IL46995A - Process for the preparation of a kanamycin derivative - Google Patents

Description

46995/2 Hovel process for the preparation of a kanamycin derivative BRISTOL-MYERS COMPAHY :44887 This invention relates to a new and novel and substantially more efficient process for the preparation of 1-[L- (-) - Ύ* -amino-a-hydroxy-butyryl] -kanamycin A (IV)·, which compound has the formula Kanamycin A possesses four primary amine functions at the 1, 3, 6' and 3" positions of the molecule. It has been established that the 6' -amine function is the most reactive and the 1-amine function is second most reactive when treated with an electrophylic agent. Both the 3 and 3" positions are less reactive than either the 1- or 6'- amine functions but do react to give low percentages of undesired acylated materials.

Accordingly, it was the object of the present invention to discover a more satisfactory process than that previously employed by our co-workers in the Israel Patent Specification Serial No. 39872 and the published Netherland application No. 7,401,517, said process being more selectively directed to acylation of the The objective of the present invention has been achieved by the process of preparing the compound having the formula which process comprises the consecutive steps of A) treating the compound having the formula with an aldehyde selected from the group comprised of benzaldehyde, salicyladehyde, p-nitrobenzaldehyde, p-methoxybenzaldehyde and pivaldehyde, in a ratio of one mole of compound II to at least three moles of aldehyde to produce the compound having the formula in which Z is a radical of the formula B) treating compound III in situ with the compound having the formula in a ratio of one mole of compound III to at least 0.5 mole of compound XX, and then hydrogenating the residue in situ to produce a compound of formula IV.

In particular. Step A is preferably carried out in a water miscible solvent such as an alcoholic solvent, e.g. absolute ethanol, methanol, n-propanol, isopropanol, i n-butanol, sec-butanol, tet-butanol; an ether solvent, e.g. tetrahydrofuran, dioxane, or other polar aprotic or protic solvents such as, for example, dimethylformaraide and acetone, or mixtures thereof, or mixtures thereof with water at a temperature range of about 5° C. to about reflux, for a period of about 30 minutes to about 5 hours.

Step B is preferably carried out with a ratio of compound III to compound XX of about 1:1, at a temperature range of about -10°C. to about +35°C, but more preferably in a range of +5°C. to about 25°C, for a period of time until acylation is complete, but for at least one to three hours; then removing the organic solvent prior to hydrogenation. The in situ hydrogenation is carried out with hydrogen in the presence of a metal catalyst selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but more preferably palladium, and most preferably palladium on charcoal in water or a water-water from the group comprising wa er an oxane, tetra y ro uran^ ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether and the like, but more preferably water at a pH of 3-5, but most preferably 4.

A more preferred embodiment is the above process for the preparation of compound IV which comprises the consecutive steps of A) treating the compound having the formula with an aldehyde selected from the group comprised of benzaldehyde, salicylaldehyde, p-nitrobenzalde-hyde, and p-methoxybenzaldehyde, in a ratio of one mole of compound II to at least three moles of aldehyde, in absolute ethanol, methanol, n-propanol, sec-butanol, tert-butanol , methyleneehloride, tetrahydrofuran, dioxane, dimethylformamide or acetone, or a mixture thereof with water, in a temperature range of 5° C. to 40° C. for a period of time of about 30 minutes to about 5 hours, to produce the compound having the formula in which Z is a radical of the formula B) treating compound III in situ or after isolation with the compound having the formula XX in a ratio of one mole of conpound III to about 0.5 to 1.0 mole of compound XX, in a range of +59C. to 35°C. for one to three hours, in a solvent system as described in step A; then removing the organic solvent and hydrogenating the residue in situ 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 more preferably palladium on charcoal, in water or a water-water miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran , ethylene-glycol dimethyl ether and propyleneglycol dimethyl ether, but preferably water, at a pH of 3-5 , but preferably 4, to produce the compound of formula IV.

A more preferred embodiment is the process for the preparation of the compound having the formula which comprises the consecutive steps of A) treating the compound having the formula with an aldehyde selected from the group consisting of benzaldehyde i salicylaldehyde and p-nitrobenzaldehyde, in a ratio of one mole of compound II to about three moles of aldehyde, in absolute ethanol, methanol, n-propanol, isopropanol, n-butanol, sec- butanol, tert-butanol , tetrahydrofuran , methylene chloride, dioxane, dimethylformamide or acetone, or a mixture thereof with water, at about 5° C. to 40° C. for a period of time of about 30 minutes to about 5 hours, to produce the compound having the formula B) treating compound III in situ or after isolation with the compound having the formula XX in a ratio of one mole of compound m to about 0.5 to 0.85 mole of compound XX, in a range of +15° C. to 30° C, for one to three hours, in a solvent system as described in step A; then removing the organic solvent and hydrogenating the residue in situ 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 more preferably palladium on charcoal, in water or a water-water miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether and propyleneglycol dimethyl ether, but preferably water, at a pH of about 4 to produce the compound of formula IV.

A most preferred embodiment of the present invention is the process for the preparation of the compound having the formula which comprises the consecutive steps of treating the compound having the formula with benzaldehyde in a ratio of one mole of compound II per three moles of benzaldehyde , in about a 1:1 mixture of water and tetra-drofuran, at about 20° C. to about 30° C. at pH of about 10, for about two to four hours, produce the compound having the formula B) treating compound Ilia in situ or after isol XX in a ratio of one mole of compound II¾ to 0.6 to 0.75 mole of compound XX, at a temperature in the range of about 20° C. to about 30° C. for a period of time of one to three hours, in a solvent system as described in Step A, a mixture of methylene chloride, methanol and water or a mixture of dimethylformamide, acetone and water; then removing the organic solvent in vacuo, adjusting the pH to 4 with ammonium hydroxide and hydrogenating the residue in situ with hydrogen at atmospheric pressure in the presence of palladium on carbon to produce the compound of formula iv.

The most preferred embodiment of the present invention is the process for the preparation of the compound having the formula IV A) treating the compound having the with salicylaldehyde in a ratio of one mole of compound II per three moles of salicylaldehyde in about a 1:1 mixture of water and tetra-hydrofuran, at about 20° C. to about 30° C.» at a pH of about 8, for about two to four hours, to produce the compound having the formula B) treating compound Illb in situ or after isolation with the compound having the formula in a ratio of one mole of compound Illb to about 0.6 to 0.75 mole of compound XX, at a temperature in the range of about 20°C. to about 30°C. for a period of time of one to three hours, in a solvent system as described in step A or a mixture of dimethylformamide, acetone and water; then removing the organic solvent in vacuo, adjusting the pH to 4 with ammonium hydroxide and hydrogenating the residue in_¾itlL with hydrogen at atmospheric pressure in the presence of palladium on carbon to produce the compound of formula IV.

For the purpose of this disclosure, the term (lower) alkyl is an alkyl residue, straight or branched chain, of 1 to 6 carbons. (Lower) -alkanol is a saturated alcohol, straight or branched chain, of 1 to 6 carbons containing one OH function.

Another object of the present invention is a modification of the above-described process using an even more in situ ) process yet affording the same superior yields of the compound of formula IV and at less expense.

