DK147075B - METHOD OF ANALOGY FOR THE PREPARATION OF CANAMYCIN A OR CANAMYCIN B DERIVATIVES OR SALTS THEREOF - Google Patents

Description

(19) DENMARK

| J | da) PRESENTATION WRITING (i1) 147075 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Patent Application No. 4811/75 (51) Int.CI.3: C07H 15/22 (22) Filing Date: Oct 24, 1975 (41) Alm. available: 27 Apr 1976 (44) Submitted: 02 Apr 1984 (86) International Application No :-( 30) Priority: 26 Oct 1974 GB 46412/74 (71) Applicant: 'PFIZER CORPORATION; Colon, PA.

(72) Inventor: James William * Moore; GB.

(74) Plenipotentiary: International Patent Office (54) Analogous process for the preparation of kanamycin A or kanamycin B derivatives or salts thereof

The invention relates to an analogous process for the preparation of novel kanamycin A or kanamycin B derivatives of the general formula I

CH.NHR

HO-- HO - NH2 i

R, OH

in

CH 2 OH

1 '^ τή / N HO I / * O 7 r- *

ISLAND

2 147075 wherein R. | represents H or CH 2, R 2 represents NH 2 or OH, and n is 1, 2 or 3, or pharmaceutically acceptable salts thereof.

Many naturally occurring 2-oxystreptamine aminoglycosides have in common a ring structure that can be reproduced by the general formula

Vv _A.

4 »^ A y- ° ~ 4 \ B / NH2 3 · 2 * 5 |

ISLAND ISLAND

K J

---

- ©, H

wherein the ring A is the skeleton of a hexapyranose group having an amino group at the 2 'and / or 6' positions, the ring B is the 2-deoxystreptamine group and the ring C represents a glycosyl group attached with a glycoside bond to either the 5- or 6-position of the streptamine ring, the second position being taken up by a hydroxyl group.

The compounds of formula I and their salts have antibacterial properties and are effective in treating infections of Gram-positive or Gram-negative bacteria, including urinary tract infections, in animals, including humans, and have advantages in use over 2-deoxystreptamine-aminoglycosides with an unsubstituted amino group. at the 1-position of the 2-deoxystreptamine ring B, such as the naturally occurring compounds kanacycin A and B, neomycins and ribostamycin.

A particular group of compounds prepared according to the invention include those wherein R is a hydrogen atom and n is 1 or 2.

Also preferred are compounds wherein the 3-hydroxy-u-aminoalkyl group at the 1-N position has (S) configuration and n is 2 or 3.

Particularly preferred single compounds of the invention comprise 1N- [j (S) -4-amino-2-hydroxy-butyl] -canamycin A and 1N - [(S) -5-amino-2-hydroxy-penethyl] - kanamycin A.

Pharmaceutically acceptable acid addition salts of the subject compounds are those formed with acids which form non-toxic acid addition salts containing pharmaceutically acceptable anions such as hydrochloride, hydrobromide, hydroiodide, sulfate or bisulfate, phosphate or acid phosphate, acetate, maleate, fumarate, oxalate , lactate, tartrate, citrate, gluconate, saccharate, p-toluenesulfonate and carbonate.

The process of the invention is characterized by reducing a compound of formula II

, 147075 3 CH ^ HRj B0— ^ L ° ΗΟ- ^ Τή 'NH2

R2 II

c "2oh ° · —fJ-Λ ° ™ H ° -" BC CH | CH2> n ™ 2

ISLAND

wherein R 2 g has the above meaning, or an acid addition salt thereof, and isolates the compound of formula I, which if desired is transferred before or after isolation into a pharmaceutically acceptable salt.

