KR800001590B1 - Process for the preparation of 1-(l-(-)-r-amino- -hydroxy-butyryl)-kanamycin a - Google Patents

Abstract

Title antibiotic compd.(I; R = MECOCH(OH)CH2CH2NH2; R1 = NH2; R2 = H)(II) was prepd., e.g., by reacting 1 mole I(R = R1 = NH2; R2 = CO2CH2Ph)(III) with >3 moles BzH, o-HOC6C6H4CHO, p-NO2C6H4CHO, p-NO2C6H4CH:N, p-MeOC6H4CH:N, tert-BuCH:N, R2 = CO2CH2Ph) with >0.5 mole IV, and hydrogenating. Thus, 6.19 g III in 0% aq. THF were stirred with 3 ml BzH 3 hr at room temp., 4g IV added at 5↿, the mixt. stirred hr at 5≰C, made pH 2,stirred 1 hr at room temp., concd., the aq. residue washed with CH2CL2, made pH 4, hydrogenated on 1.25 g 10% Pd-C overnight at room temp., and chromatographed to give 483mg II.

Description

1- [L-(-)-[gamma] -amino- [alpha] -hydroxy-butyryl] -manufacturing method of kanamycin A

The present invention relates to a method for preparing 1- [L-(-)-γ-amino-α-hydroxy-butyryl] -kanamycin] A of the following structural formula (IV) as a novel antibiotic.

Kanamycin A has four primary amine groups at the 1,3,6 ′ and 3 ″ positions of the molecule. When reacted with an electrophilic material, the amine group in the 6'-position is the most active and the amine group in the 1-position is the second most active. The amine groups in the 3- and 3 ″ -positions are less active than the amine groups in the 1- or 6′-positions, but react with each other to produce a smaller proportion of unnecessary acylated materials.

Accordingly, the object of the present invention relates to a more satisfactory manufacturing method than the manufacturing method of US Patent Nos. 221,378 and 330,377 already invented by our collaborators and others. The above-mentioned US patent formulation was to selectively acylate only amine groups in the 1-position, leaving other amine groups in the molecule.

The object of the invention relates to a process for the preparation of the following general formula (IV) compounds, which are prepared by the following reaction steps.

A) The following general formula (II) compound is reacted with an aldehyde such as benzaldehyde, silyl aldehyde, P-nitrobenzaldehyde, P-methoxybenzaldehyde and fibaldehyde, and the aldehyde ratio for compound (II) is at least 1 Reaction with 3: yields the following structural formula (III) compound.

In the formula, Z represents a compound of the following structural formula.

B) The compound of formula (III) is reacted with the following general formula (XX) without separating the compound, wherein the ratio is reacted at a ratio of 1: 0.5 molar ratio and then hydrocracked to produce the compound of formula (IV). Obtained.

In particular, as the solvent miscible with water in the first step, that is, the alcoholic solvent, anhydrous ethanol, ethanol, n-propanol, isopropanol, n-butanol, secondary-butanol, tert-butanol; As ether solvent it is suitable to react in tetrahydrofuran, dioxane or other polar aprotic or protic solvents such as dimethylformamide and acetone or mixtures thereof or mixtures of these with water, at about 5 ° C. at reflux temperature. It is appropriate to react for 30 minutes-5 hours.

In step (b), the ratio of the compound (XX) to the compound (III) is about 1: 1 molar ratio, and the acylation in the temperature range of about −10 ° C. to 35 ° C., more preferably about 5 ° C. to 25 ° C. React until finished but at least 1-3 hours is appropriate; Hydrocracking includes metal catalysts such as palladium, platinum, rani-nickel, rhodium, rudenium and nickel, but palladium is suitable and most suitable is a solvent that blends well with water or water in the presence of palladium on activated carbon, ie pH3-5 Is reacted with hydrogen in water, most preferably at pH 4 water.

General preparation of the compound of general formula (IV) is as follows.

The following structural formula (II) compound,

Aldehydes such as benzaldehyde, silyl aldehyde, P-nitrobenzaldehyde, and P-methoxyaldehyde in an amount of 1: 3 molar ratio, with anhydrous ethanol, methanol, n-propanol, secondary-butanol, tert-butanol, methylene chloride And reacted for about 30 minutes to 5 hours in tetrahydrofuran, dioxane, dimethylformamide or acetone, or water and a mixture thereof at a temperature of 5 ° C.-40 ° C. to obtain the following compound of formula (III).

In said formula, Z represents the functional group of the following structure.

B) The compound (III) produced in the above reaction is left as it is or separated, and then reacted with the following structural formula (XX).

The organic solvent was removed after reacting the molar ratio of the compound (XX) to the compound (III) to about 0.5: 1.0 at a temperature of 5 ° C.-35 ° C. for 1-3 hours in the solvent system described in step (1). And the residue is in the presence of a metal catalyst such as palladium, platinum, rani-nickel, rhodium, rudenium and nickel, suitably palladium, more suitably palladium on activated carbon, and is well mixed with water or water, i.e. dioxane, PH 3-5 of tetrahydrofuran, ethylene glycol dimethyl ether and propylene glycol dimethyl ether are suitably reacted in water at pH 4 to obtain compound (IV).

Another suitable process for the preparation of compound (IV) according to the present invention is as follows.

A) the following general structural formula (II) compound,

It reacts with aldehydes, such as benzaldehyde, salicylic aldehyde and P-nitro benzaldehyde, in an aldehyde ratio of compound (II) in a 1: 3 molar ratio of anhydrous ethanol, ketanol, n-propanol, isopropanol, n-butanol, 2 Reacted for about 30 minutes-5 hours at about 5 ° C.-40 ° C. in a tert-butanol, tert-butanol, tetrahydrofuran, methylene chloride, dioxane, dimethylformamide or acetone or a mixture of these with water (III) A compound is obtained.

In said formula, Z is a group of the following structure.

