DK142991B - PROCEDURE FOR THE PREPARATION OF 1- (L - (-) - Gamma-AMINO-ALFA-HYDROXY-BUTYRYL) -CANAMYCINE A OR ACID ADDITION SALTS. - Google Patents

Description

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\ r & / (11) PUBLICATION WRITING 142991 DENMARK (bi) in «.ci.3 c 07 h 15/22 '(21) Application No 591 6/74 (22) Filed on 1 5 · DOV · 1 9 74 (24) ) Running day 15 »HOV. 1974 (44) The application submitted and in the petition published on 9 * ®8Τ · yc r

DIRECTORATE OF

PATENT AND TRADE MARKET (30) Priority requested from it

Nov 14 1975 »415589, US

May 25, 1974, 472780, US

(71) BRISTOL-MYERS COMPANY, 545 Park Avenue, New York, N.Y. 10022, US.

(72) Inventor: Takayuki Naito, 4-24-19, Nishi-shinjuku, Shinjuku-ku, Tokyo, JP: Susumu Nakagawa, 1-14-7, Nakai, Shinjuku-ku, Tokyo, JP: MasaMsa Oka, 2- 15-1H, Shimo-meguro, Meguro-ku, Tokyo, JP.

(74) Plenipotentiary in the proceedings:

Th. Ostenfeld Patentbureau A / s.

(54) Process for the preparation of 1- (L - (-) - gamma-amino-alpha-hydroxy-butyryl) -canamycin A, or acid addition salts thereof.

The present invention relates to a particular process for the preparation of 1- [L - (-) - γ-amino-α-hydroxy-butyryl] -canamycin A

(IV), there is a known compound of the formula: 6 '41 CH 2 NH 2 HO-K 1 W5 «K0 Λ_ ^ nh2 6? vs HO ^ CJO «H2M \ h 7x \ /

2 5 S '/ Atl' · / HO-CK

° J / ψ2.

CEy-NHg or pharmaceutically acceptable salts thereof.

2 142991

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 that the 1-amine function has the second highest reactivity when treated with an electrophilic agent. Both the 3 and 3 "positions are less reactive than both the 1- and 6'-amine functions, but react with acylating agents to form materials with little content of unwanted acylated materials.

From the specification of Danish Patent Application No. 3461/72, it is known to prepare 1- [L - (-) - γ-amino-α-hydroxybutyryl] -canamycin A or B by blocking the amino group of the 61-carbon atom in kanamycin A or B, then selectively acylates the amino group at the 1-position with a derivative of L - (-) - γ-amino-α-hydroxybutyric acid in which the amino group is protected, and finally removes the protecting groups from both the 6 'position and the substituent in the the 1-position. When this known method is used to prepare the kanamycin A derivative (IV), on the basis of Example 3 of the said application, a total yield of approx. 10% relative to the amount of the starting material used kanamycin A.

The present invention provides an improved process for preparing the subject compound (IV) whereby yields greater than 30% can be obtained.

Further, from the specification to Danish Patent Application No. 593/74, it should be considered known to prepare the subject compound (IV) by first converting 6-carbobenzoxy-kanamycin A (II) to a Schiff base (III) and then treating the latter with the N-hydroxy-succinimide ester of L - (-) - γ-benzyl-oxycarbonylamino-ct-hydroxybutyric acid (VI). The process of the present invention is based on this technique but differs by using a different N-hydroxyimide ester of the acid (VI).

Thus, the process of the invention comprises treating (A) 6-carbobenzoxy-kanamycin A of Formula 3 142991 B0 ^ H0 ώ_ 13¾ V ^ = A / HO 1 / o

II

with benzaldehyde, salicylic aldehyde, p-nitrobenz aldehyde, p-methoxybenzaldehyde or pivaldehyde, using at least three moles of aldehyde per mole of compound II, to prepare the compound of formula H? CH 2 -Pf-C-O-CH 1 -CrHc-EO

III

Wherein Z is a group of the formula JJ JJ _ 'pj -h = c - / ^ - Ho2, - * cQ, -nc - (^^ _ and3, hHX h / "3 -" -' O or -Nc ' c ~, CH, upon which (B) is treated the compound of formula III in situ or after isolation with an N-hydroxyimide ester of the acid of formula 0?

ii II

H0-C-CH-CH 2 'CH 2 -NH-C-0-CH2-C6H5

OH

WE

whereupon the resulting product is hydrogenated in situ to prepare the compound of formula IV which, if desired, is converted to a pharmaceutically useful salt thereof, and the process of the invention is characterized in that the N-hydroxymide ester of said acid is optionally formed in situ, and that it has the formula

O Q

{ΥΛ - O-C-CH-CH 2 -CH 2 -NH-C-O-CH 2 -C

XX

and that at least 0.5 mol of compound XX is used. or, when compound (XX) is formed in situ, to form in an amount equal to at least 0.5 mole per ml. mole of compound (III).

5 142991

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, n-butanol, sec-butanol, tert-butanol, an ether solvent, e.g. tetrahydrofuran, dioxane or other polar aprotic or non-aprotic solvents such as e.g. dimethylformamide and acetone or mixtures thereof, or mixtures thereof with water, at a temperature in the range of approx. 5 ° C to approx. reflux temperature> preferably 5-40 ° C, for a period of from approx. 30 minutes to approx. 5 hours.

