surgery.

The compounds IV are effective in the treatment of systemic - bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per day in divided doses three or four times a day. Generally the compounds are effective when administered at a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Compound IVb, 1-[L-(—)-y-ajnino-a-hydroxybutyryl]-kanamycin B, possesses excellent antibacterial activity that appears superior to kanamycin A. Illustrated below is Table 4 showing the mini- mal inhibitory concentrations (MIC's) of kanamycin A and compound IVb (BB-K26) against a variety of gram-positive and gram-negative bacteria. The MIC's are obtained by the Steers agar-dilution method (Table 4) on nutrient agar medium.

TABLE 4 MIC (mg./ml.) Bristol Resist, Organism Code No. to BBK-26 Kanamycin E. coli N1HJ 0.4 0.8 " Jufal A15119 1.6 0.8 II AI 169 1.6 1.6 II A20363 KM 0.8 50 II A98 4 0.8 0.8 A20365 KM 0.4 >50 11 K-12 0.8 0.4 " K-12 A20664 KM 0.8 3.1 ' " K-12 A20665 KM 0.8 50 K. pneumoniae D l 0.2 0.2 II A9678 0.8 0.8 S. marcescens A20019 1.6 1.6 Ps. aeruginosa D15 3.1 12.5 " D113 KM 50 >50 It A9923 1.6 25 II A9930 0.8 6.3 II A15150 6.3 25 11 AI519 1.6 12.5 II A20479 KM 1.6 50 II A206l6 KM 3.1 25 It A20653 KM 25 50 II A983 1.6 12.5 Pseudomonas sp. A20355 1.6 6.3 ·· A20358 KM 3.1 12.5 II Δ20368 GM 50 25 II A20598 GM' 6.3 25 II A2 6 TABLE 4 (Continued) MIC (mg./l. ) Bristol Organism Code No. to BBK-26 Kanamycin A Pseudomonas sp. A20603 GM 6.3 > 50 II A206l8 GM 50 50 P . vulgaris A9436 1.6 0.8 II A9526 0.8 0.8 Pr. mirabilis A9554 1.6 1.6 II A9900 1.6 1.6 Pr. morganii A9553 1.6 1.6 II A20031 6.3 3.1 Pr. rettgeri AI5167 0.8 0.8 S. aureus 209P 1.6 0.8 " Smith 0.8 0.4 " 209P 1.6 0.8 II A20239 KM 1.6 25 S. lutea PCl-lOOl 1.6 3.1 M. flavus 0.4 0.8 B. subtills PCl-219 0.1 0.1 St. pyogenes S-23 25 25 " Dick 1.6 1.6 " Digonnet A9604 12 .5 25 II A20065 25 25 D. pneumoniae 50 25 Mycobacterium 607 6.3 0.8 " 607 D105 KM > 100 > 100 " 607 D107 KM > 100 > 100 " phlei 1.6 1.6 II ' ranae . 6.3 1.6 0.8 0.4 *determined by tube dilution method.

KM is kanamycin A; GM is genta ycin.

The above MIC data show that compound IVb (BB-K26) is superior to kanamycin A in activity, particularly against kanamycin A-resis-tant organisms.

An in vivo evaluation of compound IVb was carried out against both kanamycin B-sensitive and resistant strains of E. coli and Ps. aerugenosa.

The organisms are designated as follows: Code No. Kanamycin Sensitivity E. coli Juhl A15119 sensitive E. coli ML- 1630 A20363 resistant (phosphorylation) Ps . aeruginosa D15 - · moderately resistant Ps_. aeruginosa D113 - highly resistant (phosphorylation) Compound IVb (BB-K26) and kanamycin B were equally effective against E. coli Juhl AI5119 at a dose of 6 .3 mg./kg. in mice. Com-pound IVb was most effective against kanamycin B resistant JJ. coli ML-J.630 at a dose of 6 .3 mg./kg. vs. 100-4-00 mg./kg. for kanamycin B.