The process comprises, as an optional initial step, the formation of a suitable acid addition salt to render the compounds of formula II soluble in organic solvents. Such a reaction can e.g. is carried out by dissolving the compound of formula II in anhydrous trifluoroacetic acid, the latter being used in excess at a temperature usually between room temperature and 0 ° C. Excess acid is removed by evaporation to dryness in vacuo. The salt is then dissolved in an anhydrous, for the inert organic solvent reaction, e.g. tetrahydrofuran or dimethoxyethane and treated with the reducing agent, e.g. diborane, which is conveniently added as a solution of diborane in tetrahydrofuran, usually in excess at a temperature usually between room temperature and reflux temperature, depending on the nature of the particular reactants and the particular solvent used. The reaction is practically completed within 24 hours if carried out in tetrahydrofuran at 50 ° C with an excess of diborane. The product is then conveniently isolated by adding water to decompose unreacted diborane and remove the organic solvent by evaporation under vacuum. The pH of the remaining aqueous solution is adjusted to pH 5 and the crude product can then be purified from unreacted starting material and by-products by conventional chromatography techniques.

Many of the compounds of formula II are known antibiotics, e.g.

N-1- (4-amino-2-hydroxy-butyryl) kanamyc in A, also designated BB-K8, is disclosed in U.S. Pat. Nos. 3,781,268, 3,541,078 and 3,860,574 and in the disclosed West German patent applications Nos. 2,350,203 and 2,322,576. The 1-N- (5-Amino-2-hydroxy-valeryl) and 1-N- (3-amino-2-hydroxy-propionyl) derivatives of kanamycin A and B are described in West German Patent Application No. 2.408666 and in J. Antibiotics 1974, 2ί7, 851. The 6'-N-alkyl derivatives are disclosed in published 4 147075 West German Patent Application No. 2,350,169 and in J. Antibiotics, 1975, 28_, 483.

Compared to these known compounds, the compounds of formula I have a superior antibacterial effect as suggested by the test results set forth below.

The compounds of formula I may exist in different conformations, so the 8-hydroxy (d-aminoalkyl group at N-1 can be found in the S or R configurations or there may be a mixture of both optical isomers.

In vitro evaluation of the compounds of the invention as antibacterial agents is performed by determining the minimum inhibitory concentration (M.I.C.) of the test compound in a suitable medium in which the growth of the particular microorganism just does not occur. In practice, agar plates in which each sample compound is incorporated at a particular concentration are inoculated with a standard number of cells of the sample microorganism and each plate is then incubated for 24 hours at 37 ° C. The plates are then examined for presence or absence of bacterial growth and the associated M.I.C. value determined. Microorganisms used in such experiments have included strains of Escherichia coli, Klebsielle pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis.

In vivo evaluation of the compounds is also performed for the more active compounds, by administering the compounds subcutaneously to mice exposed to the strain Escherichia coli. Each compound is administered in a series of different doses to groups of mice and the activity of the compound is determined as the dose that provides 50% protection against the lethal action of the Escherichia coli organism over 72 hours.

For human use, the subject antibacterial compounds can be administered e.g. orally or parenterally, e.g. intravenously, intramuscularly or subcutaneously.

For human administration, it is believed that the daily dose of the antibacterial compounds in question will be of the same order of magnitude as the dose usually used by antibacterial aminoglycoside agents, e.g. from 0.1-50 mg / kg (in divided doses) by parenteral route or from 10-100 mg / kg (in divided doses) by oral route.

The preparation of the subject compounds is further illustrated by the following examples in which the temperatures are given in ° C.

Example 1 150 mg of 1N - [(S) -4-amino-2-hydroxybutyryl] -canamycin A (BB-K8, prepared as described in U.S. Patent No. 3781268) was dissolved in 10 ml of anhydrous trifluoroacetic acid at 0 ° C. The solution was evaporated to dryness in vacuo and dried under high vacuum at 20 ° G for 15 minutes to give a glassy solid. This was taken up in 5 ml of dry tetrahydrofuran and 20 ml of a 1-N solution of diborane in tetrahydrofuran was added portionwise under an atmosphere of nitrogen. The resulting clear solution was heated to 50 ° C for 3 hours, allowed to stand at room temperature for 16 hours and heated for an additional 3 hours to 50 ° C. Excess diborane was degraded to gently add a few drops of water and the organic solvent was then removed by evaporation under reduced pressure. The residue was taken up in 10 ml of water and acidified with 1/10 N aqueous sodium hydroxide solution. The pH of the resulting solution was adjusted to 5 by the addition of 2N hydrochloric acid. The solution was then chromatographed on a column containing "Amberlite" CG 50 ion exchange resin (50 ml) in the ammonium ion form, eluting first with distilled water to remove inorganic salts and then with aqueous ammonium hydroxide solution of 0 The fractions containing the product (as determined by thin layer chromatography) were combined and evaporated in vacuo to give 1N - [(S) -4-amino-2-hydroxybutyl] -canamycin A (75 mg, 50 % yield).