B) Compound (III) as it is or isolated, and then reacted with the following general structural formula (XX), but the molar ratio of compound (XX) to compound (III) is about 0.5: 0.85 moles, and the temperature range is about 15 ° -30 ° C. For about 1-3 hours at a) reacting in the solvent system mentioned in step a), removing the organic solvent, and then palladium, platinum, rani-nickel, rhodium, rudenium and nickel, suitably palladium, more preferably activated carbon In the presence of a metal catalyst such as palladium on the water, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether and propylene glycol dimethyl ether are preferably hydrolyzed with hydrogen at pH about 4 in water to obtain the compound of formula (IV).

The most suitable method for preparing the compound of formula IV is as follows.

A) For 1 mole of the compound of the following general structural formula (II)

In a molar ratio of benzaldehyde 3, it is reacted in a mixture of water and tetrahydrofuran (about 1: 1) at a temperature of about 10 to 30 ° C. at a pH of about 10 for about 2-4 hours to obtain the following structural formula (IIIa).

B) Compound (IIIa) as it is or isolated and reacted with the following general structural formula (XX),

The molar ratio of (XX) to (IIIa) is about 0.6: 0.75 and reacted at about 20 ° C-30 ° C for 1-3 hours in the solvent system mentioned in step (A). Solvents include mixtures of methylene chloride, methanol and water or mixtures of dimethylformamide, acetone and water. The solvent is removed from the reaction mixture under reduced pressure, adjusted to pH 4 with ammonium hydroxide, and the residue is hydrocracked with hydrogen in the presence of palladium on carbon at atmospheric pressure as is to obtain a compound of formula IV.

The most suitable method for preparing the compound of formula IV according to the present invention is as follows.

A) the following general structural formula (II) compound,

React with salicylic aldehyde at a molar ratio of about 1: 3 in a mixture of water and tetrahydrofuran (1: 1) at about 20 ° C.-30 ° C. for 2 hours to 4 hours at the pH of about 8 hours. Obtain the compound.

B) Compound (IIIb) is left as it is or isolated, and then reacted with the following general structural formula (XX) compound, with the molar ratio of about 0.6: 0.75 at about 20 ° C.-30 ° C. for about 1 hour-3 hours, The reaction is carried out in the solvent system or the compound of dimethylformamide, acetone and water mentioned in step), and then the organic solvent is removed under reduced pressure, adjusted to pH 4 with ammonium hydroxide, and then hydrocracked with hydrogen in the presence of palladium on carbon at atmospheric pressure. To obtain the compound of formula (IV).

In the present invention, (lower) alkyl refers to a linear or branched alkyl residue of 1-6 carbon atoms, and (lower) alkanol refers to a straight or branched saturated alcohol having one -OH group.

It is a further object of the present invention to transform the preparation process as described above into a process which yields a higher yield of structural formula (IV) compounds.

For example, the compound of formula (XX) is prepared and reacted with the compound of formula (III) in any convenient step. Thus, the compound of formula (IV) can be prepared as described in step (b), and the reaction is carried out by the general formula (IV) after isolating or isolating compound (III) obtained in step (a) as already mentioned. ) And (XIX) compounds and then

Dicyclohexyl carbo diimide is added. At this time, about 0.5-0.1 mol of compound (IV) and dicyclohexylcarbodiimide are reacted with respect to compound (III), about 0.005-0.25 mol of compound (XIX) is reacted, and about -10 degrees-35 degreeC Reaction in the solvent system described in step (a) at a temperature of about 5 ℃ to 25 ℃ for at least 10 hours, and then the organic solvent is removed, the residue is hydrogenated and the structural formula ( IV) to generate the compound.

In the above modification, the reaction is carried out at a ratio of about 0.5-0.85 moles and about 0.05-0.425 moles of compound (XIX) to 1 mole of compound (III), respectively, in a temperature range of 5 ° C-25 ° C. Reacting in the solvent system mentioned in step (a) for 1-3 hours at < RTI ID = 0.0 > C, < / RTI > or, if necessary, compound (IV) and dicyclohexylcarbodiimide per mole of compound (III) About 0.5-0.8 mol each, and about 0.05-0.45 mol ratio of compound (XIX), it can also react for 15-20 hours at 12 degreeC-30 degreeC temperature. The most suitable reaction conditions were about 0.6-0.75 mol ratio of compound (VI) and dicyclohexyl carbodiimide, respectively, with respect to 1 mol of compound (III), and about 0.06-0.375 mol of compound (XIX), and the temperature range 20 degreeC- The reaction is carried out at 30 ° C. for 15-25 hours.

The present invention also encompasses new and improved methods for the preparation of compounds of formula II as starting materials.

That is, the free base of kanamycin A is reacted with N-benzyloxy-5-norbornene-2,3-dicarboxyimide, with about 0.5-1.5 moles of N-benzyloxycarbonyloxy-5 per mole of kanamycin A. The reaction is carried out at a ratio of norbornene-2,3-dicarboxyimide. Particularly suitable molar ratio is about 1: 1, extracted with a solvent such as n-butanol, and the aqueous layer is purified by chromatography.

The reaction consists of an organic solvent that is well miscible with water, for example tetrahydrofuran-water, more preferably in a temperature range of about 0 ° C.-25 ° C., more preferably about 5 in about 40-60% tetrahydrofuran. The reaction is carried out at a temperature of 10 ° C. for 6 hours, more preferably about 4 hours.

Compound (IV), 1-N- [L-(-)-[gamma] -amino- [alpha] -hydroxybutyryl] canamycin A (BB-K8) has better antibacterial activity than cannamycin A itself. In the two tables below, the minimum inhibitory concentrations of kanamycin A and compound (IV) against Gram-positive and Gram-negative bacteria are shown by the steers agar-dilution method (Table 1) and the double dilution method (Table 2). . Table 1 used Mueller-Hinton agar medium and Table 2 used Heart-Infusion medium.

TABLE 1

TABLE 2

The MIC data showed that Compound IV (BB-K8) is superior to kanamycin A, and is particularly active against kanamycin A resistant bacteria.