Compound5 IVb showed slightly better activity against moderately resistant Ps_. aeruginosa D15 than kanamycin B. Compound IVb showed substantially better activity against highly resistant Ps_. aeruginosa D-113 than kanamycin B (see Table 5 below).

TABLE 5 E. coll Juhl A15119 E. coli ML-l6^0 dose (sc) BB-K26 KM-B * BB-K26 KM-B ^00 mg./kg. - ' - . - 2/5 100 r 5/5 5/5 . 5/5 2/5 5/5 5/5 5/5 0/5 1 TABLE 5 (Continued) E. coli Juhl A15119 E. coli ML-I63O dose (sc) BB-K26 KM-B BB-K26 KM-B 1.6 0/5 .0/5 2/5 CD5o (mg./kg.) 3.1 3.1 1.8 ca 300 Ps. aeruginosa D15 Ps. aeruginosa D-1 dose (sc) BB-K26 KM-B doses (sc) BB-K26 KM-B 100 mg./kg. 5/5 5/5 400 mg./kg. - 0/5 1/5 0/5 200 3/5 - 6.3 1/5 0/5 50 1/5 - CDcjo (mg./kg.) 3 388 50 CD50 (mg./kg.) 145 > 400 *KM-B is kanamycin B. The figures shown as 0/5, 1/5, 5/5, etc., indicate the number of surviving animals per five animals challenged; for example, 5/5 indicates 5 of 5 animals surviving the lethal dose of challenge organism when treated with Kanamycin B or BB-K26 .

EXAMPLES Example 1 Preparation of L-(-)-Y-Benzyloxycarbonylamino-a-hydroxybutyric acid (VI).

L-(-)-7-amino-a-hydroxybutyric acid (7.4 g., O.062 mole) was added to a solution of 5 *2 g. (0.13 mole) of sodium hydroxide in 50 ml. of water. To the stirred solution was added dropwise at 0- 5° C. over a period of 0.5 hour, 11.7 g. (0.068 mole) of carbo- benzoxy chloride and the mixture was continued to stir for one hour at the same temperature. The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four 80-ml. portions of ether. The ethereal extrac s were combined, washed with a small amount of saturated ■15 sodium chloride solution, dried with anhydrous sodium sulfate and 16 filtered. The filtrate -was evaporated in vacuo and the resulting 17 residue was crystallized from benzene to give 11.6 g. (7 $) of 18 colorless plates; melting point 78.5-79 ·5° C., [a]D=4.5[ (c=2 , !9 CH3OH). Infrared (IR) [ KBr] : IR(KBr) Tc=o 1740, 1690 cm"1.

Nuclear Magnetic Resonance (MR) (acetone-dg) £(in ppm from TMS) 21 2.0 (2H,m), 3 .29 (2H,d-d, J=6.7andl2 Hz), 4.16 (lH,d-d, J=4.5 22 and 8 Kz), 4. 9 (2H,s), 6.2 (2H, broad), 7-21 (5H,s). 23 Anal, calc'd. for C12H15 05: C, 5 .91; H, 5 -97; N, 5 -53 - 24 Found: C, 56.66; H, 5 -97; N, 5. 7- 25 Example 2 26 N-Hydroxysuccinimide ester of L-(-)-7-benzyloxycarbonylamino-a- 27 hydroxybutyrlc acid (VII). 28 A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 29 mole) of N-hydroxysuccinimide1 in 200 ml. of ethyl acetate was cooled to 0° C. and the 8.6 . . 42 e clohex lcarbo- diimide was added. The mixture was kept overnight in a refrigerator. The dicyclohexylurea which separated was filtered off and the filtrate was concentrated to about 50 ml. under reduced pressure to give colorless crystals of VII which were collected by filtration; 6.4 g., .p. 121-122.5° C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 ml. of a benzene-n-hexane mixture to give an additional amount of VII. The total yield was 13.4 g. (92#). [a]^ I.50 (c=2, CHC1 ) 'lR(KBr) c=o 1810, 1755* 1740, l680 cm"1. NMR (acetone-dg) 5"(in pptn from TMS) 2.0 (2H,m), 2.83(4H,s), 3.37(2H,d-d, J=6.5 and 12.5 Hz), 4.56(lH,m), 4.99(2H,s), 6.3(2H,broad), 7.23(5H,s).