Thin layer. Rf = 0.6 (The electrolyte was a mixture of equal parts acetic acid and formic acid having a pH of 2, and a potential difference of 900 volts was applied across the ends of the 20 cm plate coated with silica gel, for 40 minutes. occurred by drying the plate, spraying it with a cyclohexane solution of tertiary butyl hypochlorite and subsequent drying, cooling and developing the starch-potassium iodide solution plate. Under these conditions, reference standard BB-K8 gave an Rf of 1.0 and kanamycin A Rf value of 0.9).

Infrared spectrography showed that the amide carbonyl absorption band observed in BB-K8 at 1635 cm-1 disappeared.

Optical rotation [α] 25 + 73 ° (c 1.0, H 2 O).

Mass desorption (field desorption) showed a strong P + 1 peak at m / e 572.

A sample was converted to the volatile penta-N-acetyl-octa-O-trimethyl-silyl derivative by treatment with acetic anhydride in methanol at room temperature for 24 hours, followed by reaction with a 2: 1 mixture of hexamethyldisilane and trimethylchlorosilane at room temperature. for 24 hours. M + determined for 1357.

C56H119N5 ° 17Si8 answers t: L ^ M + l357 ·

Analysis:

Found: C 40.1 H 6.7 N 9.6% C22H45N5012.2 1 / 2H2CO3 calculated: C 40.5 H 6.9 N 9.6% 6 147075

Example 2 0.35 g of 1Nf (S) -5-amino-2-hydroxyl-valeryl] -canamycin A was converted to the trifluoroacetate salt, reduced and chromatographed as described in Example 1 to give 0.12 g, 30% yield. , 1N - [(S) -5-amino-2-hydroxy-pentyl] -canamycin A.

Thin layer. Rf = 0.7 (The conditions as described in Example 1, starting material were used s cm reference standard with an Rf value of 1.0).

Example 3 0.15 g of 1-N- (3-amino-2-hydroxy-pr opionyl) kanamycin A was reduced in a similar manner as described in Example 1 to give 0.04 g, 277 yield, 1- N- (3-amino-2-hydroxy-propyl) -canamycin A.

Thin layer. Rf = 0.6 (The conditions as described in Example 1, starting material were used as reference standard with an Rf value of 1.0).

Example 4 1-N - (S) -4-Amino-2-hydroxy-butyryl] -canamycin B was reduced in a similar manner as described in Example 1 to give 1N - [(S) -4-amino-2-hydroxy -butyl] -canamycin B.

Example 6 6-N-Methyl-1N - [(S) -4-amino-2-hydroxy-butyryl]-kanamycin A (prepared as described by H. Umezawa et al. In J. Antibiotics, 1975, 28, 483) was reduced as described in Example 1 to give 6'-N-methyl-1Nf (S) -4-amino-2-hydroxy-butyl] -canamycin A.

Thin layer. Rf = 0.7 (The conditions described in Example 1, starting material were used as reference standard with an Rf value of 1.0 and kanamycin A gave an Rf value of 1.03).

Results of testing the compounds prepared according to the Examples for antibacterial activity in vitro by the methods described previously are given in Table I below: 147075-7

Table I

In vitro activity.

Example _ M.I.C. / g / ml ___ No. * E. coli Klebsiella Proteus Pseudomonas Staphylococcus pneumoniae mirabilis aeruginosa aureus_ 1 6.2 3.1 3.1 1.6 1.6 1.6 2 6'2 3.1 12.5 3.1 1.6 3 12.5 6.2 12.5: 3.1 3.1 l ----- i «- - - - - * -

Furthermore, the compound prepared according to Example 1 has been tested for in vivo activity by the methods previously described. PDjq against E. coli in mice was 3.8 mg / kg.

