IE42179B1 - Kanamycin derivatives - Google Patents

Abstract

Semisynthetic derivatives of kanamycin A and B of the formula IV in which R3 and R have the meaning stated in Claim 1 are prepared. The process starts from corresponding compounds with R = hydrogen and comprises acylation with an acylating agent of the formula VII in which W and M have the meaning stated in Claim 1, and subsequent elimination of the amino protective group W. A preferred acylating agent of the formula VIIa is obtained in situ by mixing the appropriate N-protected carboxylic acid with equimolar amounts of N-hydroxy-5-norbornene-2,3-dicarboximide and dicyclohexylcarbodiimide as dehydrating agent. The said compounds have antibacterial activity and can therefore be used in medicines or as additives to animal feedstuffs.

Description

The present invention relates to semi-synthetic derivatives of kanamycin A and B.

According to the present invention there is provided a compound of the generic formula in which R3 is -OH and R is L-(—>{3-amino-a-hydroxypropionyl or L-(—)iS-amino-a-hydroxy-valeryl, or R is -HHj and R is L-,)-β-amino-a- hydroxypropionyl; or a pharmaceutically acceptable acid addition salt thereof.

These compounds are known as l-N-[L-(—)-13-amino-ahydroxypropionyl]-6'-N-methylkanamycin A or B, and 1-N-£L(—) -δ-amino-a-hydroxyvaleryl]-61-N-methylkanamycin A.

It is known that some R-factor mediated kanamycinresistant organisms, for example E. coll K-12R-5 and Ps.

X5 aeruginosa GN 315.inactivate the kanamycins by 6'-Nacetylation. The compounds of the present invention are derivatives of kanamycin A and B which are resistant to this type of enzymatic inactivation but still retain the majority of their biological activity and spectra.

The compounds of the invention are the compounds of formula IV in which o 1) R is L-(—)-β-amino-a-hydroxypropionyl and R is OH (IVc); 2) R is L-(—)-β-amino-a-hydroxypropionyl and R is NH2(IVd); - 2 42179 3) R is L(-)-δ-amino-a-hydroxyvaleryl and R is OH (IVe); or a pharmaceutically acceptable acid addition salt thereof.

By the term pharmaceutically acceptable acid addition salt as used herein is meant a mono-, di-, tri- or tetrasalt formed by the interaction of 1 molecule of compound IV with from 1 to 4 molecule of a pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine-containing pharmaceuticals.

The preferred salts are the sulfate, hydrochloride, acetate, maleate, citrate, ascorbate, nitrate and phosphate salts of the compounds of formula IV, the most preferred salts being the mono- and di-sulfate salts.

The present invention included within its scope a pharmaceutical composition which comprises at least one compound of formula IV, or a pharmaceutically acceptable acid addition salt thereof, together with a pharmaceutically acceptable diluent or carrier.

The present invention also provides a process for the preparation of a compound having the formula HO HO IV 1^2179 wherein R is as above defined or L- (-—) γ-amino-cthydroxybutyryl and R3 is -OH or -NH2 which comprises the consecutive steps of A) acylating the compound having the formula in which R^ is -OH'or with an acylating agent having the formula OH W-NH-(CH2)n-CH-C-M (VII) in which W is wherein X is a halogen atom, M is and n is an integer of from 1 to 3; in a ratio of at least 0.5 mole of compound VII per mole of compound II, W in which n, R and W are as above defined; and B) removing the blocking group W from compound III by a method known per se, i.e. a method in actual use or described in the chemical literature.

The acylating agent having the formula CII 0 I II W-NH-(CH2) -CH-C-M used in this process is preferably a compound in which W is M is and n is an integer of from 1 to 3. 43179 Preferably the mole ratio of acylating agent to the compound of Formula II is in the range of from 0.5 to 1.4 and most preferably is in the range of 0.8 to 1.1.

The solvent used in process step (A) is preferably a mixture of water and ethyleneglycol dimethyl ether, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran or propyleneglycol dimethyl ether, aqueous tetrahydrofuran being particularly preferred.

When W is a group of the formula the removal of this group in step B is preferably effected by hydrogenating compound III, e.g. in the presence of a metal catalyst, generally a palladium, platinum, Raney nickel, rhodium, ruthenium or nickel catalyst, preferably palladium, and most preferably palladium on charcoal, in a water-water miscible solvent system, generally water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether or propyleneglycol dimethyl ether, but preferably at 1:1 water-dioxane mixture, and most preferably in the presence Of a catalytic amount of glacial acetic acid, to produce the compound of Formula IV, The most preferred process for the preparation of compounds having formula IV comprises the consecutive steps of A) acylating the compound having formula II 5 with an acylating agent having the formula OH 0 I |) -NH- (CII„) „ -C—C — 0 — η h in which n is an integer of 1 to 3,in an aqueous tetrahydrofuran or aqueous dimethylformamideacetone solvent system, at about ambient temperature to produce the compound having the formula - 8 42179 I in which n is as above defined; and B) hydrogenating compound III' in aqueous tetrahydrofuran in the presence of palladium on charcoal at about atmospheric pressure to produce compound IV.

Alternatively, in step A, the acylating agent VII may be generated in situ by mixing the compound having the formula \yCH2-O-C-NH-(CH2)n-C—C-0H with equimolar quantities of N-hydroxy-5-norbornene2,3-dicarboximide and dicyclohexylcarbodiimide e.g. in a small quantity of anhydrous tetrahydrofuran. The - 9 1 resultant mixture is filtered and the filtrate added to an aqueous tetrahydrofuran solution of the 6'-Nkanamycin A or B to produce the appropriate compound III.

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 Gramnegative bacteria.

The compounds IV, when administered orally, are useful as an adjunctive treatment for preoperative sterilization of the bowel. Both aerobic and anaerobic flora which are susceptible to these drugs are reduced in the large intestine. When accompanied by adequate mechanical cleansing, they are useful in preparing for colonic surgery.

The present invention includes within its scope a method of combatting a bacterial infection in a non-human animal which method comprises administering to the animal an anti-bacterially effective amount of at least one compound of Formula IV or a pharmaceutically acceptable acid addition salt thereof, or of a pharmaceutical composition containing a compound of Formula IV or a pharmaceutically acceptable acid addition salt thereof.

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 to four times a day. Generally the compounds are effective when administered to a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

The compounds of formula IV, all possess substantially improved activities . against a wider spectrum of microorganisms as compared to the parent compounds from which they are derived, i.e., kanamycin A and B.

Illustrated below is Table I showing the minimal inhibitory concentrations (MIC's) of kanamycin A and B and BB-K28, 142, 148, 162 and 163 against a variety of Gram-positive and Gram10 negative bacteria as obtained by the Steers agar-dilution method on Mueller-Hinton Agar Medium.