Anal, calc'd. for Ο^Η-^^Ογ: C, 54.85; H, 5.18; N, 8.00.

Pound: C, 54.79, 54.70; H, 5-21, 5.20; N, 8.OA, 8.12. 1. G. W. Anderson et al., J. Am. Chem. Soc, 86, 1839 (1964).

Example 3 Preparation of 1-[ L- (- )-γ- benzyloxycarbonylamlno-a-hydroxybutyryl] - 6 ' -carbobenzoxykanamycin A (Ilia).

A solution of 1.6 g. (4.6 mmoles) of VII in 40 ml. of ethylene- glycol dimethyl ether (DME) was added dropwise to a stirred solu- tion of 2.6 g. (4.2 mmoles) of 6 · -monobenzyloxycarbonylkanamycin A (II) in 40 ml. of 0$ aqueous ethyleneglycol dimethyl ether and the mixture was stirred overnight. The reaction mixture was eva- porated under reduced pressure to give a brown residue of Ilia which was used for the next reaction without further purification.

· Example 4 Preparation of 1 -[L-(-) -γ-amino-ct-hydroxybutyryl ]-kanamycin A (IVa) .

The crude roduct Ilia from Exam le was disso ved in 40 ml. ^ of 0$ aqueous dioxane and a small amount of insoluble material 2 was removed by filtration. To the filtrate was added 0.8 ml. of 2 glacial acetic acid and 1 g. of 10$ palladium-on-charcoal and the ^ mixture was hydrogenated at room temperature for 24 hours in a Parr ^ hydrogenation apparatus. The reaction mixture was filtered to g remove the palladium catalyst and the filtrate was evaporated to γ dryness in vacuo. The residue was dissolved in 30 ml. of water g and chromatographed on a column of CG-50 ion exchange resin (NH+^ 9 type, 50 cm X 1.8 cm). The column was washed with 200 ml. of water and then eluted with 800 ml. of 0.1 N H 0H, 500 ml. of 0.2 N NH^OH n and finally 500 ml. of 0.5 N H OH. Ten-milliliter fractions were 12 collected and fractions 146 to 154 contained 552 mg. (22$, based 13 on carbobenzoxy kanamycin A, II) of the product IV which was desig- 14 nated BB-K8 lot 2. M.p. 187° (dec). Relative potency against 1 B. su tilis (agar plate) « 560 mcg./mg. (standard: Kanamycin A ΐβ free base) . 17 A solution of 250 mg. of BB- 8 lot 2 in 10 ml. of water was 18 subjected to chromatography on a column of CG-50 (NH+ type, 30 19 cm X 0.9 cm). The column was washed with 50 ml. of water and then eluted with 0.2 N NH OH. Ten milliliter fractions were collected. 21 Fractions 50 to 63 were combined and evaporated to dryness under 22 reduced pressure to give 98 mg. of the pure product, K8 lot 2-1. 23 M.p. 194° (dec.) [a]21 +85° (c=2, ¾0) . Relative potency against 24 B. subtilis (agar plate) = 960 mcg./mg. (standard: kanamycin A 2 free base) . 26 Anal, calc'd. for -2I½C¾ ' C, 40.62 ; H, 6.68; 27 N, 9.87. 28 ' Found: C, 40.21, 39-79; 29 H, 6.96, 6.87; N, 9.37, 9. 9.

Example 5 Preparation of N-(Benzyloxycarbonyloxy) sueclnlmide.

N-Hydroxysuccinimide"1 (23 g., 0.2 mole) was dissolved in a solution of 9 g. (0.22 mole) of. sodium hydroxide in 200 ml. of water. To the stirred solution was added dropwise 3 g. (0.2 mole) of carbobenzoxy chloride with water-cooling and then the mixture was stirred at room temperature overnight to separate the carbobenzoxy derivative which was collected by filtration, washed with water and air-dried. Yield 4l.l g. (82$).. Recry-stallization from benzene-n-hexane (10:1) gave colorless prisms melting at 78-79° C. 1. G. W. Anderson et al., J. Am. Chem. Soc, 86, 1839 (196 ). ■ r Example 6 Preparation of 6 ' -Carbobenzoxykanamycin A.