For example, the compound of formula XX can be prepared in situ and reacted with the compound of formula III in one convenient step. Thus, the compound of formula IV can be prepared as above described wherein in Step B, the reaction comprises treating compound III obtained in Step A as above-described either .in_ situ or after isolation with the compounds of the formulae VI followed by the addition of dicyclohexylcarbodi- imide, in a ratio of one mole of compound III to about 0.5 to 1.0 moles each of compound Viand dicyclohexylcarbodiimide and about 0.005 to 0.25 moles of compound XIX, in a temperature range of about -10° C. to about +35° C, but preferably in a range of +5° C. to about 25* C, for at least ten hours in a solvent system as described hydrogenating the residue in situ as previously described to produce the compound of formula IV.

A preferred embodiment of the above modification involves the use of a ratio of one mole of compound III to about 0.5 to 0.85 moles each of compound VI and dicyclohexylcarbodiimide and about 0.05 to 0.425 moles of compound xix, in a range of +5°C. to 25°C, for one to three hours in a solvent system as described in Step A, or, alternatively a ratio of one mole of compound III to about 0.5 to 0.8 moles each of compound VI and dicyclohexylcarbodiimide and about 0.05 to 0.425 moles of compound XIX in a range of +15°C. to +30°C, for 15 to 25 hours is employed. Most preferred conditions are a ratio of one mole of compound III to about 0.6 to 0.75 moles each of compound VI and dicyclohexylcarbodiimide and about 0.06 to 0.375 moles of compound XIX in a range of 20° C. to +30° C. for 15 to 25 hours.

Also included as part of the instant invention is a new and improved process for the preparation of the starting material, the compound of the formula Such process comprises treating kanamycin A free base with N-benzyloxycarbonyloxy-5-norbornenev2, 3-dicarboximide, in a ratio of about 0.5 to about 1.5 moles of N-benzyloxy-carbonyloxy-5-norbornene-2, 3-dicarboximide per mole of kanamycin A, or especially in a ratio of about 1:1, followed by purification, for example, by organic solvent extraction with a solvent such as n-butanol and chromatography of the aqueous phase.

The above reaction is preferably carried out in an aqueous-water miscible organic solvent system, such as, for example, tetrahydrofuran-water and more preferably in a concentration of about 40 to 60% tetrahydrofuran, in a temperature range of about 0° to about 25° C. or, more preferably about 5° to about 10° C, for at most 6 hours, but, more preferably, for about 4 hours.

Compound IV, l-u- [L- (-) -7-amino-a-hydroxy- butyryl J kanamycin A (BB-K8) , possesses excellent antibacterial activity that appears superior to kanamycin A itself. Illustrated below are two tables showing the minimal inhibitory concentrations ( IC's) of kanamycin A and compound IV (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.

Compound IV Kanamycin A [BB- 8] Organisa (6-8198) lot No. k Alk. faecalis A-9½3 16 8 II " A-206 8 >125 Ent. cloacae A-0656 » species A-2030A >125 2 n hafniae • 1 A-2C-674 1 1 E. coll A-0636 2 1 1» Α-20β6½ 16 . k n n A-20507 32 2 » " A-2052O > 125 k n n A-20365 >125 1 n " Α-2θβ8÷÷ 2 2 11 n A-20582 >125 2 It n A-20683 >125 8 II ?r A-20o8l . >125 2 n " A-15119 4 K. pneumoniae A-0967 A It species A-20328 >125 2 It " A-20 0 32 32 II " A-2003½ >125 II pneuaoniae Λ-20080 >125 ir " A- 0077 1 1 Pr . c_ira¾ilis Α-99ΌΟ 2 2 - TABLS I (MIC mg./ml.) Cocripound IV Kanarnycin A [BB-K8] Organism (6-8198) lot No.4 Pr. morganii A-I5153 2 2 " vulgaris A-9555 2 1 n refctgeri A-9636 Ο.25 Ο.25 " nirabilis A-206 5 4 Alk. faecalis A-9 23 16 8 " " A-20048 >125 >125 Ent. cloacae A-O656 4 4 " species A-20364 >125 2 " hafniae 1 Α-2θ6γ4 1 1 E. coli A-O636 2 1 It tt A-20664 16 . 4 A-2066'5 >125 1 if ti A-20507 52 2 II II A-20520 >125 n n A-203 5 >125 1 11 A-20684 2 2 tt 11 A-20682 >125 2 A-20683 >125 8 ir A-20681 >125 2 A-15119 4 TABLE I (Continued) Compound IV Kanamycin A [ BB- S ] Organism (6-8198) lot No. *f K. pneumoniae A-0967 ^ " species A-20328 >125 2 " " A-20330 32 32 " " A-2063^ >125 " pneumoniae A-20680 >125 4- " N A-0077 1 1 Pr. mirabilis A-9900 2 2 Pr. norganii A-15153 2 2 " vulgaris A-9555 2 1 " rettgerl A-9636 0.25 0.25 " mirabilis A-206 5 4 4 Pr. mirabilis A-20 5½ 2 2 Provldencia stuartii A-20615 2 1 "· alkalifaciens 1 1 A-20676 Ps. aeruginosa A-2022 32 2 " " A-0 ¾A 125 16 " " A-20653 >125 · 32 " species A-20601 125, 6 16 N " A-2062I >12 >125 maltophilia A-20620 32 >125 Sal. enteritidis A-0531 1 0.5 " derby A-20037 ' >125 1 TABLE I (Continued) Compound IV Kanam c n A Organise! [BB-K8] . lot Wo- ½ Ser. marcescens A-20019 2 4 n n . A- 33 8 " " A-20½0 >125 » 0 A-20 59 16 Shlg. flexneri A-968 k Aeroconas sp. A-2O070 2 2 Arizona sp. A-20671 2 1 Citrobacter sp. A-20673 Edwardsiella sp. A-20678 Staph, aureus A-9606 1 1 " " A-^7 9 0.5 1 A-9537 2 1 " " A-206i0 > 125 ■ 2 " . " A-20240 >125 8 " " A-15197 1 2 Mueller-Hinton Medium +4$ sheep blood Str. faecalis A-985^ 63 63 11 ". A-9575 125 >125 Str. pyogenes A-20200 2, 15 32 " " A-SoOif 125 125 " " A-150^0 125 125 D. pneuaoniae A-9585 63 , 32 63 " " A-2015 125 >125 • TABLE 2 (MIC tng./ml. ) Comoound IV Kanamycin A [BB-K8] Organism (6-8196) lot No.

D. pneumoniae ±5 serum 63 63 A-0585 Str. pyrogenes±5# serum A-9o0f Staph, aureus Saith A-9537 Staph, aureus A-^7 Staph, aureus A-20239 Staph, aureus Α-202* > Enter, cloacae A-0656 Enter, species A-20364 K. pneumoniae A-9807 E. coli K-12 ΜΙΟ ΙΟ A-20361 E. coli'K-12 MLI630 A-20363 E. coli K-12 A-9632 E. coli A-2066 .