’I 42179 VO VO vo m vo H H’O o H vo o h o o o non Ο O O O o o o Η H H Η Η H H A . Λ Λ A Λ mmoooooooo ΝΓΊΟΙΩΟΙΓΙΐηοΟΟ xr co m χτ H ooovoomooooo o in o o o Η H H H A A A cm vo in co m * · o t · ooh cMvoocMotnmooo O f-i OHONNiGOO H Η Η Η H Λ Λ A A tt I I m| m vo| ra hi mminminmmm H io m in h vocMCMvoCMvovovoooom ho vo cMmocMinocvioo ο Η ϊμ m h in o H Λ tt I cm! fQ ID I iQH| m m in h m m η h H co in m m H lovocMmioiommooom o cm vo vomocMininvomo in in o H Ο H CM CM W τ «Η HHHCMxT-TTxTinm ·*··*···«· mmmooooocMCMO h H in * A co vd h xt m in m in in o in h m ο o vo cm vo cm cm o c Η H in in A tt I I cm! q *t| CQ HI *3’03iocM^’^p*j'Comm cm in vd H’i OOHOOOOOVOVOO* in CO CM H H moocMincMCMNoo OHCMHHHWO Η H 1 *3* co co cm *T xf T co co m XT VO CD H CM m m h i—ί in CO I o o o o o o o o o o vo o H O m o m vo vo m m CM CM 1 CM H m m in •*4· in «Τ m VO co O σι O MO in H VO vo CM VO VO co co vo cn CO H o in η H CM O H mm m VO VO VO VO C- 03 VD o m η Γ- r* m vo in ο o io o o o o o o σ o σι in co o o o H CM CM cn CM CM CM CM CM CM CM CM m σι H CM CM CM CM < < rf < < < < < «; < H ft H < σι ft < ft < ft 1 Q tt u (ft . in d nj1 m K ( Φ CM Φ ω b.-t«> I tt nJ CM o n w -g h s Γ- O Γ*· H w c κ Pi H 3 1 2 O r* CM C 0 •H I VO VO VO U) n α o tn 2 £ , S £ o O >,1 d O CJ H X nt U ·*-* *·· H CM m r- VO CM (U β GJ Ο H H Γ m r* VO H c nJ ϋ 1 & tt vo tt 1 II • R « tt z b 2£ b tt • V) • • a) ω H W5 * S c X s X X X w x ω tt X c = c - C O CM m cn o m in vo o nJ η m in r* co cn in in in vo in H co H m *T in η H CM CM nv 1 I 1 1 '1 1 1 1 1 1 1 t I 1 1 I M ο ϋ ϋ ο 0 ϋ 0 n □ ϋ ϋ o Pj r nJ nJ m nJ nJ nJ (0 nj O W W W W M tt tt w «WWW tt tt w tt tt tt tt tt tt tt tt w I I 42179¾ I HO Ο O in o H H H KO • · · ΓΠ ΟΟσι H OO r-l H Λ Λ tt I <*i mto tt H ιΛ xj* oo co CM co O 00 co · · • • « • • · O G O o o o o o o o o o o to o o o H Η H Λ V Λ Λ TABLE I (oon't) tt I CM tt VO WH tt I CO tt«* ttH tt I CM cw WH tt I co tt CM H m KO to CO H ΚΟ Η • · • * • • • · n vo H to CM o o moOHrt CM H IO oo HH Λ Λ η ώ VO to to *a* Η ΚΟ Η • · • • · • « • · CO VO * H CM CM o o nooHtn Η H to ο ο rid Λ Λ. to CM 00 KO Ό H 00 KO to o 00 XT co « · · • • · • · • • · OHH O Ο H CM O Ο Ο Ο ο ο Η V to to Λ Λ IO xj· KO 00 xf KO KO CO o 00 «Μ* οο • · · • • · • · • • · OHO o Η H K0 o ο ο ο ο ο V Ο Ο ΙΟ cm co ω CM 00 ΚΟ Η Ο 00 CM 00 • · · * • · • · • • · ο ο ο ο Ο Η η ο ο ο ο ο ο ‘V ο ο Η H Λ Λ 03 O νο χΐ< η KO Π xi· ro to to H CM CM xr to to to O O 03 Cl CB Ol H CM CM H ri ri ri CM tt I ·*· £ tt cn i * x W to ή Η H ΒΊΟ _ HMM 3H 0 >6E • · · « M k A A & tt Η H i I I > E tn 04 Cu 0» •H 04 Ο «Μ* •rl G) •H 04 *** 41 id c 03 (X to h c to ο i •r4 o Λ Id 03 CJjCiMg H KO s n Οι M Ί·Η 4J tt CO tt CM I id CO 3 © 03 <ί H H I I 1 I rt Λ o >1 gs S 3 S H CM Pl Η H I I I I I KO VO VO Ol w aaass - 13 1 From our vast experience with kanamycin Λ and B and its chemical reactivity, it was expected that of the 4 or 5 primary amine functions found in kanamycin Λ and B, respectively, that the 6’-amino function is the most reactive -for steric reasons. It was, therefore, postulated that when the 6'-amino function was converted to a 6’-N-methyl secondary amine, its reactivity with an acylating agent would be substantially I decreased and the 1-N primary amine function would be the most reactive of the remaining primary amino functions for steric reasons. Therefore, fi’-N-methylkanamycin A or B was directly acylated with an appropriate side chain acylating agent without first blocking the other amine functions of the molecule. Control of the molar quantities of acylating agent used determines the degree of acylation occurring at other positions. .-„41 STARTING MATERIALS Reparation of L-(-)-Y~benzyloxycarbonylamino-g-hydroxybutyric acid (VI). 1) L-(-J-y-amino-a-hydroxybutyric acid 5 (7,4 g., 0,062 mole was added to a solution I 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 carboben£0 zoxy chloride and the mixture was stirred 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 extracts were combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g. (74%) colorless plates; melting point 78.579.5® C., [a]D — 4.5® (c. = 2, CHjOH). Infrared (IR) (KBr]; IR (KBr) yc=o 1740, 1690 cm.-1 Nuclear magnetic resonance (NMR) (acetone-dg) 6 25 (in p.p.m. from TMS) 2.0 (2H, m.), 3.29 (2H, d-d, J=6.7 and 12 Hz.), 4.16 (1H, d-d, J=4.5 and 8 Hz.), 4.99 (2H, s.), 6.2 (2H, broad), 7.21 (5H, s.). ’ 42179 Analysis - Calcd. for ('^2ΙΙ15Νθ5 1Percent): C, 56.91; Π, 5.97; N, 5.53. Found (percent): C, 56.66; II, 5.97; N, 5.47. 2) N-hydroxysuccinimide ester of L-(-)-ybenzyloxycarbonyl ajnino-a-hydroxybutyric acid (VII). - A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 mole) of N-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to 0° C. and then 8.6 g. (0.042 mole) of dicyclohexylcarbodiimide 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., M.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%). [ 2, CIIC13). IR (KBr) yc=o 1810, 1755, 1740, 1680 cm-1. NMR (acetone-dg). 6(in p.p.m. from TMS) 2.0 (2H, m.), 2.83 (4H, S.), 3.37 (211, d-d, J=6, ,5 and 12.5 Hz.), 4.56 (IK, m.), 4.99 (211, s.)r 6.3 (2H, broad), 7.23 (411, s.). - 16 42179 Analysis. - Calc'd. for ci6HiaN2°7 (percent): C, 54.85; H, 5.18; N, 8.00. Found (percent): C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12. 3, Preparation of N-(benzyloxycarbonyloxy)5 succinimide. - N-Hydroxysuccinimide2 (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 34 g. (0.2 mole) of carbobenzoxy chloride with water10 cooling and then the mixture was stirred at room temperature overnight to separate the earbobenzoxy derivative which was collected by filtration, washed with water and air-dried. Yield 41.1 g. (82%). Recrystallization from benzene-n-hexane 15 (10:1) gave colorless prisms melting at 78-79° C. 4) Preparation of 6'-carbobenzoxykanamycin A. A solution of 42.5 g. (90 mmoles) of kanamycin A free base in 450 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 mmoles) 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.