A solution of 42.5 g- (90 mmoles) of kanamycin A free base in 50 ml. of water and 500 ml. of dimethylformamide (DMF) was cooled below 0° C. and stirred vigorously. To the solution was added dropwise over a period of about two hours a solution of 22.4 g. (90 mmole) of N-(benzyloxycarbonyloxy)succinimide in 500 ml. of DMF, The mixture was stirred at -10° to 0° C. overnight and then at room temperature for one day. The reaction mixture was eva-porated under reduced pressure below about 50° C. The oily residue was dissolved in a mixture of 500 ml. water and 500 ml. butanol, the mixture being filtered to remove insoluble material and separated into two layers. The butanol and aqueous layers were treated with butanol-saturated water (500 ml. X 2) and water-saturated butanol (500 ml.' X 2), respectively, using a technique similar to counter current distribution. The three aqueous layers were combined and residue, a part of which crystallized on standing at room temperature. To the residue including the crystals was added about 100 ml. of methanol, which dissolved the oil and separated it from the crystals. After adding about 300 ml. of ethanol, the mixture was kept at room temperature overnight to give a crystalline mass which was collected by filtration. It weighed 44 g.

The product contained a small amount of kanamycin A as indicated by thin layer chromatography using n-propanol-pyridine-acetic acid-water (15:10:3:12) as the solvent system and nin-hydrin as the spray reagent.

The crude product was dissolved in 300 ml. of water and chro-matographed on a column (30 mm. diameter) of CG-50 ion-exchange resin (WH^+ type, 500 ml.). The column was irrigated with 0.1 N ammonium hydroxide solution and the eluate was collected in -ml. fraction. The desired product was contained in tube numbers 10-100, while kanamycin A recovered from slower-moving fractions and the position isomer(s) of the product seemed to be contained in the faster-moving fractions. The fractions 10-110 were combined and evaporated to dryness under reduced pressure to give 24.6 g. ( 5$) of a colorless product 6-carbo-benzoxykanamycin A (II) [ 61 -Cbz-kanamycin A], which began to melt and color at 204° C. and decomposed at 212° C. with gas evolution. [a]D +106° (c=2, HgO) .

TLC (silica gel F2 4J ninhydrin) Rf value Solvent System 6 '-Cbz-Kanamycin A Kanamycin n-PrOH-Pyridine -AcOH-HgO 0.42 0.33 0.1 0.04 (15 :10 :3 :12 ) (main) minor Acetone-AcOH-HgO 0.24 0.14 (Continue TLC (silica gel ninhydrin) Rf value Solvent System 6' -Cbz-Kanamycin A Kanamycin A CHClj-MeOH-c Ο.76 O.5O (1: 4 :2:1) AcGMe-n-PrOH-c.NH^OH 0.22* 0.04* . (45:105:60) *Detected by anthrone-sulfuric acid .

The final product was found to be accompanied by two minor components by TLC with one of the solvent systems tested. However, the final product was used without further purification for the preparation of BB-K8 (I).

Example 7 Preparation of L-(-)-7-amino-q-hydroxybutyric acid from ambutyrosin A or B or mixtures thereof.

Ambutyrosin A (5.0 gm. ) [U. S. Patent No.3,541,078, issued Novemb 17, 29 0] was refluxed withl60ml. of 0.5 N sodium hydroxide for one hour. The hydrolysate was neutralized with 6N HCl and chromatographed on a column of CG-50 type). The desired L-(-)-7-amino-a-hydroxybutyric acid was isolated by developing the column with water and removing the water by freeze drying. The L-(-)-7-amino-a-hydroxybutyric acid is characterized as a crystalline material having a m.p. of 212.5-214.5° C. [Column 2, lines 31-38, U. a Patent No. 3,541,078].