E. coli A-20665 Pr. tnirabilis A-9900 Pr. taorganii A-15153 Pr. vulgaris A-9^6 Ps. aeruginosa A-20227 Ps. species A-20^99 Ps. aeruginosa A-20653 TABLE 2 (MIC mg./tnl.) (continued) Compound XV Kanamycin A [BB-K8] Organlsn (6-8196) lot No. ^ Ps. species A-20621 125 125 Ser. ntarcescens A-20019 2 mareescens Α-201Ψ1 16 16 The abov** MIC data show * ha rompound IV (BB-K8) is superiox to kanamycin Λ in activity, particularly against kanamycin Λ resistant organisms The MIC data also correlate well with the in vivo results for all three organisms against which kanamycin A and compound IV were tested.

Compound IV and kanamycin A were equally effective in infections of mice caused by kanamycin A-sensitive strains of E. coli A-15119 and Staph. aureus A-9537. Although the CD5Q values (currative dose in 50% of mice lethally infected) for Staph, aureus A-9537 suggest that compound IV is slightly less active than kanamycin A; this small difference is probably not significant because the dose levels were far apart (5X dilution).

Against the kanamycin-resistant strain of E. coli A-20520, kanamycin Λ as expected, was not very effective in vivo, whereas compound IV demonstrated a marked protective action. Compound / IV was approximately 10 times morn active against this E. coli strain when administered in a -treatment regimen rather than in a 2-treatment one.

TABLE 3 A comparison of In Vitro and In Vivo Activities of Com Test Staphylococcus Escherichia coli Compounds Numbe aureus Α-15Ϊ19 Compound IV 1 1 2.0 X 2 . '. 2 2 X 2 .·<»·. 2 Kanamycin A 1 2 0.5 2 ; 4 X 2 2 minimum inhibitory concentration (ug./ml.). Tests cond Chisholm et al., (Antimicrob. Agents and Chemotherapy- using Mueller-Hinten agar as the test medium. curative dose, 0$ (mg./kg./treated X number of treatme subcutaneously at 1 and 4 hours post-infection when 2 t stered and at 0, 2 , 4 and 6 hours post-infection when 4 Other aspects of the test were carried out as described Antibiotics 22 :1. 1969) . not tested.

Compound IV is valuable as an antibacterial agent, nutritional supplement in animal feeds, therapeutic agent in poultry and animals, including man, and is especially valuable in the treatment of infectious diseases caused by Gram-positive and Gram-negative bacteria.

Compound IV, when administered orally, is useful as an adjunctive treatment for preoperative steriliza-tion of the bowel. Both aerobic and anaerobic flora which are susceptible to this drug are reduced in the large intestine. VJhen accompanied by adequate mechanical cleansing, it is useful in preparing for colonic surgery.

Compound IV is effective in the treatment of systemic bacterial infections in man when administered parenterally in the dosage range of about 250 mg. to abou 30OO mg. per day in divided doses three or four times a day. Generally the compound is effective when administered at a dosage of about 5.0 to 7-5 mg./kg. of body weight every 12 hours.

Description of the Preferred Embodiments Example 1 5-Norborncne-endo-2 , 3-dicarboxylic anhydride (XVIII) .

Preparation was carried out by the procedure of 0. Diels and K. Alder, Ann. 460, 98 (1928). To a stirred suspension of 98.1 g (1 mole) of maleic anhydride in 500 ml of dry b«nzene was added dropwise 66.1 g (1 mole) of cyclopentadiene under cooling. The mixture was stirred to afford a crystalline precipitate vhich was collected by filtration and recrystallized from ligroin (essentially n-hexane) -benzene (1:1) to yield 116 grams (71%) of the title product; m.p. 164-166° C. (lit. m.p. 164-165° C).

Example 2 N-Hydroxy-5-norbornene-endo-2 , 3-dicarboximide (HONB) (XIX) .

Preparation was carried out by the procedure of L. Bauer and S. V. Miarka, J. Org. Chem. , 24 , 1293 (1959). The norbornene anhydride (XVIII), 115 g, 0.7 mole) was added to a solution of hydroxylamine prepared by adding 47.5 g (0.45 mole) of sodium carbonate to a solution of 60.5 g (0.87 mole) of hydroxylamine hydrochloride in 140 ml of water. The mixture was heated at 60-70° C for 1 hour and kept to stand overnight in a refrigerator. The crystals were collected by filtration, washed with 120 ml of cold 5 N HC1 and dried to give 84.9 g (70%) of XIX melting at 172° C. The mother liquor was acidified to pH 2 and extracted with CHCl-j (70 ml x 10) . Evaporation of the CHCl^ extracts gave the second crop of XIX (7.2 g, 6%); m.p. 170-172eC.

Example 3 N-Hydroxy-5-norbornene-2 , 3-dicarboximide ester of L-T-benzyloxycarbonylamino-a-hydroxybutyric acid (XX) .

Dicyclohexylcarbodiimide (DDC) (2.06 g, 0.01 mole) was added at 10° C. to a stirred solution of 2.53 g (0.01 mole) of L-7-benzyloxycarbonylamino-a-hydroxybutyric acid and 1.79 g (0.01 mole) of XIX in 50 ml of dry THF (tetrahydrofuran) . The mixture was stirred overnight at room temperature. The precipitated dicyclohexylurea was removed by filtration and washed with THF. The filtrate and washings were combined and evaporated in vacuo . The oily residue (4.5 g) was passed through a silica gel column and eluted with ethyl acetate to give 4.0 g (96%) of the active ester XX as a colorless oil.

Infrared spectra (IR; Heat): 3300, 1820, 1780, 1735, 1720, 1695 cnT^. Nuclear Magnetic Resonance spectra (NMR (CDCI3 + 1 drop D20) ] : 6 (in ppm) 1.63 <2H, q, J=9Ilz), 2.15 (2H, in), 3.40 (6H, m) , 4.60 (1H, d-d, J»7 and 5 Hz), 5.13 (2H, s) , 6.20 (211, broad) , 7.40 (5H, s) .

Example 4 N-Benzyloxycarbonyloxy-5-norbornene-2 , 3-dicarboxi-mide (XVII).

To a stirred solution of 2.64 9 (0.066 mole) of. sodium hydroxide in 50 ml of water was added 3.58 g (0*02 mole) of N-hydroxy-5-norbornene-2 , 3-dicarboxi-mide XIX at 10° C. To the solution was added drop-wise 6.82 g (0.04 mole) of benzyloxycarbonyl chloride over a period of 40 minutes at 0-5° C. and the mixture was stirred for 4 hours at room temperature. To the reaction mixture was added 20 ml of n~hexane to give white precipitate which was collected by filtration and air-dried. The crude product was crystallized from benzene-n-hexane to give 5.1 g (81%0 of colorless prisms XVII; M.p. 121-122° C.

IR (I .r) : 1810, 1780, 1740, 1630 cm~l. NMR (acetcne-d6) : fi(in ppm), 1.66 (2H, broad s) , 3.45 (411, m) , 5.37 (211, s) , 6.12 (211, t, J=1.5Hz), 7.48 CM, s) .

Anal. Calc'd. for ci7Hi5N05: c> 65.17; H, 4.83; N', 4.47.

Found: C, 65.50; H, 4.73; N, 4.44.