G. W. Anderson et al., J. Am. Chem. Soc., 86, 1839 (1964).

The reaction mixture was evaporated under reduced pressure below about 50° C. The oily residue I was dissolved in a mixture Of 500 ml. water and 500 ml. butanol, the mixture being filtered to 1 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 lo current distribution. The aqueous layers were combined and evaporated to dryness under reduced pressure to give an oily 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 ninhydrin as the spray reagent.

The crude product was dissolved in 300 ml. of water and chromatographed on a column (30 mm. diameter) of Amberlite CG-50 ion-exchange resin (NH^+ type, 421 it500.). (Amberlite is a Registered Trade Mark).

The column was irrigated with 0.1 H ammonium hydroxide solution and the eluate was collected in 10-ml. fraction. The desired 5 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. (45%) of a colorless product 6carbobenzoxykanamycin A (II) [6·-Cba-kanamycin A], which began to melt and color at 204? c. and decomposed at 212® C. with gas evolution, Iu)D + 106® (e.-2, H20). i 42179 c Ή υ § β *r rH ^J· o © co in l-i O CM* XT CM VO f* CM CM o σι © co CM o rH . I 1 i 1 1 1 ( t t) a—* 1 1 1 β β 1 I 1 ♦H 1 1 O k 1 1 1 •H TJ 1 1 1 k >« 1 1 & Λ 1 1 1 MM β 1 Ό· 1 H *4 o 1 1 β X CM aa in a 10 o i ax. 1 «· CM Φ *r a o a K β in o CM 1 O 0 CM o *—* a v k Cm < o S3 xi 1 O m· a 4J K 0) CM a a β O g β a a o ti CP Φ •H I • 1 4> *u CM a o S3 a** >i rtf W •H r4 o 1 o o X3 U > k •a 0 X —n VO •H in >1 ro < o ·» Ό H CU a· 1 0 ·» I in Φ **4 •U 1 © a) X NC σ • «Ρ « β i 5* r4 β 1 ·· 1 H • k ϋ Φ O ·· Q COO· 0 ·· β 0 Φ > k in •U r-4 S in •H β 4-» U r4 CU r4 Φ O --IO •rl 0 P o ( *-* 0 a *-O a a Ω t* tt β < o < H CM CO The final product was found to be accompanied hy 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 compound IV.

) Preparation of L-(-)-γ-amino-ct-hydroxybutyric acid from ambutyrosin A or B or mixtures thereof.

Ambutyrosin A (5.0 gm.) [U. S, Patent No. 3,541,078, issued November 17, 1970] was refluxed with 160 ml. of 0.5 N sodium hydroxide for one hour. The hydrolysate was neutralized with 6 N HC1 and chromatographed on a column of CG-50 (NH4+type), The desired L-(-)-Y-amino~ct-hydroxy15 butyric acid was isolated by developing the column with water and removing the water by freezedrying. The Ii-(-)-γ—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, 0. S. Patent No. 3,541,078]. 6) Preparation of 6'-carbobenzoxykanamycin B.

To a chilled solution of 8.1 g. (0.0168 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. (0.0168 mole) of N-(benzyloxycarbonyloxy)succinimide in 40 ml. of 1,2-dimethoxyethane. 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 aqueous layer was separated and adsorbed on a column of 100 ml. of Amberlite CG-50 (NH^+type). The column was washed with 200 ml. of.water, eluted with 0.05 N NHjOH. The eluate was collected in 10-ml. fractions. Fractions 121 to 180 were collected, evaporated and freeze-dried to gxve 1.58 g (15%) of the desired product.

Fractions 1 to 120 were evaporated and re-chromatographed on CG-50 (NH4+) to give 1.21 g; (12%) 15 of the product. M.P. 151-152“ C. (dec.). ta)D24 + 104° ,(c. = 2.5, H20). yc=o 1710 cm.·1.

Analysis. - Calc’d. for C26H43N5°12 (Pereent)· C, 50.56; H, 7.02; N, 11.34. Found (percent); 2Q C, 50.71; H, 7.38; N, 11.48.

TLC (silica gel F254), Rf 0.03 in n-PrOH-pyridineAcOH-HjO (15:10:3:12); Rf 0.16 in acetone-AcOH-Hp (20:6:74). 7) Preparation of L-(-)-Y-amino-a-hydroxybutyric acid from DL-g-hydroxy-y-phthalimidobutyric acid.

A) Dehydroabietylammonium L-a-hydroxy-γphthalimidobutyrate: To a solution of 25 g. (0.1 mole) of a-hydroxy-y-phthalimidobutyric acid3 in 200 ml. of ethanol was added a solution ·— Μι Λ 42178 Of 29 g. (0.1 mole) of dehydrobietylamine in 130 ml. of ethanol. The solution was shaken vigorously for a minute and stood at room temperature for five hours,during which time fine needles crystallized out. The crystals were collected by filtration, washed with 50 ml. of ethanol and air-dried to obtain 30.1 g. (56%) of a diastereomer of the dehydrobiefchylaraine salt. M.P. 93-94’ C. £„124 + 15 D (c, « 2.5, MeOH). Recrystallization from 300 ml. of ethanol gave 23.2 g. (43%) of the pure product, M.P. 94-95“ C. (α]ρ24 + 10.8“ (c. = 2.5, MeOH). Further recrystallization did not change the welting point and the specific rotation. analysis - Calc’d. for C32H42N2°5’H2° (percent)s C, 69.54» H, 8.02» N, 5.07. Found (percent): C, 69.58» H, 8.08» N, 5,07.