Example 8 c Preparation of 6 ' -Carbobenzoxykanamycin B.

To a chilled solution of 8.1 g. (O.OI68 mole) of kanamycin B in 120 ml. of water and 80 ml. of 1,2-dimethoxyethane was added dropwise with stirring a solution of 4.2 g. (O.OI68 mole) of N- 1 The reaction mixture was stirred overnight and evaporated under reduced pressure. The residue was dissolved in 100 ml. of water and shaken twice with 50 ml. of water-saturated n-butanol. The 4 aqueous layer was separated and adsorbed on a column of 100 ml. of CG-50 (NH^+type). The column was washed with 200 ml. of water, 6 eluted with 0.05 N NH^OH. The eluate was collected in 10-ml. j fraction. Fractions 121 to l80 were collected, evaporated and 8 freeze-dried to give I.58 g. (15$) of the desired product. 9 Fractions 1 to 120 were evaporated and re-chromatographed on 0 CG-50 (NH^+) to give 1.21 g. (12$) of the product. M.p. 15I- 24 T 1 152° C. (dec). [a]D +104° (C. 2.5, ¾0). γ0=0 1710 cm." . 2 Anal, calc'd. for ¾6¾5 4012 : C, 50.56; H, 7.02 j N, 11.34. 3 _ Found: C, 50.71; H, 7-38; N, 11.48. 4 TLC (silica gel F254), Rf 0.03 in n-PrOH-AcOH-I^O (15:10:3: 12); Rf O.16 in acetone-AcOH-I^O (20:6:74). 16 Example 9 17 l8 Preparation of l-[ L-( -) -γ-benzyloxycarbonylamino-a-hydroxybutyry·'.] - g 6 ' -carbobenzoxykanamycin B (Illb).

To a stirred solution of Ί.85 g. (3.0 m.' moles) of 6'-carbo- 21 benzoxykanamycin B i .40 ml. of HgO and 0 ml. of 1,2-dimethoxy- 22 ethane was added all at once at 5° C. 1.1 g. (3·1 m. moles) of 23 N-(L-Y-carbobenzoxyamino-a-hydroxybutyryloxy)succinimide . The 2 reaction mixture was stirred overnight at room temperature and subjected to hydrogenolysis without isolating the carbobenzoxy 26 derivative (Illb). TLC (silica gel F254), Rf O.06 (starting 27 material), 0.41, 0.57 28 Rf 0.11 (starting materi 29 20:6:74). 1 Example 10 2 Preparation of l-[L-(-»)-y-amino-a-hydroxybutyryn-kanamycln B ( TTb ) . 3 To the solution obtained in Example 9 was added 0.2 g. of 10$ palladium on charcoal. After the mixture was hydrogenated under atmospheric pressure for five hours, an additional amount of 10$ 6 palladium on charcoal (0.1 g.) and 10 ml. of water were added. 7 Hydrogenation was continued overnight. The reaction mixture was 8 filtered, the filtrate was evaporated under reduced pressure, the 9 residue was dissolved in 50 ml. of water and chromatographed on a column of CG-50 (ΝΗ^+* 1,2 cm« x 5° οπι·)· Tne column was washed 11 with 200 ml. of water and then eluted with 500 ml. of 0.1.N NH^OH, 12 500 ml. of 0.2 N NH^OH, 900 ml. of 0.5 N NH OH and 500 ml. of 13 I NH^OH. The effluents were, collected in 10-ml. fraction.

Kanamycin B was recovered from fractions 60 to 76 in 3 $ recovery ! (45 mg.). Fractions 128-138 were collected, evaporated under 1o" reduced pressure and freeze-dried to give 3l8 mg. (.17#, based on 17 carbobensoxy kanamycin B) of BB-K26 (IVb). M.p. 186-1870 C. 18 (dec), [a] 9 +78° (C. 1.15, ¾0). 7c=0l64o cm."l. 19 Anal, calc'd. for C22¾ 6012. H-CDS. C, 42.72; H, 7-17; N, 13.00. 21 Pound: C, 42.23; H, 7.1 J 22 N, 12.37. 23 TLC (silica gel F254 , ninhydrin), Rf 0.11 in acetone-AcOH-HgO 24 (20 :6 :74 ) ; Rf 0 .19 in CHCl^-MeOH-coneentrate Η^ΟΗ-Η^Ο (1:4:2:1).