In vivo activity relationships for the three strains for kanamycin A and compound (IV) are shown in the MIC data.

Compound (IV) and kanamycin A were equally effective in the infection of mice by kanamycin A-sensitive strains, namely, E. coli A-15119 and Staphylococcus aureus A-9537. Although CD ₅₀ levels (50% therapeutic dose of lethal infectious mice) against Staphylococcus aureus A-9537 were less active than compound (IV) than kanamycin A, this minor difference was associated with Is incredible because of its distance (5x dilution).

As expected for kanamycin resistant bacteria, i.e., Coli A-20520, kanamycin A showed no effect in an in vivo experiment compared with the superior antibacterial action of compound (IV). Compound (IV) had about 10-fold activity against E. coli.

TABLE 3

MIC = Minimum Inhibitory Concentration (ug./ml.) Tested by the method by Kisolm and its co-investigators (Antibiotic and Chemotherapy-1969, page 244, 1970) using Müller-Hinton agar pear as test medium.

CD ₅₀ = Treatment, 50% (mg./kg. Trial x Dosing) Two mice administered subcutaneously at 1 and 4 hours post infection and four doses at 0, 2, 4 and 6 hours post infection It was injected subcutaneously. Others were tested by Purice's method (Anti-Hairy 22: 1,1969).

C- = Not tested.

Compound (IV) is very useful as an antibiotic, a nutritional additive in animal feed, a therapeutic agent for poultry and livestock, including humans, and particularly for infectious diseases caused by Gram-positive and Gram-negative bacteria.

When orally administered Compound (IV), it is useful as a preoperative sterile treatment aid of the colon. Both aerobic and basic bacteria susceptible to the drug are reduced in the gut. When used in combination with a suitable mechanical cleanser it is useful for preparing for enteral surgery.

Compound (IV) is effective for treating systemic bacterial infections in humans when intravenously injected about 5mg-300mg 3-4 times a day. Generally, the compound is effective when about 5.0-7.5 mg / kg body weight is administered every 12 hours.

Example 1

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

Preparation of the compound is O. Dills and K. Alder. Prepared by the method of A.460,98, 1928, 66.1 g (1 mol) of cyclopentadiene are added dropwise under cooling with stirring a suspension of 98.1 g (1 mol) of maleic anhydride in 500 ml of dry benzene. The mixture is stirred, filtered and recrystallized with ligroin (especially n-nucleic acid): benzene (1: 1) to give 116 g (17%) of the above compound in crystalline form.

Melting point 164-166 ° C (the melting point is 164-165 ° C in literature)

Example 2

N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide (HONB) (XIX)

Preparation of the compound is L. Bauel and S. V. By the method of Miarka (organic chemistry magazine, 24, 1293, 1959). Norbornene anhydride (XVIII), (115 g, 0.7 mol) is added to a hydroxyl amine solution prepared with a solution of 47.5 g (0.45 mol) of sodium carbonate and 60.5 g (0.87 mol) of hydroxylamine hydrochloride in 140 ml of mol. The mixture is heated to 60 ° -70 ° C. for 1 hour and left overnight in the refrigerator. The crystals were filtered off, washed with 120 ml of 5N cold hydrochloric acid and dried to yield 84.9 g (70%) of XIX having a melting point of 172 ° C. The mother liquor is acidified to pH ₂ and extracted with chloroform (70mlX ₁₀ ). Evaporation of the chloroform extract yielded 1 × IX compound (7.2 g: 6%) having a melting point of 170 ° C.-172 ° C.

Example 3

N-hydroxy-5-norbornene-2,3-dicarboxyimide ester of L-γ-benzyloxycarbonylamino-α-hydroxybutyric acid (XX)

Dicyclonucleosilcarbodiimide (DDC) (22.06 g, 0.01 mol) at 10 ° C., 2.53 g (0.01 mol) of L-γ-benzyloxycarbonylamino-α-hydroxybutyric acid and dry THF (tetrahydro) Furan) 50 ml. It is added to 1.7 g (0.01 mol) of stirring solution in XIX. The mixture is stirred overnight at room temperature. The precipitated dicyclonucleus urea is filtered off and washed with THF. The filtrate and washings are combined and evaporated under reduced pressure. The oily residue (4.5 g) is passed through a silica gel column which is then dried with ethyl acetate to give 4.0 g (96%) of the colorless oily active ester.

IR spectra (IR; knit): 33000, 1820, 1780, 1735, 1720, 1695 ml ^-1 electromagnetic resonance spectra NMR (CDCl ₃ drop of water: δ (ppm), 1.63 (2H, q, J = 9 Hz), 2.15 (2H) m, 3.40 (6H, M), 4.60 (1H, dd, J = 7 and 5 Hz), 5.13 (2H, S), 6.20 (2H, broad), 7.40 (5H, s).

Example 4

50 ml of N-benzyloxycarbonyloxy-5-norbornene-2,3-dicarboxyimide (XVII) water. 3.58 g. (0.02 mol) of N-hydroxy-5-norbornene-2,3-dicarboxyimide XIX is added at 10 degreeC, stirring the solution of 2.64 g. (0.066 mol) of sodium hydroxide in it. . 6.82 g. (0.04 mol) of benzyloxycarbonyl chloride was added dropwise to the mixture at 0-5 ° C. for 40 minutes, followed by stirring at room temperature for 4 hours. To the reaction mixture, 20 ml of n-nucleic acid is added to form a white precipitate which is filtered and air dried. Crystallization of the crude product with benzene-n-nucleic acid gave 5.1 g of colorless prism XVII. Obtained. : Melting point 121-122 ° C,

IR (KBr): 1810, 1780, 1470, 1630 cm ^-1 , NMR (acetone-b6): δ (ppm), 1.66 (2H, broad), 3.45 (4H, m), 5.37 (2H, s), 6.12 ( 2H, t, J = 1.5Hz), 7.48 (5H, s),