Example 5 6 '-N-Benzyloxycarbonylkanamycin A (II) [6 ' -Cbz-kanamycin A] .

To a stirred solution of 4.84 g (0.01 mole) of kanaroycin A free base in 50 ml of 50% aqueous THF was added 3.14 g (0.01 mole) of XVII in several portions _ over a period of an hour at 7° C. The mixture was continued to stir for 4 hours and then evaporated in vacuo to remove---the The resulting aqueous solution was diluted with 150 ml of water and extracted with two 100-ml portions of water-saturated butanol. The butanol layer was washed with 100 ml of water and evaporated in vacuo to give 1.78 g of poly-Cbz-kanamycin A. The aqueous layer and t»e was ing* were combined and concentrated in vacuo to ca 50 ml.

The aqueous solution was passed through a column of CG-50 ion-exchange resin (NH^ +, 140 ml) , which was developed successively with 200 ml of water, 1.9 L of 0.05 N NH4OII , 1.9 L of 0.1 N NH4OH , and finally 1 L of 0.3 N NH4OH . The eluate was collected in 20-ml fractions and divided into the following two fractions on the basis of Rf value of thin layer chromatography [TLC (S-114; 6» -Cbz-kanamycin Rf 0.23, kanamycin Rf 0.07)]. Each fraction was evaporated in vacuo and the residue was dried in vacuo over phosphorus pentoxide.

Fraction Tube No. eluted with Amount Identity 1 106-144 0.1 N NH4OH 3.44 g 6 * -Cbz-kana- (56%) mycin (II) 2 203-212 0.3 N NH4OH 0.54 g kanamycin (11%) recovered 6 · -Cbz-kanamycin (II) thus prepared is very pure and homogeneous by TLC on a silica gel plate; Mp 217-219° C. The physico-chemical properties are consistent with the authentic sample.

Example 6 1-N- [L- (-) -y-amino-a-hydroxybutyryl]kanamycin A (BB-K8, IV) ia benzaldehyde Schiff base To a stirred solution of 6.19 g (0.01 mole) of 6*-Cbz-kanamycin (II) in 50 ml of 50% THF was added 3 ml (0.03 mole) of benzaldehyde. The mixture was stirred for 3 hours at room temperature, cooled to 5e C. and then treated with a solution of 4 g. (0.01 mole) of XX in 50 ml THF. The mixture was stirred for an hour at 5° C. , adjusted to pH 2 with 6 N HC1, stirred for an additional hour at room temperature and then evaporated in vacuo to remove the organic solvent. The resulting aqueous solution was washed with 20 ml of CH2Cl2» adjusted to pH 4 with 10% NH4OH and hydrogenated overnight with 1.25 g of 10% palladium on carbon under atmospheric pressure at room temperature. The catalyst was filtered off and washed with 20 ml of water. The filtrate and washings were combined, adjusted to pH 8 with 10% NH40H and passed through a column of CG-50 (NII4+, 200 ml) , which was eluted with 0.4 L of water, 1.2 L of 0.1 N H40H, 2.06 L of 0.3 N NH^OH, 0.92 L of 0.5 N NH OH and finally 1.2 L of 1.0 N NH40H. The eluate was collected in 20-ml fractions and cut into the following fractions on the basis of Rf value of TLC on silica gel plate (S- 10, ninhydrin) and disc assay using B. subtills PCI 219 and P. aerugenosa A 9843. Each fraction was evaporated in vacuo and freeze-dried.

Fraction Tube No* aq NH 0H eluted wt-yield bi 1 90-99 0.3 N 1603mg(33%) 149 2 152-161 0.3 N 483mg(8%) 3 162-169 0.3 N 376mg(6%) 196 4 170-197 0.3N-0.5N 1877mg (30%) 135 5 198-203 0.5 N 100mg(2%) 6 238-257 1.0 N 146mg(2%) *1 assayed vs. B. subtilis PCI 219 assayed vs. K^ pneumoniae A 20680 which is sensitiv resistant to kanamycin, BB-!-K 11 and BB-K 29.

Fraction 4 (1.877 g) was dissolved in 10 ml of deionized water. The solution was adjusted to pH 6 ■ + with 10% !ICl and adsorbed on a CG-50 column (NH , 15 ml), which was eluted with 100 ml of 0.3 N NH^OH and 200 ml of 0.5 N NH4OHi The eluate was collected in 20-ml fractions. Tube nos. 6 and 8 through 17 were ninhydrin positive. Tube nos. 8 to 17 were combined and concentrated iri vacuo to approximately 1 mL The concentrate was treated with 3 ml of MeOH and filtered. The filter was washed with 0.5 ml of 75% aq MeOH. The filtrate and washings were combined, diluted with an additional 10 ml of MeOH and allowed to stand overnight at room temperature. The amorphous precipitate being filtered, the filtrate was seeded to give colorless crystals of IV, which was identical with an authentic sample. The filtrate was evaporated in vacuo and lyophilized. The yield and assay of each fraction are given in the following table.

IV Amount Assay subtilis pneu amorphous powder 70 mg 737 u/mg 927 u crystals 1098 mg 1163 u/mg 1087 u lyophilized powder 180 mg 608 u/mg 504 u BB-K 11 is 3"-N-[L-(-)-7-amino-a-hydroxybutyryl] kanamycin A.

BB-K 29 is 3-N-[L-(-)-T-amino-a-hydroxybutyryl]-kanamycin A.

Example 7 Preparation of L- (-) -y-Benzyloxycarbonylamino-a-hydroxybutyric acid (VI).

L- (-) -γ-aniino-o-hydroxybutyric acid (7.4 g. 0.062 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 drop-wise at 0-5° C. over a period of 0.5 hour, 11.7 < (0.068 mole) of carbobenzoxy chloride and stirring continued for one additional 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 n vacuo and the resulting residue was crystallized from benzene to give 11.6 g. (74%) of colorless plates; melting point 78.5-79.5° C, [a]D-4.5° (c»2, CH3OH). Infrared (IR) [KBrl : IR (KBr) c»o 1740, 1690 caT^, Nuclear Magnetic Resonance ( MR) (acetone-d6) δ (in ppm 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).

, Anal, calc'd. for ci2Hi5N05: c» 56.91; H, 5.97; N, 5.53.

Found: C, 56.66; H, 5.97; N, 5.47.

Example 8 N-Hydroxysuccinimide ester of L- (-) -γ-benzyloxy-carbonylamino-tt-hvdroxybutyric acid tvil) .

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 dicyclohexylcarbo-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. 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%). [a]D 1.5° (c=2, CHC13) IR (KBr) vc=o 1810, 1755, 1740, 1680 cm"1, NMR (acetone-d6) δ (in ppm from TMS) 2.0 (2H, m) , 2.83 (4H, s) , 3.37 (2H, d-d, J»6.5 and 12.5 Hz), 4.56 (1H, m), 4.99 (2H, s) , 6.3 (2H, broad), 7.23 (5H, s).

Anal, calc'd. for ci6H 8N2°7: C' 54.85; H, 5.18; N, 8.00.

Found: C, 54.79, 54.70; H, 5.21, 5.20; M. 8.14, 8.12. 1. G. W. Anderson et al., J. Am. Chem. Soc.r 86, 1839 (1964).