Fractions 201 to 222 were combined, evaporated under reduced 26 pressure and freeze-dried to give 209 mg. (12%) of another active 27 component which was designated. BB-K27. M.p. 183-184° C. (dec.) 28 Yc=0175O cm."1. . - ■' 29 Anal, calc'd. for C22H]|iN6012.^CO^ : C, 42.72; H, 7.17 N, 13.00. .* 1 Pound: C, 2.25; H, 6 .93 , 2 N, 12 .18. 3 TLC (silica gel F254 ) , Rf 0.15 in acetone-AcOH-I^O (20 :6 :74) ; 4 Rf 0.07 in CHClj-MeOH-concentrate NH^OH-HgO (1 : 4 :2 :1 ) .

. Example 11 7 Preparation of L(-)-Y-araino-a-hyd.roxybutyrlc acid from DL-a- 8 hydroxy-y-phthalimidobutyric acid . 9 (A.) Dehydroabietylammonium L-a-hydroxy-y-phthalimidobutyrate : To a solution of 25 g. (0.1 mole) of 2-hydroxy-7-phthalimidobutyric 11 acid^ in 200 ml. of ethanol was added a solution of 29 g. (0.1 mole) 12 of dehydroabietylamine in 130 ml. of ethanol. The solution was Ό shaken vigorously for a minute and stood at room temperature for 1 five hours during which time fine needles crystallized out. The 1 crystals were collected by filtration, washed with 50 ml. of 16 ethanol and air-dried to obtain 30.1 g. (56$) of a diastereomer 24 17 of the dehydroabietyla ine salt. .p. 93-94° C. [a'J +Ί5° lft (c . 2.5, eOH) . Recrystallization frorr 300 ml. of ethanol gave 19 23-2 g. (43JS) of the pure product. M.p. 94-95° C. [α]^ +10.8° (C. 2.5j HeOH) . Further recrystallization did not change the 21 melting point and the specific rotation. 22 Anal, calc'd. for , H2° Cj 69- 4; H, 8.02; 23 N. 5.07. 24 Found: C, 69 .58; H, 8 .08'; N, 5.07. 26 1. Y. Saito et al., Tetrahedron Letters, 1970> 4863;. 27 B.) L-(- ) -y-Amino-a-hydroxybutyric acid : To a solution of 28 1.5 g. (0.014 mole) of sodium carbonate in 40 ml. of water were 29 added 5-3 g. (0.01 mole) of dehydroabietylammonium-L-a-hydroxy- shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with -ml. portions of ether and evaporated to 15 ml. under reduced pressure. To the concentrate was added 10 ml. of concentrated , hydrochloric acid and the mixture was refluxed for ten hours.

After cooling, separated phthalic acid was removed by filtration.

The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The residual syrup was dissolved in 10 ml. of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was adsorbed on a column of IR-120 (H+, 1 cm. x 35 cm.), the column was washed with 300 ml. of water and eluted with 1 N ammonium hydroxide solution. The eluate was collected in 15-ml. fraction. The ninhydrin posi- tive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually.

The crystals were triturated with ethanol, filiore-'i and dried 9 in a vacuum desiccator to give 0.78 g. (66>¾) of L-(-)-7-amino-0 a-hydroxybutyric ac.d M.p. 206-207°C. [a]* -29° (C. 2 .5 , 1 ¾0) . The IR spectrum was identical with the authentic sample 2 which was obtained from ambutyrosin. 3 ^ Exam le 12 Preparation of the Monosulfate Salt of 1 -[L-(-)-7-amino-a-hydroxy-6 butyryl]kanamycin A or B. 7 One mole of 1 -[L-(-) -7-amino- -hydroxybutyryl]kanamycin A or 8 3 is dissolved in 1 to 3 liters of water. The solution is filtered 9 to remove any undissolved solids. To the chilled and stirred solu- water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate salt.