Analysis: C ₁₇ H ₁₅ NO ₅

Calculated: CC, 65.17; H, 4.83; N, 4.47

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

Example 5

6'-N-bentioxycarbonyl kanamycin A (II)

[6'-Cbz-Kanamycin A]

50 ml of 50% aqueous THF solution. To a solution of 4.87 g. (0.01 mol) of kanamycin A free base in water is added 3.14 g. (0.01 mol) of XVII at 7 ° C. for several hours with stirring. The mixture is stirred for 4 hours and evaporated to remove the organic solvent under vacuum. The resulting aqueous solution was diluted with 150 ml of water and 100 ml of water-saturated butanol. Extract twice each. Butane was washed with 100 ml of water layer and concentrated under reduced pressure to give 1.78 g of poly-Cbz-kanamycin A. The aqueous layer and water-wash solution are combined and concentrated to 50 ml under reduced pressure. The aqueous solution is passed through a column of CG-50 ion exchange resin (NH ₄ +140 ml), followed by 200 ml of water, 0.05 NNH ₄ OH 0.91. 0.1 N NH ₄ OH 1.91., And finally 0.3 NNH ₄ OH 1. The developments are collected in 20-ml fractions and divided into two fractions based on the Rf value of thin layer chromatography (s-114; 6'-Cbz-kanamycin A Rf 0.23, kanamycin Rf 0.07). Each fraction is evaporated under reduced pressure and the residue is dried over phosphorus pentoxide under reduced pressure.

So manufactured

6′-Cbz-kanamycin (II) is very pure and homogeneous by TLC on silica gel plates; Melting Point 217-219 ° C. The water-chemical properties were the same as the confirmed samples.

Example 6

1-N- [L-(-)-γ-amino-α-hydroxybutyryl] kanamycin A (BB-K8, IV) from benzaldehyde Schiff base

To a 6.19 g, (0.01 mol) solution of 6'-Cbz-kanamycin (II) in 50 ml of 50% THF, 3 ml (0.03 mol) of benzaldehyde is added with stirring. The mixture was stirred at room temperature for 3 hours and cooled to 5 ° C; THF 50 ml. It is treated with a solution of Compound (XX) 4 g. The reaction mixture is stirred at 5 ° C. for 1 hour, adjusted to pH 2 with 6N hydrochloric acid, stirred at room temperature for 1 hour more, and evaporated under reduced pressure to remove the organic solvent. The resulting aqueous solution was washed with 20 ml of CH ₂ Cl ₃ , adjusted to pH 4 with 10% aqueous ammonia, and then hydrocracked with 1.25 g of 10% carbon-palladium. The catalyst is filtered off and washed with 20 ml water. Combine the filtrate and the wash, adjust to pH 8 with 10% ammonia water and pass through a column of CG 50 (NH ₄ +200 ml). The pre-drying agent is developed with eyes of 0.4 I, 0.1 N ammonia water 1.2 I., 0.3 N ammonia water 2.06 I., 0.5 N ammonia water 0.92 I. and finally 1.0 N ammonia water 1.2 I. The development was pooled into 20-ml fractions and based on the Rf value of the TLC on the silica gel plate, the next fraction was cut off (s-110, ninhydrin), Bacillus severlylis PCI 219 and pneumoniae erogenosa A 9843 Discs are identified using strains. Each fraction is evaporated under reduced pressure and cold-dried.

* 1 Identification of Bacillus Sevtilis PCT 219

* 2 Identification of A 20680 for Klebsia pneumoniae susceptible to BB-K8 and highly resistant to cannamycin, BB-K 11 and BB-K 29.

Fraction 4 (1.877 g) is dissolved in 10 ml of de-ionized water. Adjust to pH 6 with 10N hydrochloric acid and absorb on CG-50 column (NH ₄ +15 ml). As a developer, 100 ml of 0.3 N ammonia water and 200 ml of 0.5 N ammonia water are used. The developments are collected in 20-ml fractions. In vitro numbers 6 and 8 to 17 were shown to be nin hydrin positive and in vitro 8 to 17 were combined and concentrated to about 1 ml under reduced pressure. The concentrate is treated with 3 ml methanol and filtered.

The filtrate is washed with 0.ml of 75% methanol, the filtrate is combined with the washings, diluted with the addition of 10ml of methanol and left to stand overnight at room temperature. Amorphous precipitate is filtered off and the filtrate is seeded to give IV of colorless crystals. This was the same as the positive sample. Yields and identification of each fraction are shown in the following table.

BB-K8 11 is 3 ″ -N- [L-(−)-γ-amino-α-hydroxybutyryl] -kanamycin A.

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

Example 7

L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid (VI)

L-(-)-γ-amino-α-hydroxy butyric acid (7.4 g, 0.062 mol) is added to 5.2 g (0.13 mol) sodium hydroxide in 50 ml of water. While stirring the solution, 11.7 g (0.068 mol) of carbobenzoic chloride is added dropwise at 0-5 ° C. for 30 minutes, and then the stirring is continued at the same temperature for another hour. The reaction mixture is washed with 50 ml ether, adjusted to pH 2 with dilute hydrochloric acid and extracted four times with 80 ml ether. The ethereal extracts are combined, washed with the same amount of saturated brine and dried over anhydrous sodium sulfate. The filtrate was evaporated under reduced pressure and the resulting residue was recrystallized from benzene to give 11.6 g (74%) of colorless platelet:

Melting point 78.5-79.5 ° C., αα _D −4.5 ° (c = 2, CH ₃ OH).

IR (KBr) rc = 0 1740,1690 cm ^-1 . Electromagnetic Resonance (NMR) (acetone-d6) δ (ppm from TMS) 2.20 (2H, m), 3.29 (2H, dd, J = 6.7 and 12 Hz), 4.16 (1H, dd, J = 4.5 and 8 Hz), 4.99 (2H, s), 6.2 (2H, broad), 7.21 (5H, s).