Example 9 Preparation of N-(Benzyloxycarbonyloxy)succinimide.

N-Hydroxysuccinimide (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 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-dr±ed. Yield 41.1 g. (82%) .

Hecrystallization 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 (1964).

Example 10 Preparation of 61 -Carbobenzoxykanamycin A (II).

A solution of 42.5 g. (90 m. moles) of kanamyci A free base in 450 ml. of water and 500 ml. of di-methylformamide (DMF) was cooled below 9° C. and stirred vigorously. To the solution was added dropwise over a period of about two hours a solution of 22.4 g. (90 m. mole) 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 evaporated 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 evaporated to dryness under reduce 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 CG-50 ion-exchange resin (NH^"1" type, 500 ml.). The column was irrigated with 0.1 N ammonium hydroxide solution and the eluate was collected in 10-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. (45%) of a colorless product 6-carbobenzoxy-kana ycin A (II) [6 ' -Cbz-kanamycin A], which began to melt and color at 204° C. and decomposed at 212° C. with gas evolution [u]D +106° (c=2 H20).

TLC (silica gel F254; ninhydrin).

Rf value Solvent System 6 '-Cbz-Kanamycin A Kanamycin A n-PrOH-Pyridine-AcOH-H20 0.42 0.33 0.15 0.04 (15:10:3:12 (main) (minor) Acetone-AcOH-H20 0.24 0.14 (20:6:74) CHCl3-MeOH-C.NH4OH-H20 0.76 0.50 (1:4:2:1) AcOMe-n-PrOH-C. NHAOH 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 (IV).

Example 11 Preparation of L- (-) -τ-amino-a-hydroxybutyric acid from ambutyrosin A or B or mixtures thereof.

Ambutyrosin A (5.0 gau ) [U. S. 3,541,078, issued November 17, 19701 was refluxed with 160 ml. of 0.5 N sodium hydroxide for one hour. The hydrolysate was neutralized with 6N HCl and chroma- + tographed on a column of CG-50 (NH^ type) . The desired L- (-) -7-amino-u-hydroxybutyric acid was isolated by developing the column with water and re- moving the water by freeze drying. The L-(-)-7-amino-o-hydroxybutyric acid is characterized as a crystalline material having a m.p. of 212.5-214.5° C. [Column 2, lines 31-38, U. S.

Patent No. 3,541,078].

Example 12 Preparation of L- (-) -7-amino-tt-hydroxybutyric acid from DL-u-hydroxy—r-phthaiimidobutyric acid.

A. ) Dehydroabietylammonium L-a-hydroxy-γ-phthalimidobutyrate ; To a solution of 25 g. (0.1 mole) of 2-hydroxy-7-phthalimidobutyric acid in 200 ml. of ethanol was added a solution of 29 g* (0.1 mole) of dehydroabietylamine 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-dired to obtain 30.1 g. (56%) of a diastereomer of the dehydroabietylamine salt. M.p. 93-94° C. [a]24 +15· (c. 2.5, MeOH) . Recrystall zation from 300 ml. of ethanol gave 23.2 g. (43%) of the pure product. M.p. 94-95° C. [a] §4 +10.8° (c.2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation.

Anal, calc'd. for c32H42N205*H20i C' 69*5 ? H , 8.02; N, 5.07.

Found: Cr 69.58; H, 8.08; N, 5.07. 1. Y. Saito et al.. Tetrahedron Letters, 1970 , 4863.

B.) L- (-) -γ-Amino-a-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 dehydroabiet 1ammonium L-u-hydroxy- -phthalimido-butyrate 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 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 fractions.

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 and dried in a vacuum desiccator to give 0.78 g. (66%) of L- (-) -7-amino-u-hydroxy-butyric acid. M.p. 206-207° C. [α]δ4 -29° (C. 2.5, H20) . The IR spectrum was identical with the authentic sample which was obtained from ambutyrosin.

Example 13 Preparation of 6 ' -Carbobenzoxy-1 , 3 , 3"-tri-p-nitrobehzalkanamvcin A (IIIc) . 6 * -Carbobenzoxykanamycin A (9.0 g., 14.5 m. moles) was suspended in 500 ml. of absolute ethanol at 24° C. and 8.7945 g. (58.2 millimoles) of p_-nitrobenzaldehyde was added. The mixture was heated to reflux. During this time the mixture changed to a clear, yellow solution, but at reflux a white solid quickly began to form. The mixture was heated at reflux for three hours, then cooled, and the product collected by filtration and washed with a little absolute ethanol. The yield was 14.0 g. (94.5%) of white crystalline solid, m.p. 233-234° (uncorrected) , of the title product IIIc .

Anal, calc'd. for C 7H51N7019i c» 55.46; H, 5.05> N, 9.63.

Pound: C, 55.39? H, 5.08 N, 9.60.

Example 14 Preparation of 1-N- [L- (-) - -amino-a-hydroxybutyryl] -kanamycin A (IV) . 6 ' -Carbobenzoxy-1 ,3,3" -tri-p_-nitrobenzalkana-mycin A (5.0 g., 4.91 m. moles) was dissolved in 50 ml. of dimethylformamide (DMF) at 24° C. L-(-)-7-ben2yloxycarbonylamino-o-hydroxybutyric acid N-hydroxy-succinimide ester (2.064 gt 5.895 ra.moles) was dissolved in 20 ml. of DMF at 24° C. and added slowly with vigorous stirring to the solution of the Schiff's base (III c) over 75 minutes. The solution was stirred at 24° overnight. Using steam-ejector vacuum, the solution was flashed dry at about 40° C. It was then flashed repeatedly with methanol (100 ml.), to yield a viscous oil. The oil was dissolved in 100 ml. of 1:1 dioxane K^O and 10 ml. of glacial acetic acid was added. The solution was placed in a 500 ml. Parr bottle, together with 2.5 g. of 5% Pd/C (Engelhard) and reduced at 45 psi H for four hours at 24° C. The total pressure drop in this time (closed bottle) was 87 psi, including periodic repressurizations . The mixture was filtered through a pad of dia-tomaceous earth which was then washed with 3X50 ml. of 50% aqueous dioxane. The combined filtrates were flashed dry and azeotroped with n-butanol (100 ml.) and finally flashed repeatedly with methanol, giving a viscous oil. It was dissolved in 30 ml. of methanol and slowly poured into 1000 ml. of cold (5-10° C.) diethyl ether with vigorous stirring. After stirring the resulting suspension for 1/2 hour in an ice bath, the precipitate was collected by filtration and immediately dried in a vacuum desiccator over P2°5* Tne Pro<*uct mixture weighed 4.9620 g. TLC (thin layer chromatography) showed it to consist mainly of compound IV, kanamycin A, and some traces of di and trisubstituted kanamycin A, and including 4-amino-2-hydroxybutyric acid.