Step B is preferably carried out with a ratio of compound III to compound XX of approx. 1: 1, more preferably 0.5 - approx. 1.0 mole of XX per mol III, at a temperature in the range of approx. -10 ° C to approx. + 35 ° C, and preferably in a range of from + 5 ° C to approx. 25 ° C, for such a time that the acylation is complete, but for at least 1-3 hours, after which the organic solvent is removed before the hydrogenation. The hydrogenation in situ is carried out with hydrogen in the presence of a metal catalyst in the form of palladium, platinum,

Raney nickel, rhodium, ruthenium or nickel, but preferably palladium, and especially palladium on charcoal in water or a water-water miscible solvent system in the form of water and dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether and the like, but preferably in water value of 3 - 5, and preferably 4.

According to an embodiment of the process according to the invention, during step (A), compound II is treated with benzaldehyde, salicylaldehyde or p-nitrobenzaldehyde, whereby step (B) preferably uses 0.5 to 0.85 moles of compound XX per ml. mole of compound III, at a temperature of 15 - 30 ° C for 1-3 hours, in a solvent system as described above in step A.

According to another embodiment, compound (A) is treated with compound II with benzaldehyde, using 3 moles of benzaldehyde per ml. mole of compound II, in a ca. 1: 1 mixture of water and tetrahydrofuran, at approx. 20 to 30 ° C and at a pH of approx. 10 for approx. 2-4 hours, to prepare the compound of formula 6 142991 H il

“* 1_ ^« -O

ho—: h2oh \ vJ

Ησ I / and

Ilia hr B) treats compound Ilia in situ or after isolation with the compound of formula XX using 0.6 - 0.75 moles of compound XX per ml. mole of compound Illa, at a temperature of ca. 20 to 30 ° C for a period of 1-3 hours, in a solvent as referred to in step A), a mixture of methylene chloride, methanol and water or a mixture of dimethylformamide, acetone and water, whereupon the organic solvent is removed in vacuo, The pH is adjusted to 4 with ammonia water and the residue is hydrogenated with hydrogen at atmospheric pressure in the presence of palladium-on-carbon to prepare the compound of formula IV.

According to a further embodiment of the process according to the invention, the compound of formula II is treated with salicylaldehyde, using 3 moles of salicylaldehyde per ml. mole of compound II, in a ca. 1: 1 mixture of water and tetrahydrofuran at approx. 20 to 30 ° C and at a pH of approx. 8 for approx. 2-4 hours to prepare the compound of formula 7 142991

ISLAND

HO ^. CH2-N-i-0-CH2-CgH5 ηοΛ_Λ HO

h N = C ~ f J

HO-CH.OH \ q- ^ 7 ^ OH HO] / rr and

Hib B) treats Compound Illb in situ or after isolation with the compound of Formula XX using approx. 0.6 - 0.75 compound XX per mole of compound Illb, at a temperature of approx. 20 to 30 ° C for a period of 1-3 hours and in a solvent as referred to in step A) or a mixture of dimethylformamide, acetone and water, whereupon the organic solvent is removed in vacuo, the pH is adjusted to 4 with ammonium chloride. hydroxide, and the residue is hydrogenated in situ with hydrogen at atmospheric pressure in the presence of palladium-on-carbon to prepare the compound of formula IV.

According to a preferred embodiment of the process of the present invention, the compound of formula XX is formed in situ and reacted directly with the compound of formula III in one step. Hereby, the same excellent yields of the compound of formula IV are obtained and the compound of formula XX is not isolated. This embodiment is characterized in that the compound of formula XX is formed in situ by reacting compounds of formulas 8 H-0-C-CH-CH 2 -CH 2 -NH-C-0-CH 2 -C 6 H 5

OH

WE

and then dicyclohexylcarbodiimide is added, using 0.5 - 1.0 mol of both compound VI and dicyclohexylcarbodiimide and ca. 0.005 to 0.425 moles of compound XIX per mole of compound III.

Preferably a temperature in the range of from -10 ° C to approx. + 35 ° C, but preferably from ca. 5 to 25 ° C for at least 10 hours in a solvent system as described above under step A), whereupon the organic solvent is removed and the residue hydrogenated in situ as described above to prepare the compound of formula IV.

An example of the above embodiment comprises the use of 0.5 to 0.85 moles of both Compound VI and dicyclohexylcarbodiimide and ca. 0.05 to 0.425 moles of compound XIX per mole of compound III at a temperature of 5 - 25 ° C for 1-3 hours in a solvent system as described in step A), or alternatively 0.5 to 0.8 mole of both compound VI and dicyclohexylcarbodiimide are used, and ca. 0.05 to 0.425 moles of compound XIX per mole of compound III at a temperature of 15 - 30 ° C for 15 - 25 hours. It is most preferred to use approx. 0.6 to 0.75 moles of both compound VI and dicyclohexylcarbodiimide and ca. 0.06 to 0.375 moles of compound XIX per mole of compound III at a temperature of 20-30 ° C for 15-25 hours.

For the preparation of the starting material of formula 142991 9 Η 9 CH -'- N -'t-O-CH - "" Cf-Hc "Vi.