Elemental Analysis: C ₁₂ H ₁₅ NO ₅

Calculated: C, 56.91; H, 5.97; N, 5.53

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

Example 8

N-hydroxysuccinimide of L-(-)-γ-benzyloxy-carbonylamino-α-hydroxybutyric acid (VII)

A 200 ml solution of 10.6 g (0.042 mol) of IV and 4.8 g. (0.042 mol) of ethyl acetate in N-hydroxysuccinimide was cooled to 0. C., and 8.6 g (0.042 mol) of dicyclonuclear carbodiimide was added. do. Mix the mixture overnight in the refrigerator. Filtration of the dicyclonuclear urea produced by precipitation and concentration of the filtrate to about 50 ml under reduced pressure yields VII of colorless crystals. Yield 6.4 g; Melting point 121-122.5 ° C. The filtrate is evaporated to dryness under reduced pressure and the crystal residue is washed with 20 ml of benzene-n-nucleic acid to give more VII. Total yield: 13.4 g (92%). [Α] _D 1.5 ° (c = 2, chloroform) IR (KBr) rc = 0,1810,1755,1740,1680 cm ^-1 . (Acetone-d6) δ (ppm from TMS) 2.0 (2H, m), 2.83 (4H, s), 3.7 (2H, dd, J = 6.5 and 12.5 Hz), 4.56 (1 Hm), 4.99 (2H, s) , 6.3 (2H, Broad), 7.23 (5H, s).

Elemental analysis: C ₁₆ H ₁₈ N ₂ O ₇

Calculated: C, 54.85; H, 5. 18; N, 8.00

Found: C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14,8.12

G. Dowel You. Andersen and his collaborators, J. Am. Chem. Soc., 18, 1839 (1964)

Example 9

Preparation of N- (benzyloxycarbonyloxy) succinimide

N-hydroxysuccinimide (23 g, 0.2 mol) is dissolved in a solution of 9 g (0.22 mol) of sodium hydroxide in 200 ml of water.

While stirring the solution, 34 g (0.2 mol) of carbobenzoxyl chloride was added dropwise under water cooling, and then the mixture was stirred overnight at room temperature to form a carbobenzox derivative which was washed with water and air dried. Yield 41.1 g (82%).

Recrystallization with benzene-n-hexane (10: 1) yields a melting point of 78-79 ° C. on the colorless prism.

Example 10

The manufacturing method of 6'-carbobenzone kanamycin A (II)

A solution of 42.5 g (90 m.moles) of kanamycin A free base in 450 ml of water and 500 ml of dimethylformamide (DMF) is cooled below 9 ° C. and stirred vigorously. To this solution is added dropwise a solution of 22.4 g (90 m.moles) of N- (benzyloxycarbonyloxy) -succinimide in 500 ml of DMF for 2 hours. The reaction mixture is stirred overnight at −10 ° -0 ° C., then at room temperature for one day, and then evaporated under about 50 ° C. under reduced pressure. The oily residue is dissolved in 500 ml of water and 500 ml of butanol, and the mixture is filtered to remove insoluble matters and then divided into two layers. The butanol and water layers are treated with butanol-saturated water (500 ml X ₂ ) and water-saturated butanol (500 ml X ₂ ), respectively, by conventional countercurrent distribution. When the three aqueous layers are combined and evaporated to dryness under reduced pressure, an oily residue is produced, and when left at room temperature, part of it becomes a crystal. When the residue containing crystals is added to about 100 ml of butanol, the oily substance dissolves, the crystals are separated therefrom, then 300 ml of ethanol is added, and the mixture is allowed to stand overnight at room temperature. This crystal mass weighs 44 g. TLC using n-propanol: pyridine: acetic acid-water (15: 10: 3: 12) as the solvent system and ninhydrin as the spraying reagent indicated that the product contained very small amounts of kanamycin A.

The crude product was dissolved in 300ml of water and chromatography in Kalam (30ml diameter) of the CG-50 ion exchange resin (NH ₄ ⁺ type, 500ml). The column is soaked with 0.1 N ammonia water and the development is collected in 10 ml fractions. The target is contained in test tubes No. 10-100, while kanamycin A is recovered in the slower-moving fraction, and the position-isomers of the product are contained in the faster-moving fraction. It is thought to be. When fractions 10-100 were combined and evaporated to dryness under reduced pressure, 24.6 g of colorless 6-carbobenzoxy-kanamycin A (II) 6′-Cbz-kanamycin A was produced, melted at 204 ° C. and colored. Decomposes as they occur.

[α] _D 106 ° (c = 2H ₂ O). TLC (silica gel, F 254; ninhydrin)

Antron-Detected by sulfuric acid.

In some solvent systems, TLC found that the final product was accompanied by traces of the other two substances. However, the final product is no longer purified and used for the preparation of BB-K ₈ (IV).

Example 11

L-(-)-[gamma] -amino- [alpha] -hydroxybutytic acid preparation from ambutytocin A or mixtures thereof

5.0 g Ambutyrosine A (US Pat. No. 3,541,078, published November 17, 1970) is refluxed with 160 ml of 0.5 N sodium hydroxide for 1 hour. The hydrolyzate is neutralized with 6N hydrochloric acid and chromatographed on a column of CG-50 (NH4 ⁺ type). The column is developed with water and the water is removed by cold drying to isolate the target L-(-)-γ-amino-α-hydroxybutyric acid. This is a crystal having a melting point of 212.5-214.5 ° C. (Cal. 2, Line 31-38, US Pat. No. 3,541,078)

Example 12

Preparation of L-(-)-γ-amino-α-hydroxybutyric acid from DL-α-hydroxy-γ-phthalimidobutyric acid

15°(C, 25, 메탄올) 에탄올 300ml로 재결정하면 순수한 목적물 23.2g(43%)이 생성된다. 융점, 94-95℃,

10.8°(C, 2.5, 메탄올). 더 재결시켜도 융점 및 입체회전은 변하지 않는다.A) Dehydroabiethylammonium L-α-hydroxy-γ-phthalimidobutyrate: 2-hydroxy-γ-phthalimidobutyric acid ^* 25 g of a ethanol 200 ml solution of 130 g of dehydroabiethylamine 29 g of ethanol Add vigorously for 1 minute and stir at room temperature for 5 hours until fine needle crystals precipitate. The crystals were filtered off, washed with 50 ml of ethanol and air dried to yield 30,1 g (56%) of stereoisomers of the dehydro abiethylamine salt. Melting point 93-94 ℃

Recrystallization with 300 ml of 15 ° (C, 25, methanol) ethanol yields 23.2 g (43%) of pure target product. Melting point, 94-95 ° C,

10.8 ° (C, 2.5, methanol). Further recrystallization does not change the melting point and steric rotation.