The various fractions were separated using a 40X100 mm. glass column packed with Amberlite IRC-50 (NH4+form, Type I, 100/200 mesh) to provide a resin bed of ca. 980 mm. The column was charged with 4.600 g. of the crude mixture dissolved in 1 ml. of water. The column was eluted with a gradient from H20 to 2 NH^OH, the eluate being collected in 15 ml. fractions. 295 fractions were collected, then the column was washed with 1.5.1 of 3 N NH OH. Fractions were pooled on the basis of optical rotations, and solids isolated therefrom by lyophylizing. Correction to Weight of Whole Fraction ComposiWeight Crude- No. tion Grams Grams Bioassay %Yield 120-150 kana A 1.1654 1.257 809 42.6 201-224 BB-K29 0.40 0.43 228-241 BB-K8 0.6523 0.7036 786+101* 39.2** ♦Average of 4 plate assays, + standard deviation. **If credit is taken for recovered kana A, the con version to BB-K8 (IV) was 33.5%.

In this run, the ratio of BB-K8/kana A was 0.45.

Compound IV, as recovered from this process, identical with authentic product obtained by the method described in co-pending application, Serial No. 221,378. M.p. 194° (dec.) [ajg1 +05° (c=2, H20 . Relative potency against B. subtilis (agar plate)=» 960 mcg./mg. (standard: kanamycin A free base) .

Anal, calc'd. for C H O ,2H CO : C, 22 43 5 13 2 3 40.62? H, 6.68; N, 9.87.

Found: C, 40.21; 39.79; H, 6.96, 6.87; N, 9.37, 9.49.

Example 15 Preparation of the Monosulfate Salt of 1-N-IL- (-) -7-araino-a-hydroxybutyryl]kanamycin A.

One mole of 1-ΪΪ- (L- (-) -7-amino-a-hydroxy-butyryllkanamycin A 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 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 16 Preparation of the Disulfate Salt of 1-N- [L-(-) -7-amino-g-hydroxybutyryllkanamycin A (BB-K8.2H2S04) .

Thirty-five grams of 1-N- [L- (-) -7—amino-o-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 Oarco 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-wash was adjusted to pH 1-1.3 with 50% V/V sulfuric acid. This solution was added with rapid stirring over a 10 minute period to 600-800 ml. of methanol (3-4 volumes of methanol) . The mixture was stirred for five minutes at pH 1-1.3, passed through a 100 mesh screen, stirred for two minutes and allowed to settle for five minutes. Most of the 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 (dihydrogen sulfate) 2 was 32-34 grams; [o]o2H20»+74.75 , decomposition at 220-23-°C.

Elemental Analysis (On Dry Basis*) % C 32.7, 33.5, 32.3 33.5 % N 8.78, 8.7, 8.2 8.8 8.97 % S 8.75, 8.9, 7.8, 8.85 8.2 % Ash nil *Karl Fisher water content: 2.33, 1.79, 2.87% (theory for monohydrate is 2.25% water). This salt is hygroscopic but not deliquescent. After storage of an aliquot in air at room temperature for 18 hours the water content increased to 9.55, 9.89% (theory for a pentahydrate is 10.33% water).

Example 17 1-N- [L- (-) -T-amino-u-hydroxybutyryl]kanamycin A (IV) via the absence of Schiff base intermediate.

To a stirred solution of 1.36 g (2.2 m moles) Of e'-N-Cbz-kanamycin (II) in 60 ml of 50% aq THF was added dropwise 930 mg (2 m moles) of the active ester XX in 10 ml THF over a period of 30 min at room temperature. The mixture was stirred overnight and then hydrogenated with 1.0 g of 10% palladium on carbon under atmospheric pressure at room temperature. The catalyst was removed and washed with water. The filtrate and washings were combined and evaporated in vacuo to remove the organic solvent. The resulting aqueous solution was passed through a column of CG-50 (NH4+, 70 ml), which was irrigated successively with 0.4 L of water, 0.5 L Of 0.1 N NH40H, 0.5 L of 0.3 N NH^OH , 1 L of 0.5 N NH4OH and finally 0.5 L of 1 II NH^OH. The eluate was collected in 20-ml fractions and divided into fractions on the basis of Rf value of TLC on silica gel plate (S-110, ninhydrin) and disc assay using subtilis PCI 219 and pneumoniae Type 22 #3030 A 20680. Each fraction was evaporated in vacuo and lyophilized.

Example 18 6*-Carbobenzoxy-1,3 ,3*-trisalicylalkanamycin A 6'-Carboben2oxykanamycin A (9.0 g., 14.5 m. moles) was suspended in 500 ml. of absolute ethanol at 24° C. and 58.2 millimoles of salicylaldehyde was added. The mixture was heated to reflux.

During this time, the mixture changed to a clear, yellow solution, but at reflux a white solid quickly began to form. The mixture was heated at reflux for three hours, then cooled, and the product collected by filtration and washed with a little absolute ethanol. The melting point upon recrystallization from 4:1:5 tetrahydrofuran-methanol-heptane, after drying was 196-198° C.

Anal. Calc'd. for C, 60.62; H, 5.86; N, 6.02.

Found: C, 58.67; H, 5.73; N, 5.98; Karl Fischer Water, 2.45 Anal. Corrected for water: Found: C, 60.14; H, 5.60; N, 6.13.

Example 1? Preparation of 1-N- [L- (-)-Y-amino-g-hydroxy-butyryl.kanamycin A via the in situ process In suitable equipment, slurry 1000 g» (1.074 moles) of 6 ·-carbobenzoxy-1 ,3,3*-tri-salicylal-kanamycin A in 11,400 ml of THF at 22-25° C. Add 600 ml. of water with stirring. Solution occurs in about 10 minutes. Add as solids 181.5 g (0.716 mole) of -benzyl-oxycarbonylamino-2-hydroxybutyric acid and 0.12 moles of N-hydroxy-5-norbornene-2 ,3-dicarboximide to the solution. The pH is in the range of 5.5 to 6.0. Maintaining the temperature at 22-25° C, add 162.3 g. (0.787 mole) of dicyclohexylcarbodiimide dissolved in 3000 ml of dry THF. The slurry was stirred for 24 hours at 22-25° C. and then cooled to 0-5° C. to crystallize out the dicyclohexyl urea for 2 hours.

The urea was removed by filtration and the cake was washed with 800 ml of 95/5 THF water. The filtrate and washes were combined and reduced in volume under vacuum to a volume of 3000 ml. 4000 ml. of methanol was added and the volume again reduced to 3000 ml. in vacuo .

TO the solution thus obtained was added i 2000 ml of absolute methanol at 40° C and then it was seeded with e'-carbobenzoxy-l^a^a"-^!-salicylal-kanamycin Λ and allowed to cool and crystallize for 0.5 to 1 hour. Then 2000 mis of water was added over one-half hour with good agitation at about 22-25° C for two hours. The temperature was then reduced to 0-5° for 1 hour.

Remove the solids by filtration and wash with 900 mis of cold 2:1 methanol-water and then 800 mis of methanol. Save the filtrates and washes. The cake is essentially recovered 6'-carbobenzoxy-l,3,3"-trisalicylalkanamycin A.

The filtrates above are diluted with 1000 ml of water. To this is added 2000 ml of methylene chloride. At 25° C, lower the pH to 1,8 - 2.0 with 6N HC1. Hold at this pH for one-half hour.

Separate the methylene chloride layer and wash the aqueous phase with an additional 2000 ml of methylene chloride. The combined organic phases are saved for possible salicylaldehyde recovery.