Table II gives the results of comparative experiments, which determined the antibacterial effect of compounds of the invention and similar effects of known derivatives of kanamycin A or kanamycin B. The microorganisms listed in this Table II resistant microorganisms.

Table II also lists the activity in MIC values. The results by which the compounds prepared according to the process of the invention were more effective than the comparative compounds are denoted by an asterisk (*). It appears that the compounds are particularly active against Pseudomonas strains.

147075 8

Table II

In vitro activity ________

Microorganism Beteg-MIC (µg / ml

Compound 1-H-HABA Compound 1-N-HAVA

according to Kanamycin A, Kanamycin A

Example 1 (Comparison (Comparison) Example 1 Equation) ___ 1 Escherichia coli E104 3.1 3.1 6.2 6.2 2 Escherichia coli 110 3.1 3.1 6.2 3.1 3 Escherichia coli 116 1.6 1.6 3.1 3.1 4 Escherichia coli E 172 1.6 1.6 6.2 6.2 5 Escherichia coli 10 3.1 1.6 6.2 *) 12.5 6 Escherichia coli 36 3.1 3.1 6.2 6.2 7 Escherichia coli 51 3.1 3.1 12.5 *) 25 8 Escherichia coli E 173 0.8 *) 1.6 3.1 3, 1 9 Proteus mirabilis P 133 3.1 3.1 12.5 6.2 10 Proteus mirabilis 8 1.6 1.6 3.1 *) 6.2 11 Proteus vulgaris P 136 3.1 1.6 1.6 *) 6.2 12 Proteus vulgaris P 124 0.8 0.8 6.2 3.1 13 Pseudomonas aeruginosa Ps 56 0.8 0.8 3.1 3.1 14 Pseudomonas aeruginosa Ps 52 0.8 0.8 1.6 ") 3.1 15 Pseudomonas aeruginosa Ps 48 0.8") 1.6 1.6 1.6 16 Pseudomonas aeruginosa Ps 169 1.6; 3.1 6.2 6.2 17 Klebsiella / Aerobacter K37 1.6 1.6 3.1 3.1 18 Klebsiella / Aerobacter K 38 0.8 0.4 1.6 ") 3.1 19 Klebsiella / Aerobacter K 33 1.6 1.6 3.1 3.1 20 Klebsiella / Aerobacter A 39 1.6 1.6 6.2 3.1 21 Klebsiella / Aerobacter A 40 1.6 1.6 3.1 3.1 22 Staphylococcus aureus S 223 0.4 "0.8 0.8 1.6 23 Staphylococcus aureus S 222 0.4 0.4 0.4" 1.6 24 Staphylococcus aureus S 202 3.1 3.1 6.2 6.2 Staphylococcus aureus S 228 0.8 0.8 1.6 1.6 26 P.seudumonas aeruginosa Ps 53 1.6 1.6 3.1 ") 6.2 (GAcT) 27 Pseudomonas aeruginosa Ps 54 1.6 1.6 6.2 6.2 (GAcT, KPT) 28 Escherichia coli (KPT) E 174 3.1 3.1 12.5 *) 29 29 Escherichia coli E 175 3.1 3.1 12.5 12.5 (KAdT, KPT) Escherichia coli (KAcT) E 176 50 12.5 100 50 HAVA = 2-hydroxy-5-amino-valeroyl HABA = 2-hydroxy-4-amino-butyryl 9

Table II (continued)

In vitro activity ... _ _ _ - - - MIC µg / ml

Microorganism Designate 1-N-HABA-Compound 6'-N-Methylation Compound Kanamycin Compound 1-HABA