Example 13 Preparation of the Disulfate Salt of l-[L-(-)-7-amino-a-hydroxy- Thirty-five grams of l-[L-(-)-7-amino-a-hydroxybutyryl]-kanamycin A (as the monobicarbonate trihydrate) was dissolved in 125 ml. of deionized water. A pH of approximately 9.0 was noted. The pH was lowered to 7-7-5 with 50^ V/V sulfuric acid.

Eight and one half grams of Darco G-60 (activated charcoal) was added and the mixture was slurried at ambient room temperature for 0.5 hour. The carbon was removed by suitable filtration and washed with 40 ml. of water. The water wash was added to the filtrate.

The combined filtrate-wash above was adjusted to pH 2-2.6 . with 50$ V/V sulfuric acid. A large amount of carbon dioxide was evolved. The solution was left at house vacuum with stirring for 20 minutes to expel additional carbon dioxide.

Eight and one half grams of Darco G-60 was added to the degassed solution. The mixture was slurried for 0.5 hour at ambient room temperature. The carbon was removed by suitable filtration and washed with 35 ml. of deionized water. The water was added to the filtrate.

The combined filtrate-washwas adjusted to pH 1-1.3 with 0$ V/V sulfuric acid. This solution- was added with rapid stirring over a 10 minute period to 60O-80O ml. of methanol 1 minutes at H 1-1.3, passed through a 100 mesh screen, stirred 2 for 2 minutes and allowed to settle for 5 minutes. Most of the 5 supernatant was decanted. The remaining slurry was suitably filtered, washed with 200 ml. of methanol and vacuum dried at 50° C. for 24 hours. The yield of amorphous BB-K8 (di- 6 hydrogen sulfate )g was 32-3 grams; [a]^2 .Hg0e-*7 .75, decom- 7 position at 220-230° C. 3 Elemental Analysis (On Dry Basis*) 9 Found Theory 0 C 32.7, 33-5, 32.3 33-5 11 N 8.78, 8.7, 8,2, 8.8 8.97 12 # S 8.75, 8.9, 7-8, 8.85 8.2 13 # Ash nil — I4* *Karl Fisher water contents 2.33, 1.79, .87# (theory for S monohydrate is 2.25# water). This salt is hygroscopic but not ^ deliquescent. After storage of an aliquot in air at room tem- Ή peraturt for l8 hours the water content increased to 9-55, x® 9·89# (theory for a pentahydrate is 10.33$ water).

Claims (21)

1. CLAIMS acceptable acid addition salt thereof; which process comprises the consecutive steps of A) acylating kanamycln A or kanaraycin B with an acylating agent selected from the compounds having the formulae 0 II X - C¾ - c - χ or a carbodlimide thereof 0 II C — CHg - CHg — COOH or a carbodiimlde thereof in which Ί and R3 are alike or different and each is H, F, CI, Br, O j OH, (lower) alkyl or (lower)alkoxy and X is chloro, bromo or iodo, or with its functional equivalent as an acylating agent, in a ratio of 1 mole or less of acylating agent per mole of kanamycin A or B, in a solvent, at a temperature below about 50° C. to produce the compound having the formula in which R^ is as above and V is a radical of the formula NO, 0 |l X - CH2 - c or in which R and RJ are as above B) acylating the compound of formula II with an acylating agent having the formula OH 0 H W - NH - CIL CH C - M said acylating agent being derived from L- (-) -V-amino-a-hydroxybutyric acid, in "which W is a radical selected from the group comprising in which Έ and BP are as above, or with its functional equivalent as an acylating agent for a primary amine, in a ratio of at least 0. 5 mole of acylating agent per mole of the compound II, in a solvent, to produce a compound of the formula in which R3, V and W are as above j and C) removing the blocking groups W and Y by methods known per se to produce the compound of formula I and, if desired, subsequently converting the product by methods known per se into a nontoxic pharmaceutically acceptable acid addition salt thereof.