The aqueous phase pH is raised to 3.5 - 4.0 with concentrated ammonium hydroxide. This solution is the hydrogenated at room temperature, at 2 SO lb/in pressure with hydrogen gas in the presence of 5% Pd/C. The bio yield of desired product in solution is 30-45% of theory.

Example 20 Preparation of Compound IV using various mole percentages of N-hydroxy-5-norbornene-2,3-dicarboximide while holding the other mole percentages constant.

Substitution in the procedure of example 20 for the 0.12 moles of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) used therein of the following various mole percentages produced the following percentage yields of compound IV» % Yield of Experiment # Mole % HONB Compound IV 1 0 · 9.33 2 2 20.07 3 10 31.72 4 50 36.52 5 100 36.01 It can be seen from the above results that the reaction will produce compound IV in the complete absence of HONB. However, it can also be seen that the best yields of compound IV are obtained when the mole percentage of HONB is 10 to 50% of the molar quantity of 4-benzyl-oxycarbonylamino-2-hydroxybutyric acid used in the acylation reaction. The precise mechanism by which the acylation of the blocked kanamycin A proceeds is unknown. However, it is observed that the presence of the HONB somehow produces better yields than previously obtained.

Example 21 Preparation of 1-N-[L- (-)-'V'-amino-a-hvdroxybutvrvl] kanamycin A (IV) 6 ' -Carbobenzoxy-1, 3, 3 ' -trisalicylalkanamycin A (18.6 g., 20.0 mmoles), prepared as described in Example 18, was dissolved in 55 ml. of dimethylformamide (DMF) at 45°C. To the above solution were added 7.3 ml of water and 102 ml. of acetone. The solution was allowed to cool to about 25° C. and a 50 ml. solution of acetone and L- (-) - T^-benzyloxycarbonylamino-a-hydroxybutyric acid N-hydroxy-succinimide ester (9.9 mmoles) was added all at once. The reaction mixture was stirred at approximately 25° C. for 40 hours. Subsequently, 150 ml. of water and a few crystals of 6 ' -carbobenzoxy-1, 3, 3 '-trisalicylalkanamycin A were added and the mixture stirred at about 25° for one hour followed by 3 hours at 0-5° C. The excess and unreacted salicylaldehyde Schiff base crystallized and was collected on a filter. The filter cake was washed with 60 ml. of 50% aqueous acetone. The cake was dried and weighed to afford 9.4 g. of recovered Schiff base, m.p. (crude) 170-185° C.

The filtrate from above was treated with 40 ml. of methylene chloride and the pH adjusted from 6.2 to 2.0 to remove further starting material. The layers were separated and the aqueous layer was extracted with 2 x 40 ml. of methylenechloride. The pH of the aqueous layer was adjusted to 3.7 and a small volume of acetone and methylene chloride was purged in vacuo. The aqueous solution was then hydrogenated with 3.0 g. of 5% Pd/C catalyst at 24° C for 15 hours at 45 ps. hydrogen. The reaction mixture was filtered through diatomaceous earth to remove the catalyst. The filtrate was adjusted to a volume of 100 ml. A one ml. sample of this solution was removed for plate and disc assay. The plate assay indicated 1936 meg./ml. of compound IV and the disc assay gave 2325 meg./ml. which corresponds to a 33.4% yield of the final product.

The remaining 99 ml. of solution were concentrated to a volume of about 30 ml. and processed through column chromatography as previously descrived in, e.g.. Example 14. The combined fractions containing compound IV, 500 ml., were concentrated to about 20 ml. wherein 45 ml. of methanol was added followed by the dropwise addition of about 25 ml. of isopropanol to crystallize the product. The crystalline material was collected on a filter, washed with 50/50 methanol-isopropanol and dried to afford 2.31 g. of BB-K8, compound of formula IV. Plate assay of the solids ga¾e 836 mcg g indicating a purity of 83.6 and an actual yield of pure compound of formula IV of 33.3% based on 9.9 mmoles of starting material.

Claims (15)

46995/2 CLAIMS: 1.4.75 1. A process for the preparation of the compound for Claims 1-11 and having the formula 13-15 except for the · modi fication claimed in Claim 12. which process comprises the consecutive s of A) treating the compound having the formula CLAIMS:

1. A process for the preparation of the compound having the formula which process comprises the consecutive steps of A) treating the compound having the formula with an aldehyde selected from the group comprised of benzaldehyde, salicylaldehyde, p-nitrobenzaldehyde, p-methoxybenzaldehyde and pivaldehyde, in a ratio of one mole of compound II to at least three moles of aldehyde o produce the compound having the formula B) treating compound III in situ with the compound having the formula XX in a ratio of one mole of compound III to at least 0.5 mole of compound XX# and then hydrogen- ating the residue in situ to produce the compound of formula IV.

2. A process as claimed in Claim 1 which process comprises A) treating the compound of the formula II with an aldehyde selected from the group comprised of benzaldehyde, p-nitrobenzaldehyde, salic laldehyde, p-methoxybenzaldehyde and pi aldehyde, in a ratio of one mole of compound II to at least three moles of aldehyde, in absolute ethanol, methanol, n-propanol, methylenechloride, isopropanol, n-butanol, sec-butanol, tert-butanol, tetrahydrofuran, dioxane, diraethylformamide or acetone, or mixtures thereof, or a mixture with water, in a temperature range of 5°C. to about reflux temperature, for a period of time of about 30 minutes to about 5 hours, to produce the compound of the formula III in which Z is a radical of the formula B) treating compound ill with the compound of the formula XX in a ratio of one mole of compound III to at least 0,5 moles of compound XX, in a temperature range of about -10° to about +35° C, for at least one hour, in a solvent system as described in Step A; then removing the organic solvent and hydrogena-ting the residue in situ with hydrogen in the presence of a metal catalyst from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, in a water or a water-water miscible solvent system selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether and propyleneglycol dimethyl ether at a pH of 3-5, to produce the compound of formula IV.

3. A process as claimed in any of Claims 1 and 2, which comprises A) treating the compound of the formula II with an aldehyde selected from the group comprised of benzaldehyde, salicylaldehyde, p-nitrobenzaldehyde and p-methoxybenzaldehyde, in a ratio of one mole of compound II to at least three moles of aldehyde; in absolute ethanol, methanol, n-propanol, isopropanol, methylene chloride, n-butanol, sec-butanol, tert-butanol, tetrahydrofuran, dioxane, dimethylformamide or acetone or a mixture thereof or a mixture thereof with water in a temperature range of 5°C. to 40°C. for a period of time of about 30 minutes to about 5 hours, to produce the compound of the formula III in which Z is a radical of the formula B) treating compound III in situ or after isolation with the compound of the formula XX in a ratio of one mole of compound III to about 0.5 to 1.0 moles of compound XX, in a range of +5° C. to 35° C., for one to three hours in a solvent system as described in Step A; then removing the solvent and hydrogenating the residue in situ with hydrogen in the presence of a metal catalyst selected from the group comprising palladium, platinum. Raney nickel, rhodium, ruthenium and nickel, Aaut- miscible solvent system, selected from the gro^ comprising water and dioxane, tetrahydrofuran ethyleneglycol dimethyl ether and propyleneglycol dimethyl ether at a pH of 3-5, to produce the compound of formula IV.