B) I,-(-)-γ-amino-g-hydroxybutyric acid: TO a solution of 1.5 g. (0.014 mole) of sodium carbonate in 40 ml. of water were added 5.3 g, (0.01 mole) of dehydroabiethyammonium Lo-hydroxy-Y-phthalimidobutyrate and 60 ml. of ether. The mixture was shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 20-ml. portions of ether and Ί ...J 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 Amberlite 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 ninhydrin positive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered off and dried in a vacuum desiccator to give 0.78 g. (66%) of L-(-)-yamino-a-hydroxybutyric acid. M.P. 206-207° C. [a]2^ -29° (c. = 2.5, H_O). The IR spectrum was D 2 identical with the authentic sample which was obtained from ambutyrosin. 8) Preparation of L-g-Benzyloxycarbonylaroino-ahydroxypropionic Acid, XX, L-p-Amino-a-hydroxypropionic acid* (8.2 g., 0.078 mole) was dissolved in a solution of 6.56 g. (0.0164 mole’) of sodium hydroxide and in 60 ml. of water. To the stirred solution was added dropwise 14.7 g. (0.086 mole) of carbobenzoxy chloride below 5° C. The mixture was stirred for an hour at room temperature, washed with 60 ml. of ether and adjusted to pH 2 with dilute HC1. The precipitate was collected by filtration, washed with water and air-dried to give 9.65 g. (52%) of XX. The filtrate was extracted with five 100-ml. portions of ether. The ethereal solution was washed with water, dried over sodium sulfate and evaporated to dryness in vacuo to give an additional 2,0 g. (11%) of XX. A total of 11.65 g. of XX was crystallized from 500 ml. of benzene-ethyl acetate (4il) to give 9.36 g. (50%) of pure XX, m.p. 128.5129.5· C. Infrared (IR) (KBr)s γΟβθ 1745, 1690 cm*1. (a]p5 + 2.9® (c 5.0, MeOH). Nuclear Magnetic Resonance Spectra [NMR (DMSO-dg)Js δ (in ppm) 3.053.45 (2H, m, CH2N), 4.05 (1H, d-d, -O-CH-CO-), .03 (2H, s, CH2Ar) 7.18 (1H, broad, NH), 7.36 (5H, spring H).

Analysis calc’d. for c, 55.23; •H, 5.48; N, 5.86. pound: C, 55.34; H, 5.49; N, 5.87.

*K. Preudenberg, Ber., 47, 2027 (1914). 9) N-Hydroxysuccinimide Ester of L-g-benzyloxycarbonylamino-a-hydroxyprOpionic Acid XXI.

To a chilled and stirred solution of 478 mg. (2 m. moles) of XX and 230 mg. (2 m.moles) of Nhydroxysuccinimide in 10 ml. of tetrahydrofuran (THF) was added 412 mg. (2 ra.moles) of dicyclohexylcarbodimi.de. The mixture was stirred for an hour at 0-5“ C., for two hours at room temperature and then filtered to remove the N,N’-dicyclohexylurea. The filtrate containing the title product was used for the next reaction without isolation.

) Preparation of L-fi-Benzyloxycarbonylamino-ahydroxyvaleric acid xxii.

To a stirred solution of 400 mg (3.0m moles) of L-6-amino-a-hydroxyvaleric acid**and 250 mg (6.5 m moles) of sodium hydroxide in 25 ml of water was added dropwise 580 mg (3.3 m moles) of carbobenzoxy chloride over a period of 30 minutes at 0-5° C. The mixture was stirred for an hour at 5-l5“C, washed with 25 ml of ether, adjusted to pH 2 2179 with hydrochloric acid and extracted with three 30-ml portions of ether. The combined ethereal solution was shaken with 10 ml of a saturated sodium chloride solution, dried over anhydrous sodium sulfate and evaporated in vacuo to give crystals which were recrystallized from benzene to yield 631' mg (78%) of XXII, mp 110111° C,; infrared spectrum [IR(KBr)]: 3460, 3350, 1725, 1685, 1535, 1280, 730, 690cm*·1.

Nuclear magnetic resonance spectrum [NMR (acetone-dg)]: ΰ (in ppm) 1.70 (4H, m) 4.14 (2H, q, J=4.5Hz), 4.19(1H, m), 4.82(2H, s), 6.2(3H, broad), 7.25(5H, s). [a]25 + 1.6 (c 10, MeOH).

Analysis calc’d. for C^H^NO,-: C, 58.42; H.6.41; N, 5.24.' Found: C, 58.36; H, 6.50; N, .27.

**S. Ohshiro et al., Yakugaku Zasshi, 87, 1184 (1967). .mA ilWBErir .....J - 42179 11) N-Hydroxysuccinimide ester of L-6-benzyloxycarbonylamino-a-hydroxyvaleric acid XVIII.

To a stirred and chilled solution of 535 mg (2.0 m moles) of XXII and 230 rag (2.0 m moles) of N-hydroxysuccinimide in 55 ml of ethyl acetate was added 412 mg (2.0 m moles) of N, N’-dicyclohexylcarbodiimide (DCC). The mixture was stirred for 3 hours at room temperature and filtered to remove precipitated N, N'-dicyclohexylurea. The filtrate was evaporated in vacuo to yield 780 mg (100%) of viscous syrup XXIII. IR(Neat): YC=O 1810, 1785, 1725 cm1. \ · · ---------Ί - 28 a III ir Description of the Preferred Embodiments Example 1 61-N-Methylkanamycin A (BB-K 25) by Method A 1. To a suspension of 610 mg of LiAlH^ in 5 10 ml of dry dioxane was added dropwise at 70° C a suspension of 618 mg of 6’-N-carbobenzoxykanamycin A (g’-N-Cbz-kanamycin A) in 30 ml of dry dioxane. The mixture was stirred at 70® C. for 20 hours and then cooled to -5 - 0° C, To the reaction mixture was added cautiously about 20 ml of cold water. After the addition was completed, the mixture was neutralized with 2 N HC1 and then evaporated into dryness under reduced pressure. The residue was washed with a large amount of EtOH to give 536 15 mg Of the crude product, which was dissolved in a small amount of water and chromatographed on a column of CG-50 ion-exchange resin (NH.+ type, i 4 ml). The column was irrigated with water, 1 L of 0.1 N NH4OH, 600 ml of 0.2 N NH^OH and finally 500 ml of 0.5 N NHjOH. Ten milliliter fractions were collected and subjected to a ninhydrin spot test, disc assay (B. subtiljs PCI 219) and TLC (thin flayer ohronotography) silica gel plate; solvent system , S-110 (CHCl3-MeOH-28% NH^OHHjO c*l:4:2:l). The fractions which gave a ninhydrin-positive and bio-active spot at Rf 421^9 0.50 by TLC wepe combined and evaporated into dryness under reduced pressure to give 137 mg (27%) BB-K 25, m.p. 183-187°C (dec.).