Elemental analysis: C ₃₂ H ₄₂ N ₂ O ₅ H ₂ O

Calculated: C, 69.54; H, 8.02; N, 5.07

Found: C, 69.58; H, 8.08; N, 5.07

Saito and his collaborators Tetra Hedron Ratters 1970, 4863.

B) L-(-)-γ-amino-α-hydroxybutyric acid:

-29°(C 2.5, H₂O). 암부티토신으로부터 얻어진 확실한 시료와 IR 분광기 치가 일치하였다.To 40 ml of an aqueous solution of 1.5 g (0.014 mol) of sodium carbonate was added a 60 ml solution of 5.3 g (0.01 mol) of dehydroabiethyl ammonium L-α-hydroxy-γ-phthalimidobutyrate and vigorously until all the solid material was completely dissolved. Stir. The ether layer is separated and the aqueous layer is washed twice with 20 ml of ether and then concentrated to 15 ml under reduced pressure. 10 ml of concentrated hydrochloric acid is added to the concentrate, and the complex is refluxed for 10 hours. After cooling, phthalic acid is filtered off and the filtrate is evaporated under reduced pressure. The residue is dissolved in 10 ml water and the solution is evaporated to dryness. This operation is repeated twice to remove excess hydrochloric acid. The remaining syrup phase is dissolved in 10 ml water and a trace of insoluble phthalic acid trace is made. The filtrate is absorbed in an IR-120 (H ⁺ , 1 cm × 35 cm) column, then the column is washed with 300 ml of water and developed with 1N ammonium hydroxide solution. The developments are collected in 15 ml fractions and the ninhydrin positive fractions 10-16 fractions are combined and evaporated under reduced pressure to form a syrupy material that slowly crystallizes. The crystals were dissolved in ethanol, filtered and dried in desiccator under reduced pressure to yield 0.78 g (66%) of L-(-)-γ-amino-α-hydroxy butyric acid. Male point 206-207 ℃

-29 ° (C 2.5, H ₂ O). The IR spectroscopy values coincident with the positive samples obtained from ambutitocin.

Example 13

6′-carbobenzox-1,3,3 ″ -tri-P-nitro-benzalkanamycin A (IIIc)

6'-Carbobenzox kanamycin A (9.0 g, 14.5 mmol) is suspended in 500 ml anhydrous ethanol at 24 ° C. and 8.7945 g (58.2 mmol) P-nitrobenzaldehyde is added. The mixture is heated to reflux, during which the mixture turns to a clear yellow solution. However, at reflux, a white solid is formed rapidly. The mixture was heated to reflux for 3 hours, cooled and the product was filtered and washed with a small amount of anhydrous ethanol to yield 14.0 g (94.5%) of white crystalline solid. Melting Point 233-234 ° C

Elemental Analysis: C ₄₇ H ₅ N ₇ O ₉

Calculated: C, 55.46; H, 5.05; N, 9.63

Found: C, 55.39; H, 5.08; N, 9.60

Example 14

1-N- [L-(-)-γ-amino-α-hydroxybutyryl] -cannamycin A (IV)

6'-Carbobenzoxy-1,3,3 "-tri-P-nitrobenzalkanamycin A (5.0 g, 4.91 mmol) is dissolved in 50 ml of dimethylformamide (DMF) at 24 ° C.

L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy-succinimide ester (2.064 g, 5.895 mmol) was dissolved in 20 ml of DMF at 24 ° C and the Schiff base (IIc) solution was dissolved. Add slowly for 75 minutes with vigorous stirring. The solution was stirred overnight at 24 ° C., spray dried at about 40 ° C. using a steam ejector reduced pressure, and then continued spraying with methanol (100 ml) to produce a viscous oil. This oily substance is dissolved in 100 ml of dioxane: water (1: 1), 10 ml of glacial acetic acid is added, and 500 ml are poured into a Par bottle, 2.5 g of 5% pd / C is added, and 45 psi. Reduction at 4 hours. The pressure before this (closed bottle) drops to 87 psi. It should be re-pressed periodically. The mixture is filtered by passing through a diatomic earth metal pod prewashed with 3 x 50 ml of 50% aqueous dioxane. The combined filtrates are spray dried, azeotropic with n-butanol (100 ml) and finally sprayed with methanol to produce a viscous oily material. This is dissolved in 30 ml of methanol and slowly poured into 1000 ml of cold (5-10 ° C.) diethyl ether with vigorous stirring. The reaction suspension is stirred for 30 minutes in an ice water bath, and the resulting precipitate is filtered off and rapidly dried to a bed in a reduced pressure desiccator. 4.9620 g of the resultant mixture TLC mainly contained compound (IV), kanamycin A and di- and tri-substituted kanamycin A, and 4-amino-2-hydroxybutyric acid.

Ambel light IRC-50 (NH ₄ ⁺ type, type I, 100 × 200 mesyu) using a 40 × 100mm glass Kalam filled with divided into several fractions to obtain a resin plate of about 980mm.. 4.6 g of crude product dissolved in 1 ml of water was placed in a column and developed in fractions from water to 2N ammonium hydroxide to collect the developments in 15 ml fractions. After collecting 295 fractions, the column is washed with 1.51 3N ammonium hydroxide and the fractions with the same optical rotation are collected and the replacement is isolated by lyophilization.