“Τή, i_« * 2

Hn for 2 ° H0 2 \ n N-o 2 -nh 2 σσ ii, free kanamycin A base can be treated with N-benzyloxycarbonyloxy-5-norbornene-2,3-dicarbox-imide using approx. 0.5 to 1.5 moles of N-benzyloxycarbonyloxy-5-norbornene-2,3-dicarboximide, or especially 1 mole thereof per mole of kanamycin A, after which the product is purified, for example, by extraction with an organic solvent such as n-butanol and aqueous phase chromatography.

The above reaction is preferably carried out in an aqueous-water miscible organic solvent system such as e.g. tetrahydrofuran water, preferably containing 40-60% tetrahydrofuran, at a temperature of 0-25 ° C, or preferably 5-10 ° C, for a maximum of 6 hours, but preferably for approx. 4 hours.

Compound IV, 1-N- [L - (-) - γ-amino-α-hydroxy-butyryl] kanamycin A (BB-K8) possesses excellent antibacterial activity which is higher than the activity of kanamycin A. The two tables below illustrates the minimum inhibitory concentrations (MICs) of kanamycin A and compound IV (BB-K8) against a number of different gram-positive and gram-negative bacteria, which MICs were obtained by Steers Agar dilution method (Table I) and the double dilution method (Table II). Mueller-Hinton Agar Medium was used in the study in Table I and cardiac infusion was used in the study in Table II.

10 142991

Table X (MIC values mg / ml)

Compound IV

Organism Kanamycin A [BB-K8] (6-8198) Sample # 4

Alk. faecalis A-9423 16 8 "" A-20648> 125> 125

Ent. cloacae A-0656 4 4 "species A-20364> 125 2" hafniae 1 A-20674 1 1 E. coli A-0636 2 1 "" A-20664 16 4 "" A-20665> 125 1 "" A-20507 32 2 "" A-20520> 125 4 "" A-20365> 125 1 "" A-20684 2 2 "" A-20682> 125 2 "" A-20683> 125 8 "" A-20681> 125 2 " "A-15119 4 4 K. pneumoniae A-0967 4 4" species A-20328> 125 2 "" A-20330 32 32 "" A-20634> 125 4 "pneumoniae A-20680> 125 4" "A-0077 1 1

Pr. mirabilis A-9900 2 2

Pr. morganii A-15153 2 2 "vulgaris A-9555 2 1" straightener A-9636 0.25 0.25 "mirabilis A-20645 4 4" "A-20454 2 2"

Providencia stuartii A-20615 2 1 "alkalifaciens A-20676 1 1 11 142991

Table I (continued)

Compound IV

Organism Kanamycin A [BB-K8] (6-8198) Sample # 4

Ps. aeruginosa A-20229 32 2 "" A-0943A 125 16 »" A-20653> 125 32 "species A-20601 125, 63 16" "A-20621> 125> 125" maltophilia A-20620 32> 125

Sole. enteritidis A-0531 1 Of5 "derby A-20087> 125 1

Ser. marcescens A-20019 2 4 Η π A-9933 4 8 "" A-20460> 125 4, 2 "" A-20459 4 16

SHIG. flexneri A-9684 4 4

Aeromonas sp. A-20670 2 2

Arizona sp. A-20671 2 1

Citrobacter sp. A-20673 4 4

Edwardsiella sp. A-20678 4 4

Staph, aureus A-9606 1 1 "" A-4749 0.5 1 "" A-9537 2 1 "" A-20610> 125 2 "" A-20240> 125 8 "" A-15197 1 2

Mueller-Hinton medium + 4% sheep blood

Str. faecalis A-9854 63 63 "" A-9575 125> 125

Str. pyogenes A-20200 32, 16 32 "" A-9604 125 125 "" A-15040 125 125 "" A-20065 125 125 D. pneumoniae A-9585 63, 32 63 "" A-20159 125> 125 12 142991

Table II (MIC values mg / ml)

Compound IV

Organism Kanamycin A [BB-K8] (6-8196) Sample No. 4 D. pneumoniae -5% Serum A-0585 63 63

Str. pyrogenes + 5% serum A-9604 125 125

Staph, aureus Smith A-9537 0.5 0.5 "" A-9497 0.5 0.5 "" A-20239 125 4 "" A-20240 125 4

Enter.cloacae A-0656 2 2

Enter.species A-20364 125 2 K. pneumoniae A-9867 2 4 E. coli K-12ML1410 A-20361 2 4 E. coli K-12 ML1630 A-20363 125 2 E. coli K-12 A-9632 2 1 E. coli A-20664 32 8 E. coli A-20665 125 8

Pr. mirabilis A-9900 2 16

Pr. morganii A-15153 4 16

Pr. vulgaris A-9436 1 2

Ps. aeruginosa A-20227 4 1

Ps. species A-20499 63 4

Ps. aeruginosa A-20653 125 4

Ps. species A-20621 125 125

Ser. marcescens A-20019 2 4 "" A-20141 16 16

The above MIC values show that compound IV (BB-K8) has greater activity than kanamycin A, especially against kanamycin A resistant organisms.

The MIC values also agree well with the results obtained in in vivo testing of kanamycin A and compound IV against three organisms.