NMR(D2O): δ (ppm), -2.51 (3 H, s, β'-Ν-Ο^), 4.95 (1H, d, 4 Hz, 1-H), 5.01 (1H, d, 3 Hz, l’-H).

• Analysis Calc’d. for cj, θΗ^Ν^Ο^.ΐ^άοj ί C, 42.85; 11,,7.19.- N, 9.99, ' Found: C, 42.97; H, 7.27; N, 9.63. 2. To a suspension of 8.8 g of LiAlH^ in 100 ml of dry dioxane was added a suspension of 9.0 g of 6’-N-Cbz-kanamycin A in 200 ml of dry dioxane and the reaction mixture was stirred for 4 days at 80® C. ‘The,reaction mixture was cooled to 15 10° C, treated cautiously with 200 ml of water and filtered to remove insoluble material. The filtrate was neutralized with N HC1 and evaporated in vacuo. The residue was washed several times with EtOH and dissolved in a small amount of water.

The aqueous solution was chromatographed on a column of CG-50 (NH4 + , 200 ml), which was washed with 100 ml of water and eluted successfully with 2.0 L of 0.1 N NH4OH and 1.5 L .of 0.2 N NI^OH.

The eluate was collected in 20-ml fractions. v .

Fractions 119 to 144 showed a bioactive and ninhydrin 1 positive spot at Rf. 0.45 in TLC (silica gel plate; CHCl3-CH-28% NH4OH-H2O =1:4:2:1). They were combined, concentrated under reduced pressure and finally lyophilized to give 1.139 g (16%) of BB-K 25 which was identical with that prepared in Method A-(l).

Example 2 C-N-Methylkanamycin A (BB-K 25) by Method B To a stirred suspension of 618 mg (1 m mole) of 6'-N-Cbz-kanamycin in 30 ml of dry pyridine were added -7 ml of trimethylchlorosilane and 14 ml of hexamethyldisilazane at 70®C, The reaction mixture was stirred overnight at the same temperature and evaporated in vacuo. The residue was treated with dry tetrahydrofuran (THF). The insoluble material was filtered off and washed with dry .THE. The filtrate and the washings were combined and evaporated in vacuo to give 1.567 g of the trimethylsilylated product, which was dissolved in 30 ml of dry THF. The solution was added to a suspension of 758 mg of lithium aluminum hydride in 70 ml of dry THF. The mixture was refluxed for 22 hrs. under stirring. After cooling to ca. 0eC., the reaction mixture was treated cautiously with 20 ml of ice-water and filtered to remove1insoluble material. The filtrate neutralized with 6 N HCl and evaporated to dryness in vacuo. The residue was washed several times with ethanol, dissolved in a small amount of water and chromatographed on a column of CG-50 (NH^ +, 50 ml). The column was washed with 50 ml of water and eluted successively with 1 L of 0.1 N NH^OH and 600 ml of 0.2 N NH^Oh. The eluate was collected in 20-tnl fractions and monitored as described in Method A-(l). Fractions 47 - 72 which gave a ninhydrin positive and bioactive spot at Rf 0.50 by TLC were combined, evaporated under reduced pressure and lyophilized to give 258 mg (52% from 6 *-Cbz-kanamycin A) of L0 BB-K 25, m.p. 183-187°C.

Example 3 1-N-(L(-)-γ-amlno-g-hydroxybutyryl)-6'-N-methylkanamycin A (BB-K 28).

To a solution of 750 mg of 6'-N-methylkanamycin A 15 (BB-K 25) in 30 ml of 60% aq THF was added 525 mg of N-hydroxysuccinimide ester of N-Cbz-L-γ -amino-πhydroxybutyric acid. The reaction mixture was hydrogenatated overnight at room temperature under atmospheric pressure in the presence of 500 mg of % palladium on charcoal. The reaction mixture was filtered and evaporated under reduced pressure.

The residue was dissolved in a small amount of water and adsorbed on a column of CG-50 (NHj +, 70 ml).

The column was washed with water and irrigated successively with 850 ml of 0.1 N ammonia, (tube nos. 1-43 were collected in 20-ml fractions), Tsrrir J 1450 ml of 0.2 N ammonia (tube nos. 44-115 in 20-mlfractions) and finally 100 ml of 0.5 N ammonia (tube nos. 116-215 in 10-ml fractions). Fractions 152 to 161 which showed a bioactive and ninhydrin positive spot at Rf. 0.17 in TLC (silica gel, CHC13-CH3OH-28% NH^OH-HjO = 1:4:2:1) were combined, evaporated under reduced pressure and lyophilized to give 149 mg (16%) of BB-K 28, mp. 187-189°(dec). infrared (IR(KBr)]: VC=O 1650 cnT1.

NMR (D2O): 2.70 ppm (3H, s, N-CH-j).

Analysis calc’d. for C23H45N5°i3*2H2CO3‘ 2H20s C, 39.52; H, 7.03; N, 9.23.

Found: C, 39.26, 39.00? H, 6.69, 15 6.54; N, 9.69, 9.20.

In order to remove a trace of BB-K 11 isdmer (3*-N-acylated isomer), BB-K 28 was subjected to column chromatography with tetramine copper (TACu) type of Amberlite CG-50 ion exchange 20 resin. BB-K 28 (73 mg) was dissolved in a small amount of water and chromatographed on a column of CG-50 (TACu type, 3 ml). The column was washed with 20 ml of water and then eluted with 100 ml of 0.2 N NH.OH, and finally 100 ml of . 1.0 N NH^OH. The eluate was collected in 7-ml fractions and monitored with ninhydrin spot test, 43179 disca'^say (B. subtillis PCI 219 and Pseudomonas aeruginosa) and TLC on silica gel ninhydrin). Fractions 21 - 24 showed a ninhydrin-positive and bio-active (against the P. aeruginosa strain) spot at Rf 0.2. They were combined and evaporated in vacuo to give 30 mg of blue powder. The bluecolored residue (30 mg) was dissolved in a small •amount of water and adsorbed on a column of CG-50 (NH^t, 3 ml), which was irrigated with 20 ml of 10 0.2 N NH4OH and 200 ml of 0.5 N NH^OH. The eluate was collected in 7-ml fractions. Fractions 19-23 showing a positive ninhydrin test were combined, evaporated in vacuo and freeze-dried to give 20 mg of pure BB-K 28; m.p. 187-189° C(dec.).

Example 4 1-N-(L (-)-8-amino-a-hydroxypropionyl]-6'-N-methylkanamycin A (BB-K 162).