* Average of 4 plate sympathy

? ? ± standard deviation

** If the signs of canna A recovery were seen, the conversion of BB-K8 (IV) was 33.5%. At this time, the ratio of BB-K8 cana A was 0.45.

85°(c=2, 물).Compound (IV) recovered by the present production method was the same as the sure product obtained by the method described in co-filed Application No. 221,378. Melting Point: 194 ° (Decomposition)

85 ° (c = 2, water).

Comparative titer on Bacillus subtilis (agar plate) = 960 MCG / MG. (Standard: kanamycin A free base).

Elemental analysis: C ₂₂ H ₄₃ N ₅ O ₁₃ 2H ₂ CO ₃

Calculated: C, 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

Sulfuric acid primary salt preparation of 1-N-L-(-)-γ-amino-α-hydroxybutyryl kanamycin A

1 mol of 1-N- [L-(-)-γ-amino-α-hydroxy-butyryl] kanamycin A was dissolved in water 1-31., The insoluble solid was filtered off and then cooled, and the solution was stirred with 500 ml of water. 1 mol of sulfuric acid dissolved in is added. The mixture is left to stir for 30 minutes and cold-ethanol is added until a precipitate is produced. Filtration of the solid yields the desired first salt of sulfuric acid.

Example 16

1-N- [L - (-) - γ- amino -α- hydroxy-butyryl] The preparation of 2 sulfate _{(BB-K8 · 2H 2 SO} 4) of kanamycin A.

Dissolve 35 g of 1-N- [L-(-)-γ-amino-α-hydroxybutyryl] kanamycin A (as monobicarbonate trihydrate) in 125 ml of de-ionized water, resulting in a pH of about 9.0. Lowered to pH 7.5 with% v / v sulfuric acid.

8.5 g of Darco G-60 (activated carbon) is added and the mixture is slurried at room temperature for 30 minutes. The carbon is removed with a suitable filter and washed with 40 ml of water. Combine the water wash with the filtrate.

The combined filtrates are adjusted to pH 2-2.6 with 50% v / v sulfuric acid. Large amounts of carbon dioxide are generated. The solution is stirred for a further 20 minutes in a vacuum tube so that carbon dioxide is completely generated.

=74.75 220-23℃에서 분해함.8.5 g of Darco G-60 is added to the gas depleted solution, and the mixture is slurried at room temperature for 30 minutes. Filter the carbon with a suitable filter, wash with 35 ml of de-ionized water, combine the wash with the filtrate and adjust to pH 1-1.3 with 50% v / v sulfuric acid. The solution is added to 600-800 ml (3-4 volumes of methanol) for 10 minutes with rapid stirring. The mixture is stirred at pH1-1.3 for 5 minutes, passed through a 100 mesh screen, stirred for 2 minutes and then left to stand for 5 minutes. Most of the supernatant was decanted, the remaining slurry was moderately filtered, washed with 200 ml of methanol and dried under reduced pressure at 50 ° C. for 24 hours. The yield of amorphous BB-K (dihydrogen sulfate) was 32-34 g.

74.75 Decomposition at 220-23 ° C.

Elemental Analysis ( ^{* in} dry matter)

^* Karl Fitzsch water content: 2.33, 1.79, 2.87 (Theoretical monohydrate is 2.25% water.) This salt is hygroscopic but not deliquescent. After storage for 18 hours in air at room temperature, the water content increases to 9.95, 9.89%.

(Theoretical value of pentahydrate is 11.33% of water).

[Example 17]

Preparation of 1-N- [L-(-)-γ-amino-α-hydroxybutyryl] chingnamycin A (IV) without Schiff base intermediate.

A solution of 1.36 g (2.2 mmol) of 6′-N-Cbz-kanamycin (II) in 60 mL of 50% THF aqueous solution is added dropwise with stirring at room temperature for 930 mg (2 mmol) of the active ester (XX) in 10 mL THF. The mixed solution was stirred overnight, and then hydrolyzed at room temperature and atmospheric pressure with 1.0 g of 10% palladium on carbon. Remove catalyst and wash with water.

The filtrate and washings are combined and evaporated under reduced pressure to remove the organic solvent. The resulting aqueous solution was passed through CG-50 (NH ₄ +, 70 ml) column and 0.4 L of water, 0.5 N of 0.1 N NH ₄ OH, 0.5 L of 0.3 N-NH ₄ OH, 1 L of 0.5 N-RH ₄ OH and 1 N. Moisten the column in the order -NH ₄ OH 0.5ℓ. Split the fractions based on TLC values on silica gel plates (s-110, ninhydrin) with 20-ml fractions of the solution, and then transfer the Bacillus subtilis PCI 219 and K. Pneumoniae Type 22 # 3038 A 20680. Complete plate was identified. Each fraction was evaporated under reduced pressure and freeze dried.

Example 18

6'-carbobenzone -1,3,3 "-trisalicyl alkanamycin A

6'-Carbobenzoxycanamycin A (9.0 g, 14.5 mmol) was suspended in 500 ml anhydrous ethanol at 24 ° C and 58.2 mmol of salicylicaldehyde was added. The mixed solution is heated to reflux, during which time the mixed solution turns into a clear yellow liquid, while white solids are rapidly produced at reflux, but the mixture is heated to reflux for 3 hours, cooled and the product is filtered and washed with a small amount of anhydrous ethanol. The melting point of the dried product, recrystallized from tetrahydrofuran-methanol-heptane (4: 1: 5), is 196-198 ° C.

Elemental analysis: C ₃₇ H ₅₄ N ₄ O ₁₆

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

Found: C, 60.14; H, 5. 60; N, 6.13

Example 19

The 1-N- [L-(-)-γ-amino-α-hydroxy-butyryl] kanamycin A preparation by simultaneous manufacture.