2. The process of Claim 1 characterized by the fact that (1) in Step A the kanamycin A or B is reacted with an acylating agent of the formula wherein R^ and R5 are as defined in Claim 1, in a solvent selected from dimethylformamide, dimethylacetamide, tetra- hydrofuran, dioxane, 1, 2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine or N-(lower) alkylpiperidines, or mixtures thereof; and (2) in Step B an acylating agent of the formula wherein R and RJ are as defined in Claim 1 is reacted with compound II in a ratio of about 0.5 to about 1.4 moles of acylating agent per mole of compound II, in a solvent ί selected from a mixture of water and ethyleneglycol dimethyl - 2 ether, dioxane, dimethylacetamide, dimethylformamide, tetra- 3 hydrofuran or propyleneglycol dimethyl ether. 4 5 6 γ 3.

3. The process of Claim 2 characterized by the fact that (1) 8 Step A is conducted in dimethylformamide as the solvent at a 9 temperature below 25° c . and (2 ) the a.cylation reaction of. 0 Step B is conducted in 1 : 1 water-ethylene glycol dimethyl ether I as the solvent in a molar ratio of about 0.8 to about 1. 1 moles of 2 acylating agent per mole of compound II. 3 5 6 4.

4. The process of Claim 2 or Claim 3 characterized by the 17 fact that the W and Y blocking groups are removed in Step C 18 by Hydrogenating compound III with hydrogen in the presence of 19 a metal catalyst in a water-water miscible solvent system. 20 21 22 23 5 .

5. The process of Claim 4 characterized by the fact that the 24 w and Y blocking groups are removed by hydrogenating compound III 25 with hydrogen in the presence of a metal catalyst selected from 26 palladium, platinum, Raney nickel, rhodium, ruthinium or nickel, 27 in a water-water miscible solvent system selected from water and 28 dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, or 29 propyleneglycol dimethyl ether, in the presence of a catalytic 3° amount of glacial acetic acid. - - 6 .

6. The process of Claim 5 characterized by the fact that the W and Y blocking groups are removed by hydrogenating compound III with hydrogen in the presence of a palladium on charcoal catalyst, in a solvent comprising 1:1 water-dioxane . .

7. A compound having the formula in -) -γ-amino-a-hydroxy- butyryl or L-(-)-7-benzyloxycarbonylamino-a-hydroxybutyryl, R^ is OH or NHg wherein Rx or R must be other than Hj or a nontoxic pharma¬ ceutically acceptable acid addition .salt thereof. 0 1 II

8. The compound of claim 7 wherein R is CgH^CH^O-C-, R^ is H and R is NH . 2 .

9. The compound of claim 7 wherein R1 is CgH^CHg-O-C-, R2 is H and Rr is OH.

10. The compound of claim 7 wherei is _ 3 L-(-)-7-benzyloxycarbonylamino-a-hydroxybutyryl and Br is OH. 0

11. The compound of claim 7 wherein R 1 is CgH^CHg-O-C11-, R2c is 3 L-(-)-Y-benzyloxycarbonylamino-a-hydroxybutyryl and R is NHg . 1 2

12. The compound of claim 7 wherein R is H, R is L-(-)-y-amino 3 a-hydroxybutyryl and R is OH; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

13. The compound of claim 7 wherein R1 is H, R2 is L-( - ) -γ-amino a-hydroxybutyryl and R^ is NHgj or a nontoxic pharmaceutically acceptable acid addition salt thereof. 1 .

14. The monosulfate salt of the compound of claim 12.

15. The monosulfate salt of the compound of claiml3«

16. The disulfate salt of the compound of c3ai! 3 .

17. The disulfate salt of the compound of c3 elm 13. 1$.

18. The mono or polyhydrates of the compound of claim 16. 1§.

19. The mono or polyhydrates of the compound of claim 17.

20. The mono or polyhydrates of the compounds of claim 12

21. The mono or polyhydrates of the compounds of claim 13 ,