A mixture of 218 mg (0.912 m mole) of N-Cb2L-isoserine, 163 mg (0.912 m mole) of N-hydroxy-520 norbornene-2,3-dicarboximide (HONB) and 188 mg (0.912 m mole) of DCC (dicyclohexylcarbodiimide) in 10 ml of THF was allowed to stand at 5° C overnight and then filtered. The filtrate was added to a solution of 441 mg (0.892 ro mole) of 6’-N-methyl25 kanamycin A in 20 ml of 50% aq THF, the mixture was - 34 1 stirred at room temperature for 5 hours and concentrated under reduced pressure to about 2 ml.

The concentrate was adsorbed on a column of Amberlite CG-50 (NH^+, 26 ml), which was washed with 40 ml of water and eluted with 500 ml of 0.1 N NH^OH, The eluate was collected in 10-ml fractions. Fractions 11-16 were combined and evaporated in vacuo to give 231 mg of the Nacylated product. From the 0.3 N NH^OH eluate was recovered 175 mg (40%) of the starting material, BB-K 25.

To a solution of the acyl derivative in 20 ml of 50% aq EtOH was added 130 mg of 10% Pd on charcoal and the mixture was hydrogenated under ordinary pressure at room temperature. The catalyst was filtered off and the filtrate was evaporated to remove the organic solvent. The resulting aqueous solution was subjected to column chromatography on CG-50 (NH^+, 25 ml). The column was eluted successively with 40 ml of water, 240 ml of 0.1 N NHjOH, 500 ml of 0.2 N NH^OH, 300 ml of 0.4 N NH^OH and the eluate was collected in 10-ml fractions, The bio-active fractions were combined and evaporated in vacuo to afford 127 mg of the crude product, which was re-chromatographed on a column of CG-50 (tetramine copper type, 4 ml) and eluted - 35 1 ,1 4217Ό with 200 ml of 0.3 Μ NH.OH, 300 ml of 0.5 N NH.OH 4 4 and finally with 300 ml of 1 N NHjOII. was collected in 10-ml fractions. Tube The eluate No. .Eluaht Amount Rf Remarks 4 0.3 N NH4OH 27 mg 0.33 inactive 19-24 0.5 N NH^OH 38 mg 0.33 inactive, probably a position isomer 50-54 1.0 N NH.OH 4 35 mg 0.37 the desired product, BB-K162 Tube nos.' 50 through 54 were combined and evaporated to dryness to give 35 mg (6.8%) of the desired bio-active product, BB-K 162; m.p. 178-185° C (dec.). 1630 cm-1.

Example 5 1-N-[L(-)-6-amino-ot-hydroxyvaleryl)-6 *-N-methylkanamycin A (BB-K 163).

A mixture of 94 mg (0.353 m mole) of N-Cbz-Lγ-amino-a-hydroxyvaleric acid, 64.5 mg (0.353 m mole) of HONB and 74.5 mg (0.353 m mole) of DCC in 5 ml of dry THF was allowed to stand at 4° C overnight and filtered. The filtrate was added to a solution of 175 mg (0.351 m mole) of 6·N-methylkanamycin A in 10 ml of 50% ag THF and the mixture was stirred at room temperature for 5 hours. The reaction mixture was hydrogenated at room temperature in the presence of 70 mg of 10% palladium on charcoal. The catalyst was filtered off and the filtrate was evaporated to remove the THF. The resulting aqueous concentrate was chromatographed on a column of Amberlite CG-50 (NH4+type, 20 ml), which was eluted successively with water (50 ml), 0.1 S NH4OH (250 ml), 0.2 N NH40H (450 ml) and 0.5 N NH4OH (400 ml). The eluate was collected in 10-ml fractions. The bio-active fractions were combined and evaporated into dryness to afford 40 mg of the crude product, which was rechromatographed on a column of Amberlite CG-50 (tetramine copper type, 1.5 ml) and eluted with 10 ml of water and 100 ml of 0.5 N NHjOH. The eluate was collected 15 in 5-ml fractions. Tube No. Eluant Amount Rf Remark 7-8 0.5 N NH4OH 5 mg 0.21 inactive 9 0.5 N NH4OH 9 mg 0.21, 0.29 a mixture of two components 10-14 0.5 N NH4OH 24 mg 0.29 the desired product, BB-K 163 20 Tube nos. 10 to 14 were combined and evaporated into dryness to give 24 mg (11%) of the desired bioactive product, BB-K 163; m.p. 167- 174° C (dec.). /Br 1620 cm1. C=0 Example 6 6*-N-Methylkanamycin B, BB-K 140 To a 'suspension of 2.0 g (3.25 m moles) Of 61-N-Cbz-kanamycin B in 100 ml of dry pyridine were added 20 ml of trimethylsilyl chloride and ml of hexamethyldisilazane at 70° C. under Stirring and the mixture was stirred overnight at the same temperature. The precipitate was removed by filtration and washed 0 with dry THF. The filtrate was combined with the washings and evaporated to dryness. The oily residue was dissolved in 80 ml of dry THF. The solution was added dropwise to a stirred suspension of 4.4 g of lithium aluminum hydride in 200 ml of 3 dry THF and the mixture heated under reflux overnight.. After cooling, the reaction mixture was treated cautiously with ice water, adjusted to pH 7 with 2 N HC1 and evaporated to dryness under reduced pressure. After washing with ethanol thoroughly, the residue (2.7 g) was dissolved in 5 ml of water and chromatographed on a column of Amberlite CG-50 (NHj+,40 ml). The column was washed with 300 ml of water and eluted with 0.1 N ammonia. The eluate was collected with 10-ml / · fractions and monitored with a ninhydrin spot test, disc assay (B, subtilis PCI 219) and TLC (silica 4317S gel plate, CHCl3-CH3OH-28% NH4OH-H2O = 1:4:2:1). Fractions 70 - 134 which showed a ninhydrin-positive and bio-active spot at Rf 0.47 by TLC were combined and evaporated under reduced pressure to give 889 mg (55 %) of BB-K 140; m.p. 177 -181° C (dec.).

NMR (0,,0): 2.74 ppm (3H, s, N-CI^).

Analysis Calc’d. for C^gHjgNgO^g’i^COg: C, 42.93; H, 7.39; N, 12,52.

Found: C, 42.93; H, 7.13; N, 11.86.

Example 7 1-N-[L(-)-γ-Amino-a-hydroxybutyryl]-61-N-methylkanamycin B (BB-K 142). 1S A mixture of 253 mg (1 m mole) of N-CBz-Lγ-amino-a-hydroxybutyric acid, 115 mg (1 m mole) of N-hydroxysuccinimide (HOSu) and 206 mg (1 m mole) of DCC was stirred for 3 hours at 5°C. and filtered to remove the precipitate which separated during 2o the reaction. The resulting active ester solution was added to a solution of 497 mg (1 m mole) of C’-N-methylkanamycin B (BB-K 140) in 10 ml of water and the mixture stirred overnight at room temperature. >The reaction mixture was filtered and evaporated in vacuo. The residue was dissolved in 5 ml of water and adsorbed on a column of CG-50 -------- 39 i ion-exchange resin (NH^ + type, 45 ml), which was eluted successively with 200 ml of water, 1500 ml of 0.05 N ammonia and 500 ml of 0.3 N ammonia. The eluate was collected in 10-ml fractions. The desired intermediate derivative was contained in fractions 58 to 72 which were eluted with 0.05 N ammonia; while 163 mg (33%) of BB-K 140 was recovered from fractions eluted with 0.3 N ammonia.