Compound IV and kanamycin A were equally effective against infections in mice induced by kanamycin A-sensitive strains of E. coli A-15119 and Staph, aureus A-9537. Although the CD5Q values (curative dose in 50% of lethally infected mice) with regard to Staph, aureus 13-A-9537 suggest that compound IV is slightly less active than kanamycin A, this small difference is probably not significant because the dose concentrations lay far apart (5 x dilution).

As expected, kanamycin A was not particularly effective in vivo against kanamycin-resistant strains of E. coli A-20520, whereas compound IV showed a significant protective effect. Compound IV was ca. 10 times more active against this E. coli strain administered by a 4-treatment regimen than by a 2-treatment regimen.

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15 142991

Compound XV is valuable as antibacterial agent, prophylactic supplement in animal feed, poultry, mammal and human therapeutic agent and is especially valuable in the treatment of infectious diseases caused by gram-positive and gram-negative bacteria.

Compound IV is useful upon oral administration in a treatment for pre-operative sterilization of the viscera. The amount of both aerobic and anaerobic flora sensitive to this agent is reduced in the large intestine. For appropriate mechanical cleansing, the use of Compound IV is useful in the preparation of colon surgery.

Compound IV is effective in the treatment of systemic bacterial infections in humans by parenteral administration in an amount of approx. 250-3000 mg per day divided into doses three or four times a day. In general, the compound is effective in administering at a dose of ca. 5.0 - 7.5 mg / kg body weight every 12 hours.

In the following, Examples 1-9 illustrate the preparation of starting materials, while Examples 10-14 illustrate the process of the invention.

Example 1 5-norbornene-endo-2,3-dicarboxylic anhydride (XVIII).

The compound was prepared according to that of O. 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 benzene was added dropwise 66.1 g (1 mole) of cyclopentadiene under cooling. The mixture was stirred to form a crystalline precipitate which was collected by filtration and recrystallized from ligroin (essentially n-hexane) benzene (1: 1) to give 116 g (71%) of the subject product having a melting point at 164-166 ° C (according to the literature 164-165 ° C).

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

The compound in question was prepared by L. Bauer and S.V. Miarka, J. Org. Chem., 24, 1923 (1959). The norbornene anhydride (XVIII), (115 g, 0.7 mol) was added to a solution of hydroxylamine prepared by adding 47.5 g (0.45 mol) of sodium carbonate to a solution of 60.5 g (0, 87 mol) of hydroxylamine, hydrochloride in 140 ml of water. The mixture was heated at 60-70 ° C for 1 hour and refrigerated overnight. The crystals were collected by filtration, washed with 120 ml of cold 5 N HCl and dried to give 84.9 g of 16.92991 (70%) of compound XIX melting at 172 ° C. The mother liquor was acidified to pH 2 and extracted with CHCl 3 (70 ml x 10). Evaporation of the CHCl3 extracts gave a second portion of compound XIX (7.2 g, 6%) melting at 170 - 172 ° C.

Example 3

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

Ambutyrosine A (5.0 g) [U.S. Patent No. 3,541,078] was refluxed with 160 ml of 0.5 N sodium hydroxide for 1 hour. The hydrolyzate was neutralized with 6 N HCl and chromatographed on a column of "CG-50" (NH 2 + type). The desired L - (-) - γ-amino-α-hydroxybutyric acid was isolated by washing the column with water and removing L - (-) - γ-amino-α-hydroxybutyric acid is a crystalline material having a melting point of 212.5 - 214.5 ° C [column 2, lines 31-38 of U.S. Patent 3,541 0.078].

Example 4

Preparation of L - (-) - γ-amino-α-hydroxybutyric acid from DL-α-hydroxy-γ-phthalimide butyric acid.

A). Dehydroabietylammonium L-g-hydroxy-Y-phthalimide butyrate;

To a solution of 25 g (0.1 mole) of α-hydroxy-γ-phthalimide butyric acid 2 The solution is shaken vigorously in one. and allowed to stand at room temperature for 5 hours, during which time fine needles crystallized. The crystals were collected by filtration, washed with 50 ml of ethanol and air-dried to give 30.1 g (56%) of a diastereomer of the dehydroabietylamine salt. The diastereomeric o 24 had a melting point of 93 - 94 C and a specific rotation [g] D - + 15 ° (c = 2.5, methanol). Recrystallization from 300 ml of ethanol gave 23.2 g (43%) of the pure product. This product had a melting point of 94 - 95 ° C and a specific rotation [α] D = (c = 2.5, methanol). Further recrystallization did not change the melting point and the specific rotation.

Analysis calculated for: C32H42N2 ° 5 "H2 °: C, 69.54; H, 8.02; N, 5.07

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

17 142991 B). L - (-) - γ-Amino-α-hydroxybutyric acid: To a solution of 1.5 g (0.014 mole) of sodium carbonate in 40 ml of water was added 5.3 g (0.01 mole) of dehydroabietylammonium La-hydroxy-Y-phthalimide butyrate and 60 ml of ether. The mixture was shaken vigorously until all 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 10 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 evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The syrupy residue 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 "1R-120" (H +, 1 cm x 35 cm) which 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 10-16 were combined and evaporated under reduced pressure to form a syrup which gradually crystallized. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g (66%) of L - (-) - γ-amino-α-hydroxybutyric acid, mp 206-207 ° C. Specific rotation [α] j ^ = 29 ° (c = 2.5, H₂O). The IR spectrum was identical to that of the authentic sample obtained from ambutyrosine.