With a suitable device, dissolve 100 g (1.074 mol) of a slurry of 6′-carbobenzoxy-1,3,3 ″ -trisalicyl-alkanamycin A in 11,400 ml of THF at 22-25 ° C. and 600 ml of water with stirring. Add. In about 10 minutes it becomes a solution. To this solution was added 181.5 g. (0.716 mol) of 4-benzyloxycarbonylamino-2-hydroxybutyric acid and 0.12 mol of N-hydroxy-5-norbornene-2,3-dicarboxyimide, followed by pH 162.3 g (0.787 mol) of dry THF 3000ml solution of dicyclohexylcarbodiimide are added while adjusting to 5.5-6.0 and maintaining the temperature at 22-25 ° C. After stirring the slurry at 22-25 ° C. for 24 hours and cooling to 0-5 ° C., dicyclohexylurea precipitates as crystals for 2 hours.

The urea is filtered off and the filtrate is washed with 800 ml of THF 95.5% aqueous solution. Combine the urine and washings, depressurize and concentrate to 3000ml. 4000 ml of methanol was added thereto, and the resultant was concentrated under reduced pressure to 3000 ml.

To this solution was added 2000 ml of anhydrous methanol at 40 ° C., followed by the addition of 6′-carbobenzoxy-1,3,3 ″ -trisalicyl-kanamycin A, followed by cooling and crystallization for 30 minutes-1 hour. do. Stir well at about 22-25 ° C. for 2 hours and add 2000 ml of water over 30 minutes. The temperature is lowered to 0-5 ° C. for 1 hour.

The solids are removed by filtration and washed with 900 ml of methanol: water (2: 1) cold-mixture and again with 800 ml of methanol. Combine the filtrate and wash solution. The filtration material is recovered substantially as 6'-carbobenzox-1,3,3 "-trisalicyl al-kanamycin A

Dilute the filtrate with 1000 ml of water and add 2000 ml of methylene chloride. Lower the pH to 1.8-2.0 with 6N-since at 25 ° C and hold this pH for 30 minutes.

The methylene chloride layer is separated and the aqueous layer is washed with 2000 ml of methylene chloride to combine the organic solvent layers to recover salicyaldehyde as much as possible.

The pH of the aqueous layer is increased to 3.5-4.0 with concentrated ammonium hydroxide and hydrocracked with hydrogen gas in the presence of 5% Pd / c at 50 pounds per inch of pressure at room temperature. The biological yield of the target in solution was theoretically 30-45%.

Example 20

Preparation of compound IV using each mole% of N-hydroxy-5-norbornene-2,3-dicarboxyimide.

0.12 mole of N-hydroxy-5-norbornene-2,3-dicarboxyimide (HORB) was carried out in the same manner as in Example 20 using various molar percentages as follows. Was obtained.

As can be seen from the above results, HONB is produced without a total softness (IV) compound. However, the yield of compound (IV) was the best when the mole percentage of HONB was 10-50% of the molar amount of 4-benzyl-oxycarbonylamino-2-hydroxy-butyric acid used in the acylation reaction. The detailed reaction mechanism in the acylation process of the protected kanamycin A process is not yet known. However, it was observed that the yield was superior in the presence of HONB.

Example 21

The preparation method of 1-N- [L-(-)-γ-amino-α-hydroxybutyryl] kanamycin A (IV).

Dissolve in 55 ml of dimethylformamide (DMF) at 45 ° C. in 6′-carbobenzoxyl-1,3,3 ″ -trisalicyl-kanamycin A (16.6 g, 20.0 milliliters) already prepared in Example 18. . 7.3 ml of water and 102 ml of acetone were added to the solution, and the mixture was left to cool to about 25 DEG C, followed by L-(-)-L-benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy linear imide ester ( 9.9 mmol) and 50 ml of acetone are added all at once.

The reaction mixture was stirred at about 25 ° C. for 40 hours, 150 ml of water and a small amount of 6′-carbobenzoxyl-1,3,3 ″ -trisalicyl-kanamycin A were added, followed by stirring at about 25 ° C. for 1 hour. Stir at 0-5 ° C. for 3 hours. Excess and unreacted salicyaldehyde Schiff base precipitates as crystals, which are collected by filtration. The filtrate was washed with 60 ml of 50 ml acetone aqueous solution and dried to give 9.4 g of recovered Schiff base.

Melting Point (Crude Product): 170-185 ° C

The filtrate is treated with 40 ml of methylene chloride and adjusted to pH 2.0 from pH 6.2 to remove starting material. The organic layer is separated and the aqueous layer is extracted with 2 × 40 ml of methylene chloride. Adjust the pH of the aqueous layer to 3. Remove a small amount of acetone and methylene chloride under reduced pressure. The solution of the aqueous layer is hydrocracked at 45 Psi hydrogen pressure with 3.0 g of 5% Pd / C for 15 hours at 24 ° C. To remove the catalyst, it is filtered through a earth metal edide of divalent atoms. 1 ml of this solution was used as a sample, and the disk was prepared. Compound IV was 1936 mcg / ml in plate identification and 2325 meg / ml in original identification, which was equivalent to 33.4% yield of final product.

The remaining 99 ml of the solution was concentrated to about 30 ml and column chromatography as described in Example 14 above. The fractions containing compound (IV) were collected and concentrated to about 20 ml of the collected 500 ml, 45 ml of methanol was added and about 25 ml of isopropanol was added dropwise to obtain a crystalline product.

This crystalline product was filtered off, washed with methanol-isopropanol (50:50) and dried to afford 2.31 g of BB-K8 of formula (IV). The platelet was 836 mcg / ml of solid and 83.6% of pure wool, and the actual yield of pure compound was 33.3% based on 9.9 mmol of starting material.

Claims (1)

Next, 1 mole of the compound of general formula (II) is reacted with at least 3 moles of aldehyde selected from the group including benzaldehyde, salicylaldehyde, P-nitrobenzaldehyde, P-methoxybenzaldehyde and fibaldehyde to produce the following general formula (III) compound A method for preparing the compound of the general formula (IV), characterized in that, after a known reaction, 1 mol of this (III) compound is reacted with at least 0.5 mol of the following general formula (XX) compound and then hydrolyzed.

Z is a functional group of the following structural formula.