A solution of the intermediate derivative in ml of 50% THF was hydrogenated overnight under ordinary pressure in the presence of 30 mg of 10% palladium on carbon. The reaction mixture was filtered and concentrated to about 4 ml.

The concentrate was adsorbed on a column of . Amberlite CG-50 (NH^ + type, 10 ml), which was washed with 150 ml of water and eluted successively with 350 ml of 0.05 N NHjOH, 300 ml of 0.1 N NHjOB, 150 ml of 0.3 N NH^OH and 300 ml of 0.5 N NH^OH. The eluate was collected in 15-ml fractions. Fractions 67 and 68 which showed a bioactive spot at Rf 0.22 on TLC (silica gel plate, CHC13-CH3OH~28%NR4OII-H2O=1;4:2:1) were combined and re-chromatographed on a column of CG-50 (the lower part, NH^ type 1ml; the upper part, tetramine copper type 2 ml). The column was eluted with 100 ml of 0.5 N NH4OH and then with 50 ml of 1.0 N NH.OH and the eluate was collected in 5-ml fractions.. Fractions 15 through 33 were combined and evaporated in vacuo to give 29.4 mg fSS) of the desired bio-active product, BB-K 142; m.p. 188-192° C (dec.). IR(KBr): Vc_,o 1640 cm Analysis Calc’d. for C23H46N6°122H2CO3i C, 41.55; H, 6.97; N, 11.63.

Found: C, 41.52; H, 6.72; N, 10.95.

A small sample of BB-K 142 was heated at 100° C for 1 hour in 0.5 N NaOH to afford 6’-N-methylkanamycin B and L-a-hydroxy-Y-amino-butyric acid which were confirmed by TLC. Example 8 1-N-[L(-)-β-Amino-ot-hydroxypropionyl]-6'-N-methylkanamycin B (BB-K 148).

A stirred mixture of 120 mg (0.5 m mole) of N-Cbz-L-isoserine, 58 mg (0.5 m mole) of HOSu and 103 mg (0.5 m mole) of DCC in 5 ml of THF was allowed to stand at 5° C overnight. The mixture filtered, and the filtrate was added to a solution of 248 mg (0.5 m mole) of BB-K 140 in 5 ml of water and stirred overnight. After removing THF, the aqueous solution ‘was adsorbed on a column of Amberlite CG-50 (NH4+ type, 10 ml). Elution was carried out with 300 ml of 0.5 N NH4OH followed by 300 ml of 43179 0.1 Ν ΝΗ,ΟΗ and 10-ml fractions were collected.

The desired intermediate derivative (68 mg) was obtained by evaporation Of fractions 18 to 31 which were eluted with 0.05 N NH^OH while 69 mg (28%) of BB-K 140 was recovered from fractions eluted with 0.1 N ammonia. i I A solution of the intermediate derivative in 10 ml of water was hydrogenated overnight in the presence of 20 mg of 10% Pd-C and the reaction mixture was filtered and concentrated to about ml. The concentrate was chromatographed on a column of Amberlite CG-50 (NH4+, 8 ml) and eluted successively with 280 mol of 0.05 N NH^OH, 340 ml of 0.1 N NH4OH and 200 ml of 0.2 N NH^OII. The desired bio-active component (35 mg) was obtained from fractions eluted with 0.2 N NH^OH. The product which still showed two to three ninhydrin positive spots was' purified by re-chromatography on a column of CG-50 (the lower part,.NH4 type 1 ml; the upper part, tetramine copper type 2 ml), and then eluted successively with 60 ml of 0.3 N NH^OH, 110 ml of 0.5 N NH^OH and 100 ml of 1.Ό N NH^OH. 5 ml fractions were collected. The desired product, BB-K 148, was obtained from fractions 40 to 50 which were eluted with 1.0 Ν ΝΗ,ΟΗ. - 42 ---1 Yield 8.1 mg. (3 %); m.p. 190198 C. (dec.). IR(KBr): \»C=Q 1630 cm1.

Example 9 1-N-tL-(-)-6-amino-q-hydroxyvaleryl]-6'-N5 methylkanamycin B.

Substitution in the procedure of example 5 for the 6'-N-methylkanamycin A used therein of an equimolar quantity of 6'-N-methylkanamycin B produces the title product.

Amberlite CG-50 is the tradename for the chromatographic grade of a weakly acidic cationic exchange resin of a Carboxylic-polymethacrylic type.

The cupra-ammonium form of Amberlite CG-50 was prepared in the following way: to a stirred suspension of CG-50 (NH^+) in water was added 10% cupric sulfate solution to give the copper Balt of CG-50,which was filtered. The resin was washed several times with water, then treated with IN NH^OH under stirring, filtered and washed several times with water to give deep blue, cupraammonium form of CG-50. 43179 Example 10 Preparation .of the monosulfate salt of 1-[ί.-(-)-γamino-a-hydroxybutyryl]-6'-N-methylkanamycin A or B One mole of 1-[L-(-)-γ-amino-a-hydroxybutyryl]5 6'-N-methylkanamycin A or B is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution, is .added one mole of sulfuric acid dissolved in 500 ml of water. The mixture is stirred for 30 minutes, following Which I I 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 11 Preparation of the disulfate salt of 1-[1.-(-)-8amino-ct-hydroxypropionyl]-61-N-methylkanamycin A or B.

Thirty-five grams of 1-(1-(-)-8-amino-a-hydroxy20 propionyl]-6*-N-methylkanamycin A or B is dissolved in 125 ml. of deionized water. The pH is lowered to 7-7.5 with 50% v./v. sulfuric acid.

Eight and one-half grams of Darco G-60 (activated.charcoal) is added and the mixture is —-4 42178 slurried at ambient room temperature for 0.5 hour. (Darco is a Registered Trade. Mark).

The carbon is removed by suitable filtration and washed with 40 ml. of water. The water wash is added to the filtrate.

The combined filtrate-wash above is adjusted to pH 2-2.6 with 50% v./v. sulfuric acid. A large amouht of carbon dioxide is evolved. The solution is left under vacuum with, stirring for 20 minutes to expel additional carbon dioxide.

Eight and one-half frams of Darco G-60 are added to the degassed solution. The mixture is slurried for 0.5 hour at ambient temperature.