Example 5

Preparation of L - (-) - Y-benzyloxycarbonylamino-α-hydroxybutyric acid (VI).

L - (-) - γ-amino-α-hydroxybutyric acid (7.4 g, 0.062 mol) was added to a solution of 5.2 g (0.13 mol) of sodium hydroxide in 50 ml of water. To the stirred solution was added dropwise at a temperature of 0-5 ° C over a period of 0.5 hour 11.7 g (0.068 mole) of carbobenzoxy chloride and stirring continued for an additional 1 hour at the same temperature. The reaction mixture was washed with 50 ml of ether, the acidity was 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 over anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue crystallized from benzene to give 11.6 g (74%) of colorless plate crystals having a melting point of 78.5 - 79.5 ° C and a specific rotation of [a] D = - 4.5 ° (c = 2, CH 2 OH). IR spectrum (KBr): Yc_O = 1740, 1690 cm NMR spectrum (acetone-dg) cf (in ppm from TMS) = 2.0 (2H, m), 3.29 (2H, dd, 18 6.7 and 12 Hz), 4.16 (1H, dd, J = 4.5 and 8 Hz), 4.99 (2H, s), 6.2 (2H, wide), 7.21 (5H, s).

Analysis calculated for C C HH ^NOgi C, 56.91; H, 5.97; N, 5.53

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

Example 6 The N-hydroxy-5-norbornene-2,3-dicarboximide ester of L-γ-benzyloxycarbonylamino-g-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-γ-benzyloxycarbonylamino-α-hydroxybutyric acid VI and 1, 79 g (0.01 mole) of compound XIX in 50 ml of dry tetrahydrofuran. The mixture was stirred overnight at room temperature. The precipitated dicyclohexylurea was removed by filtration and washed with tetrahydrofuran. 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. IR spectrum (Neat): 3300, 1820, 1780, 1735, 1720, 1695 cm NMR spectrum (CDClCl + 1 drop D₂O): δ f (in ppm) = 1.63 (2H, q, J = 9Hz), 2.15 (2H, m), 3.40 (6H, m), 4.60 (1H, dd, J = 7 and 5Hz), 5.13 (2H, s), 6.20 (2H, wide) , 7.40 (5H, s).

Example 7 N-Benzyloxycarbonyloxy-5-norbornene-2,3-dicarboximide (XVII).

To a stirred solution of 2.64 g (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-dicarboximide XIX at 10 ° C. To the solution was added dropwise 6.82 g (0.04 mole) of benzyloxycarbonyl chloride over a period of 40 minutes and at 0 - 5 ° C, after which the mixture was stirred for 4 hours at room temperature. To the reaction mixture was added 20 ml of n-hexane to form a 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%) of colorless prismic crystals of compound XVII), having a melting point of 121 - 122 ° C.

IR spectrum (KBr) = 1810, 1780, 1740, 1630 cm NMR spectrum (acetone-dg): rf'd ppm) = 1.66 (2H, broad s), 3.45 (4H, m), δ , 37 (2H, s), 6.12 (2H, t, J = 1.5 Hz), 7.48 (5H, s).

For C forΟH₂Ο: C, 65.17; H, 4.83; N, 4.47.

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

IS-142991 61-N-benzyloxycarbonylcanamycin A (II) [6'-Cbz) -canamycin A].

To a stirred solution of 4.84 g (0.01 mole) of the free kanamycin A base in 50 ml of 50% aqueous tetrahydrofuran was added, divided into several portions, 3.14 g (0.01 mole) of compound XVII over for a period of 1 hour at 7 ° C. The mixture was still stirred for 4 hours and evaporated in vacuo to remove the organic solvent. 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 washings were combined and concentrated in vacuo to ca. 50 ml. The aqueous solution was passed through a column of "CG-50" ion exchange resin (NH4 +, 140 ml), which was eluted successively with 200 ml of water, 1.9 liter 0.05 N NH40H, 1.9 liter 0.1 N NH40H and finally 1 liter of 0.3 N NH 4 OH. The eluate was collected in 20 ml fractions and divided into the following two fractions based on the Rf value by 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 dried in vacuo over phosphorus pentoxide.

Fraction Glass No. Eluted with Quantity Contents 1 106-144 0.1 N NH40H 3.44 g 6'-Cbz-kana (56%) mycine (II) 2 203-212 0.3 N NH40H 0.54 g kanamycin (11%) recovered.

The 6'-Cbz-kanamycin (II) thus prepared is very pure and homogeneous due to thin layer chromatography on a silica gel plate and had a melting point of 217-219 ° C. The physicochemical properties were consistent with the properties of the authentic sample.

Example 8

Preparation of N- (benzyloxycarbonyloxy) succinimide.

N-hydroxysuccinimide 1 (23 ° 2 mol) ° Dissolve in a solution of 9 g (0.22 mol) sodium hydroxide in 200 ml of water. To the stirred solution was added dropwise 34 g (0.2 mole) of carbobenzoxychloride under water cooling, and then the mixture was stirred at room temperature overnight to separate the carbobenzoxide derivative, which was collected by filtration, washed with water and air-dried. The yield was 41.1 g (82%). Recrystallization from benzene-n-hexane (10: 1) gave colorless prism crystals melting at 78-79 ° C.