according to (together with kanamycin A

Example 4 Equation) Example 5 (Comparison) 1 Escherichia coli E 104 6.2 6.2 6.2 6.2 2 Escherichia coli 110 3.1 3.1 6.2 6.2 3 Eschirichia coli 116 3 , 1 3.1 6.2 3.1 4 Escherichia coli E 172 3.1 3.1 6.2 6.2 5 Escherichia coli 10 6.2 *) 12.5 12.5 12.5 6 Escherichia coli 36 3.1 3.1 6.2 6.2 6.2 Escherichia coli 51 6.2 *) 12.5 12.5 12.5 8 Escherichia coli E173 3.1 1.6 3.1 3.1 9 Proteus mirabilis P 133 1.6 *) 6.2 12.5 12.5 10 Proteus mirabilis 8 3.1 3.1 3.1 3.1 11 Proteus vulgaris P 136 3.1 3.1 3.1 *) 6.2 12 Proteus vulgaris 124 3.1 1.6 3.1 1.6 13 Pseudomonas aeruginosa Ps 56 1.6 *) 3.1 3.1 *) 6.2 14 Pseudomonas aeruginosa Ps 52 1.6 1.6 3, 1 3.1 15 Pseudomonas aeruginosa Ps 48 3.1 1.6 3.1 3.1 16 Pseudomonas aeruginosa Ps169 3.1 *) 6.2 6.2 *) 12.5 17 Klebsiella / Aerobacter K 37 3.1 3.1 6.2 6.2 18 Klebsiella / Aerobacter K 38 0.8 0.8 3.1 1.6 19 Klebsiella / Aerobacter K 33 1.6 *) 3.1 3.1 3.1 20 Klebsiella / Aerobacter A 39 3.1 3.1 6.2 * 12.5 21 Klebsiella / Aerobacter A 40 3.1 3.1 3.1 3.1 22 Staphylococcus aure us S 223 0.4 *) 1.6 1.6 1.6 23 Staphylococcus aureus S 322 0.2 *) 0.4 1.6 1.6 24 Staphylococcus aureus S 202 3.1 *) 6.2 6 , 2 *) 12.5 25 Staphylococcus aureus S 228 0.4 *) 1.6 1.6 *) 3.1 26 Pseudomonas aeruginosa Ps 53 1.6 *) 6.2 6.2 6.2 (GAcT) 27 Pseudomonas aeruginosa Ps 54 1.6 ") 6.2 6.2 6.2 (GAcT, KPT) 28 Escherichia coli (KPT) E 174 6.2 6.2 12.5 12.5 29 Escherichia coli (KAdT, KPT) E 175 25 12.5 12.5 12.5 30 Escherichia coli (KAcT) E 176 25 12.5 12.5 6.2 GAcT = Gentamicin Acetyl Transferase KPT = Kanamycin Phospho Transferase KAdT = Kanamycin adenyl transferase KAcT = Kanamycin acetyl transferase 147075 The results of further comparative experiments with a number of aminoglycoside resistant microorganisms are listed in Table III below. This Table III sets out the geometric mean of the obtained IC.IC. values of the compounds of Examples 1 and 2 in comparison with the IC. -. values of the starting material for the compound prepared according to Example 1, namely 1-N-((S) - 4-amino-2-hydroxy-butyryl 3-kanamycin-Δ (BB-K8 or Amikacin).

Table III

Geometric mean of the MIC values in μg / ml of aminoglycoside resistant bacteria.

Species examined Compound Compound BB-K8 number from Example 1 from Example 2 Comparison)

Ps. aeruginosa 23 1.4 *) 1.9 *) 2.2

Klebsiella 22 1.6 1.5 1.7

Citrobacter 5 1.4 1.4 1.6 S. aureus 3 1.2 **) 1.2 *) 2.0

These results show the superior effect of the compounds of Examples 1 and 2 against Pseudomonas aeruginosa and S. aureus relative to the action of the known compound BB-K 8.

Further studies have been carried out on the in vitro activity of the compound prepared in Example 1 and of the known compound BB-K 8 both with aminoglycoside sensitive as well as with aminoglycoside resistant isolated forms of Pseudomonas sp. The results obtained are given in Table IV below.

__Table IV __

Pseudomonas species Number of isolates - Geometric mean value of I.I.C. values prepared in l-g / ml

Compound from BB-K8 Example 1 (Comparison)

Aminoglyco Lateral Sensitivity 1.6 1.6 2.1

Aminoglycoside resistant '18 2.2 3.3

It can be seen from Table IV that the compound prepared in Example 1 has a clearly greater activity than the known Comparative Compound BB-K8 over Pseudomonas sp. for aminoglycoside therapy.