The carbon is removed by suitable filtration and washed with 35 ml. of deionized water. The wash is added to the filtrate.

. The combined filtrate-wash is adjusted to pH 1-1.3.with 50% v./v. sulfuric acid. This solution is added with rapid stirring over a 10 minute period to 600-800 ml. of methanol (3-4 volumes of methanol). The mixture is stirred for 5 minutes at pH 1-1,3, passed through a 100 mesh screen, stirred for 2 minutes and allowed to settle for 5 minutes. Most of the supernatant is decanted. The remaining slurry is suitably filtered, washed with 200 ml. of methanol and vacuum dried at 50° C. for 24 hours to yield the title disulfate salt.

Claims (29)

1. CLAIMS: Γ) L-(—)-ύ-amino-w-hydroxyValery1, or R 3 is -NH 2 and R is L-(—) -β-amino-a-hydroxypropiony1.
2. 2. A compound as claimed in claim 1 wherein R is -OH and R is L-(—)-B-amino-a-hydroxypropionyl. 3. .
3. 3. A compound as claimed in claim 1 wherein R xs -NH 2 10 and R is L- (-)-β-amino-α-hydroxypropiony1.
4. 4. A compound as claimed in claim 1 wherein R 3 is -OH and R is L- (—)-δ-amino-a-hydroxyvaleryl.
5. 5. A pharmaceutically acceptable acid addition salt of a compound as claimed in any one of the preceding claims. 15
6. 6. A pharmaceutically acceptable acid addition salt as claimed in claim 5 which is the mono- or di-sulfate salt.
7. 7. A process for the preparation of a compound of the formula. 20 in which R is as defined in claim 1 or L-(—)-y-amino~a~ hydroxybutyryl and R 3 is -OH or -NH 2 which comprises the consecutive steps of A) acylating the compound having the formula in which R is -OH or -NH 2 , with an acylating the formula agent having OH I W-NH-(CH-) -CH Z n in which W is I CH 2 -O~C-, ch 3 CII, 0 I 3 » CH 3 -C-O-C- J, or 0 0 II I C-CH 2 -CH 2 -C· wherein X is a halogen atom; M is . -η -uo 2 N°2 43179 and n is an integer of from 1 to 3; in a ratio of at least 0.5 mole of compound VII per mole of compound II, in a solvent to produce the compound having the formula in which n and W are as above defined and R is -OH or -NH 2 ; and B) removing the blocking group W from compound III ί by a method known per se to produce a compound of Formula IV
8. 8. A process as claimed in claim 7 wherein Step A is carried out with an acylating agent of the formula OH 0 I P W-NH-(CH 2 ) n -CH— C-M in which W is a radical of the formula
9. 9. A process as claimed in claim 7 or claim 8 wherein the mole ratio of acylating agent to the compound of Formula II is in the range of 0.5 to 1.4.
10. 10. A process as claimed in claim 7 or claim 8 wherein the mole ratio of acylating agent to the compound of Formula II is in the range of 0.8 to 1.1.
11. 11. A process as claimed in any one of claims 7 to 10 wherein the solvent is a mixture of water and ethyleneglycol dimethyl ether, dioxane, dimethylacetamide.
12. 12. A process as claimed in claim 11 wherein the solvent is aqueous tetrahydrofuran.
13. 13. A process as claimed in any one of claims 7 to 12 wherein the blocking group W is a group of the formula •CH 2 “O-Cand the removal of this group in Step B is effected by hydrogenation.
14. 14. A process as claimed in claim 13 wherein the hydrogenation is carried out in the presence of a metal catalyst in a water-water miscible solvent system.
15. 15. A process as claimed in claim 14 wherein the catalyst is palladium, platinum, Raney nickel, rhodium, ruthenium or nickel.
16. 16. A process as claimed in claim 14 or claim 15 wherein the solvent is a mixture of water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether or propyleneglycol dimethyl ethep.
17. 17. A process as claimed in any one of claims 13 to 16 wherein the hydrogenation is carried out in the presence of palladium on charcoal in a 1:1 water-dioxane solvent system and in the presence of a catalytic amount of glacial acetic acid. 48179 Μ in which R is as defined in claim 1 or L- (—) -γ-amino-a3 hydroxybutyryl and R is -OH or -NH 2 which comprises the consecutive steps of A) acylating the compound having formula II, given in claim 7 with an acylating agent having the formula CH 2 -O-C-NH- (CH 2 ) n -C-C-O-N. OH 0 or in which n is an integer of from 1 to 3, in an aqueous tetrahydrofuran or aqueous dimethylformamide-acetone solvent system, at about ambient temperature to produce the compound having the formula 50 4 in which n is as above defined? and B) hydrogenating compound III' in aqueous tetrahydrofuran in the presence of palladium on charcoal at about atmospheric pressure to produce compound IV.
18. 18. 19. A process as claimed in claim 18 wherein the acylating agent of Step A is generated in situ by mixing a compound of the formula A- CH 2 -0-C-NH-(CH 2 ) n OH 0 1 1 ) -C—c~ with equimolar quantities of N-hydroxy-5-norbornene~2,3dicarboximide and dicyclohexylcarbodiimide.
19. 19. 20. A process as claimed in any one of claims 7 to 19 wherein R is L-(—)-γ-amino-a-hydroxybutyryl and R^ is OH. 51 3179
20. 20. 21. A process as claimed in any one of claims 7 to 19 wherein R is L-(—)-amino-a-hydroxypropionyl or R' is OH.
21. 21. 22. A process as claimed in any one of claims 7 to 19 wherein R is L~(—)-fi-amino-a-hydroxyvaleryl and R 3 is OH.
22. 22. 23. A process as claimed in any one of claims 7 to 19, wherein R 3 is NH 2 and R is L-(—)-y-amino-u-hydroxybutyryl.
23. 23. 24. A process as claimed in any one of claims 7 to 19 wherein R 3 is NH 2 and R is 1- (—)-β-amino-a-hydroxypropionyl.
24. 24. 25. A process as claimed in any one of claims 7 to 19 wherein R is NH 2 and R is L- (—)-fi-amino-a-hydroxyvaleryl.
25. 25. 26. A process as claimed in claim 7 substantially as hereinbefore described with reference to any one of Examples 3 to 5 and 7 to 9. I {
26. 26. 27. A compound as claimed in claim 1 whenever produced by a process as claimed in any one of claims 7 to 26.
27. 27. 28. A pharmaceutical composition which comprises at least one compound as claimed In any one of claims 1 to 6 or claim 27 together with a pharmaceutically acceptable diluent or carrier.
28. 28.
29. 29. A method of combatting a bacterial infection in a nonhuman animal which method comprises administering to the animal an anti-bacterially effective amount of at least one compound as claimed in any one of claims 1 to 6 or claim 27 or of a composition as claimed in claim 28.