1. G.W. Anderson et al., J. Am. Chem. Soc., £ 6, 1839 (1964).

20 142991

Example 9

Preparation of 6'-carbobenzoxycanamycin A (II).

A solution of 42.5 g (90 mmol) of the free base of kanamycin A in 450 ml of water and 500 ml of dimethylformamide was cooled to below 9 ° C and stirred vigorously. To the solution, a solution of 22.4 g (90 mmol) of N- (benzyloxy-carbonyloxy) succinimide in 500 ml of dimethylformamide was added dropwise over a period of 2 hours. The mixture was stirred at -10 ° C - 0 ° C overnight and then at room temperature for 1 day. The reaction mixture was evaporated under reduced pressure at a temperature of less than ca. 50 ° C. The oily residue was dissolved in a mixture of 500 ml of water and 500 ml of butanol, filtering the mixture to remove insoluble material and separating it into two layers. The butanol layer and the water layer were treated with butanol-saturated water (500 ml x 2) and water-saturated butanol (500 ml x 2), respectively, using a countercurrent distribution technique. The three aqueous layers were combined and evaporated to dryness under reduced pressure to give an oily residue, part of which crystallized on standing at room temperature. To the residue, including the crystals, was added ca. 100 ml of methanol which dissolved the oil and separated it from the crystals. After adding approx. 300 ml of ethanol were kept at room temperature overnight to form a crystalline mass which was collected by filtration. The weighed 44 g. The product contained a small amount of kanamycin A, which was demonstrated by thin layer chromatography using n-propanol-pyridine-acetic acid water (15: 10: 3: 12) as solvent system and ninhydrin as a developing agent.

The crude product was dissolved in 300 ml of water and chromatographed on a column (30 mm diameter) of "cG-501 ion exchange resin (NH4 + type, 500 ml). The column was quenched with 0.1 N ammonium hydroxide solution and the eluate was collected in 10 ml The desired product was found in glass # 10-100, while kanamycin A was recovered from slower moving fractions and the position isomer (s) of the product appeared to be in faster moving fractions. pressure to form 24.6 g (45%) of a colorless product (6-carbo-benzoxycanamycin A (II) [6r-Cbz-kanamycin A], which began to melt and assume color - at 204 ° C and decompose at 212 ° C during gas evolution. Specific rotation [α1D + 106 ° (c = 2, 10 °). Thin layer chromatography (silica gel "F254" / ninhydrin).

21 U2991 _Rf value__

Solvent system 6'-Cbz-kanamycin A Kanamycline A

n-1 propanol-pyridine 0.42 0.33 0.15 / 0.04

acetic acid-H-O

(15: 10: 3: 12) (main (minor constituent) part) acetone acetic acid H 2 O 0.24 0.14 (20: 6: 74) CHCl 3 -methanol jug. 0.76 0.50 (1: 4: 2: 1) * o ηΛ * ethyl acetate-n-propanol-kc) nc 0.22 0.04 (45: 105: 60) * Detected using anthronic sulfuric acid.

The final product was found to be accompanied by two minor constituents by thin layer chromatography with one of the solvent systems tested. However, the final product was used without further purification to prepare BB-K8 (IV).

Example 1Q

1-N- [I - (-) - γ-amino-g-hydroxybutyryl] kanamycline A (BB-K8, IV) via benzaldehyde Schiff base.

To a stirred solution of 6.19 g (0.01 mole) of 6'-Cbz-kanamycin (II) in 50 ml of 50% tetrahydrofuran was added 3 ml (0.03 mole) of benzaldehyde. The mixture was stirred for 3 hours at room temperature, cooled to 5 ° C and then treated with a solution of 4 g (0.01 mole) of compound XX in 50 ml of tetrahydrofuran. The mixture was stirred for 1 hour at 5 ° C, the acidity was adjusted to pH 2 with 6N HCl, then the mixture was stirred for an additional 1 hour at room temperature and then evaporated in vacuo to remove the organic solvent. The resulting aqueous solution was washed with 20 ml of CH 2 Cl 2 / acidity adjusted to pH 4 with 10% NH 2 OH, then the mixture was hydrogenated overnight under atmospheric pressure and at room temperature using 1.25 g of 10% palladium-on-carbon. The catalyst was filtered off and washed with 20 ml of water. The filtrate and washings were combined, the acidity was adjusted to pH 8 with 10% NH 2 OH, and the mixture was passed through a column of * fcG-50 '' (NH 2+, 200 ml), eluting with 0.4 liters of water, 1 2 liters of 0.1 N NH40H, 2.06 liters of 0.3 N NH40H, 0.92 liters of 0.5 N NH40H and finally 1.2 liters of 1.0 N NH40H. The eluate was collected in 20 ml fractions and subdivided into the following fractions of 22 14299 1 based on the Rf values by thin layer chromatography on silica gel plate (S-110, ninhydrin) and slice samples using B. subtilis PCI 219 and P. aeruginosa A 9843. fraction was evaporated in vacuo and lyophilized.