4. A process according to any of Claims 1-3, which comprises A) treating the compound of the formula II with an aldehyde selected from the group consisting of benzaldehyde, salicylaldehyde . and p-nitrobenzaldehyde, in a ratio of one mole of compound II to about three moles of aldehyde; in absolute ethanol, methanol, n-propanol, isopropanol, n-butanol, sec- butanol, tert-butanol, tetrahydrofuran, dioxane, methylene chloride, dimethylformamide or acetone or a mixture thereof or mixtures thereof with water in a temperature range of 5° C. to 40° C, for a period of time of about 30 minutes to about 5 hours, to produce the compound of the formula III treating compound III i situ or after isolation with the compound of the formula XX in a ratio of one mole of compound III to 0.5 to 0.85 mole of compound XX, in a temperature range of +5° C. to 30° C, for one to three hours in a solvent system as described in step A; then removing the solvent and hydrogenating the residue in sitii with hydrogen in the presence of a metal catalyst selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, in water or a water-water miscible solvent system, selected from the group comprising water and dioxane, tetrahydro-furan, ethyleneglycol dimethyl ether and propyleneglycol dimethylether at a pH of about 4 to produce the compound of formula IV.

5. A process according to any of Claims 1-4 which comprises A) treating the compound of the formula II with benzaldehyde in a ratio of one mole of compound II to about three moles of benzaldehyde, in about a mixture of water and tetrahydrofuran, at about 20° C, about 30° C. for about two to four hours, at a pH of 10, to produce the compound having the formula B) treating compound Ilia in. situ or after isolation with the compound of the formula XX in a ratio of one mole of compound Ilia to 0.6 to 0.75 mole of compound XX, at a temperature in the range of about 20°C. to about 30° C. for a period of time of one to three hours in a solvent system as described in step A# a mixture of methylene chloride, methanol and Water or a mixture of dimethylformamide, acetone and water; then removing the organic solvent in vacuo, adjusting the pH to 4 with ammonium hydroxide and hydrogenating the residue in situ with hydrogen at atmospheric pressure in the presence of palladium on carbon to produce the compou of formula iv.

6. The process of any of Claims 1-4, which comprises A) treating the compound of the formula II with salicylaldehyde in a ratio of one mole of compound II to about three moles of salicylaldehyder in about a 1:1 mixture of water and tetrahydrofuran, at about 20° C. to about 30° C. for about two to I B) treating compound Illb in situ or after isolation with the compound of the formula XX in a ratio Of one mole of compound Illb to about 0.6 to 0.75 mole of compound XX at a temperature in the range of about 20°C. to about 30°C. for a period of time of one to three hours, in a solvent system as described in step A, or a mixture of dimeth Iformamide, acetone and water; then removing the organic solvent in vacuo, adjusting the pH to 4 with ammonium hydroxide and hydrogenating the residue in situ with hydrogen at atmospheric pressure in the presence of palladium on carbon to produce the compound of formula IV.

7. A process according to any of Claims 1-4, in which Step B comprises treating a compound of the formula III in situ or after isolation with the compounds having the formulae VI and followed by the addition of dicyclohexylcarbodi-imide, in a ratio of one mole of compound III to about 0.5 to 1.0 moles each of compound vi and dicyclohexylcarbodiimide and about 0.005 to 0.25 moles of compound XIX , in a temperature range of about -10° C. to about +35° C, for at least ten hours in a solvent system as described in Step A? then removing the organic solvent and hydrogenating the residue jn situ as described in any of Claims 1-4 to produce the compound of formula IV.

8. The process of Claim 7 wherein in Step B a compound of formula III is treated in situ or after isolation with the compounds having the formulae - H-0-C ?-CH-CH2-CH2-NH-C ϊ-0-CH2 OH VI and followed by the addition of dicyclohexylcarbodiiraide in a ratio of one mole of compound III to about 0.5 to 0.85 moles each of compound VI and dicyclohexylcarbodiimide and about 0.05 to 0.425 moles of compound XIX, in a range of +5° C. to 25° C, for one to three hours in a solvent system as described in Step A.

9. A process according to any of Claims 7 and 8 wherein in Step B, a compound of formula III is treated in situ with the compounds having the formulas followed by the addition of dicyqlohexylcarbodiimide in a ratio of one mole of compound III to about o.5 to 0.8 moles each of compound VI and dicyclo-hexylcarbodiimide and about 0.05 to 0.425 moles of compound XIX in a range of +15° C. to +30° C, for 15 to 25 hours.

10. A process according to Claim 5 wherein in Step B, a compound of formula ilia is treated in situ or after isolation with the compounds having the formulae and with stirring followed by the addition of dicyclohexylcarbodiimide in a ratio of one mole of compound III to to about 0.6 to 0.75 moles each of compound VI and dicyclohexylcarbodiimide and about 0.06 to 0.375 moles of compound XIX in a range of +25° to +35° C. for 15 to 25 hours, in a 1:1 mixture of tetrahydrofuran and water* 46995/2

11. A process according to Claim 6, wherein in Step B, a compound of formula Illb is treated in situ or after isolation with the compounds having the formulae VI and with stirring, followed by the addition of dicyclohexylcarbodiimide in a ratio of one mole of compound Illb to about 0.6 to 0.75 moles each of compound VI and dicyclohexylcarbodiimide and about 0.06 to 0.375 moles of compound XIX in a range of +20° C. to 30° C. for 15 to 25 hours, in a 9:1 mixture of tetrahydrofuran and water.

12. A process according to any one of Claims 1 to 9, wherein in Step B a compound of formula III is treated in situ or after isolation with the compound of formula XX or with the compounds having the formulae VI and XIX in a solvent system consisting of a mixture of dimethylformamide , acetone and water. -71- 46995/2

13. A process according to any one of the preceding claims, wherein the compound having the formula II in Claim 1 is prepared by treating kanamycin A free base with N-benzyloxycarbonyloxy-5-norbornene- 2, 3-dicarboximide, in a ratio of about 0.5 to about 1.5 moles of N-benzyloxycarbonyloxy-5-norbomene-2, 3- dicarboximide per mole of kanamycin A, followed by purification.

14. A process of Claim 13 wherein the ratio of kanamycin to N-benzyloxycarbonyloxy-5-norbornene-2 , 3-dicarboximide is about 1:1, and the reaction is carried out in an aqueous-water miscible organic solvent system, at a temperature range of about 0° to 25° C, for at most 6 hours, followed by purification by organic solvent extraction and chromatography.

15. A process according to any of Claims 13 and 14, wherein the ratio of kanamycin to N-benzyloxycarbonyl-oxy-5-norbornene-2, 3-dicarboximide is 1:1, and the reaction is carried out in 50 to 6o per cent aqueous tetrahydrofuran at a temperature range of about 5° to about 10° C, for about 4 hours, with agitation followed by removal of the tetrahydrofuran in vacuo, extraction of the aqueous residue with n-butanol and purification of the aqueous phase by chromatography. For the^-f^piicants