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Fraction 4 (1.877 g) was dissolved in 10 ml of deionized water. The acidity of the solution was adjusted to pH 6 with 10% HCl, and it was adsorbed on a 'bG-50' column (NH 2+, 15 ml), eluting with 100 ml of 0.3 N NH 2 OH and 200 ml of 0, 5 N NH 2 OH The eluate was collected in 20-ml fractions Glass Nos. 6 and 8-17 were ninhydrin positive, Glass Nos. 8-17 were combined and concentrated in vacuo to about 1 ml. The concentrate was treated with 3 ml of methanol. The filtrate and washings were combined, diluted with a further 10 ml of methanol and allowed to stand overnight at room temperature. Compound IV, which was found to be identical to an authentic sample, the filtrate was evaporated in vacuo and lyophilized, yield and test of each fraction are shown in the table below.

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a> Η β · Ρ φ Φ φ> β Μ Η β Φ Η β φ Μ φ ft ι-Η * β φ Η ΓΗ β +> Φ -Η • Η 4-1 -Ρ, β Λ β W ft β Ο > 1 0 Ο S β> 1 ft <3 W ιβ 26 142991 —B ~ ff. - * - · * - is 3 "-N- [L - (-) - γ-amino-α-hydroxybutyryl] -canamycin A.

SB ~ ff 29 is 3-N- [L - (-) - γ-amino-α-hydroxybutyryl] -canamycin A.

Example 11

Preparation of the monosulfate salt of 1-N- [L - (-) - γ-amino-α-hydroxybutyryl] kanamycin A.

One mole of 1-N- [L - (-) - γ-amino-α-hydroxybutyryl] kanamycin A was dissolved in 1-3 liters of water. The solution was filtered to remove any undissolved solids. To the cooled and stirred solution was added one mole of sulfuric acid dissolved in 500 ml of water. The mixture was stirred for 30 minutes, then cold ethanol was added to the mixture until precipitation occurred. The solids were collected by filtration and found to be the desired monosulfate salt.

Example 12

Preparation of the disulfate salt of 1-N- [L- (~) -γ-amino-α-hydroxybutyryl] kanamycin A - (BB-K8 · 2 ^ 50 ^).

35 g of 1-N- [L - (-) - γ-amino-α-hydroxybutyryl] kanamycin A (in the form of monohydrocarbonate, the trihydrate) was dissolved in 125 ml of deionized water. The pH was found to be approx. 9.0. The pH was lowered to 7-7.5 with 50% by volume sulfuric acid.

8½ g of "Darco G-60" (activated charcoal) was added and the mixture was slurried at room temperature for half an hour. The activated charcoal was removed by appropriate filtration and washed with 40 ml of water. The wash water was added to the filtrate.

The acidity of the aforementioned combination of filtrate and wash water was adjusted to pH 2 - 2.6 with 50% by volume sulfuric acid.

A large amount of carbon dioxide was developed. The solution was allowed to stand under vacuum and stirring for 20 minutes to evaporate additional carbon dioxide.

8½ g of "Darco G-60" was added to the degassed solution. The mixture was slurried for half an hour at room temperature. The activated charcoal was removed by appropriate filtration and washed with 35 ml of deionized water. The water was added to the filtrate.

The acidity of the filtrate-wash water combination was adjusted to pH 1 - 1.3 with 50% by volume sulfuric acid. This solution was added with rapid stirring over 10 minutes to 600 - 800 ml of methanol (3-4 volumes of methanol). The mixture was stirred for 5 minutes at pH 1 - 1.3, passed through a 100 mesh sieve, stirred for 2 minutes and allowed to settle for 5 minutes. Most of the liquid above was decanted away. The residual slurry was appropriately filtered, washed with 200 ml of methanol and vacuum dried at 50 ° C for 24 hours. The yield of amorphous BB-K8 (dihydrogen sulfate) 9 was 32 - 34 g and had a specific rotation of [a) D = +74.75 (H + O) and decomposed at 220 - 223 ° C.

JFI

Elemental analysis (on a 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 zero * Karl Pisher water content: 2.33, 1.79, 2.87% (theoretical water content for the monohydrate is 2.25%), This salt is hygroscopic , but not fluent. After storing a sample in the air at room temperature for 18 hours, the water content was increased to 9.55, 9.89% (theoretical water content of the pentahydrate is 10.33%).

Example 13

A) 61-carbobenzoxy-1,3,3 "-trisalicylalkanamycin A

6'-carbobenzoxycanamycin A (9.0 g, 14.5 mmol) was suspended in 500 ml of absolute ethanol at 24 ° C and 58.2 mmol 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 formed. The mixture is heated at reflux for 3 hours, then cooled, and the product is collected by filtration and washed with a small amount of absolute ethanol. The melting point after recrystallization from 4: 1: 5 tetrahydrofuran-methanol-heptane and drying was 196 - 198 ° C.

Analysis calculated for C 47 H 54 N 4 ° C: C, 60.62; H, 5.86; N, 6.02

Found: C, 58.67; H, 5.73; N, 5.98

Karl Fisher water content = 2.45.

Found analysis corrected for water: C, 60.14; H, 5.60; N, 6.13.

B) Preparation of 1-N- [L - (-) - γ-amino-g-hydroxybutyryl] kanamycin A via the in situ process.

In a suitable apparatus, 1000 g (1,074 mol) of 6 * -carbo-benzoxy-1,3,3H-trisalicylal-kanamycin A is stored in 11,400 ml of tetrahydrofuran at 22-25 ° C. With stirring, 600 ml of water was added. The solid dissolved in approx. 10 minutes. In the form of solids, 181.5 g (0.716 mole) of 4-benzyloxycarbonylamino-2-hydroxybutyric acid and 0.12 mole of N-hydroxy-5-norbornene-2,3-dicarboximide were added to the solution. The pH value is 5.5 - 6.0. Maintaining a temperature of 22-25 ° C, 162.3 g (0.787 mole) of dicyclohexylcarbodiimide dissolved in 3000 ml of dry tetrahydrofuran was added. The slurry was stirred for 24 hours at 22-25 ° C and then cooled to 0-5 ° C to crystallize the dicyclohexylurea over 2 hours.

The urea was removed by filtration and the filter cake washed with 800 ml of a tetrahydrofuran-water mixture (95: 5). The filtrate and washings were combined and the volume reduced in vacuo to 3000 ml. 4000 ml of methanol was added and the volume again reduced in vacuo to 3000 ml.

To the solution thus obtained was added 2000 ml of absolute methanol at 40 ° C, and the solution was seeded with 6'-carbobenzoxy-1,3,3 "-trisali-cylal-kanamycin A and allowed to cool to precipitate crystals in Then, 2000 ml of water was added over half an hour with good stirring at about 22 - 25 ° C for 2 hours, then the temperature was reduced to 0 - 5 ° for 1 hour.

The solids were removed by filtration and washed with 900 ml of a cold methanol-water mixture (2: 1) and then with 800 ml of methanol. The filtrates and washings are stored. The filter cake consisted essentially of 6'-carbobenzoxy-1,3,3 "-trisalicylalkanamycin A.

The above filtrates were diluted with 1000 ml of water. 2000 ml of methylene chloride was added. The pH was lowered at 25 ° C to 1.8 - 2.0 with 6 N HCl. The pH was kept at this value for half an hour.

The methylene chloride layer was separated and the aqueous phase washed with an additional 2000 ml of methylene chloride. The combined organic phases are stored for possible salicylaldehyde recovery.

The pH of the aqueous phase was raised to 3.5 - 4.0 with concentrated ammonia water. This solution was hydrogenated at room temperature at a hydrogen gas pressure of 345 kPa in the presence of 5% Pd / C. The biologically active yield of the desired product in solution was 30 -45% of theory.

Example 14_

Preparation of Compound IV using Various N-Hydroxy-5-Norbornene-2,3-Dicarboximide Mole Ratio while Maintaining the Other Mole Ratio.

By replacing in Example 13 B the 0.12 moles of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) used with the mole percentages given below, the percent yields of compound IV obtained were obtained:

Claims (3)

29 142991 Experiment Mo1% HQNB% yield of Compound IV 1 0 9.33 2 2 20.07 3 10 31.72 4 50 36.52 5 100 36.01 From the above it can be seen that Compound IV can be prepared in complete absence of N-hydroxy-5-norbornene-2,3-dicarboximide. However, it can also be seen that the best yields of compound IV are obtained when N-hydroxy-5-norbornene-2,3-dicarboximide is used in an amount which constitutes 10 - 50 mol% of the molar amount used in the acylation reaction 4 -benzyloxycarbonylamino-2-hydroxybutyric acid. The exact mechanism by which the blocked kanamycin A is acylated is unknown. However, it is observed that the presence of N-hydroxy-5-norbornene-2,3-dicarboximide somehow yields better yields than previously obtained. A process for the preparation of 1- [L - (-) - γ-amino-α-hydroxy-butyryl] -canamycin A of the formula CHp-KH? BO Λ —H — x CHpOH HO Λ— E ° —s. I / NH yv - / c = o h With benzaldehyde, salicylaldehyde, p-nitrobenzaldehyde, p-methoxybenzaldehyde or pivaldehyde, with at least three moles of aldehyde are used per day. mole of compound II, to prepare the compound of formula K? CH 2 N-C-O-CiL-arHc sk III 31 142991 wherein Z represents a group of the formula Η H -N = lfy «° 2, -» = C-O, -N «CHØ-OCH3. "V \ H / C" 3 -Μ * ° Ό or - "* C 'C \ CH3' CBj upon which (B) is treated the compound of formula III in situ or after isolation with an N-hydroxyimide ester of the acid of formula 0? II-HO-C-CH-CH 2 -CH 2 -NH-CO-CH 2 -C 6 H 5 OH and then the resulting product is hydrogenated in situ to prepare the compound of Formula IV which, if desired, is converted to a pharmaceutically useful salt thereof, characterized wherein the N-hydroxyimide ester of said acid is optionally formed in situ and has the formula? II \ | ^ oc-ch-ch2-ch2-nh-co-ch2 ~ c6h5 OH ^ II o XX and at least 0 is used; 5 moles of compound (XX) per mole of compound (III) or, when compound (XX) is formed in situ, to form in an amount at least equal to 0.5 mole per mole of compound (III).