FI56388C - REFERENCE TO A FRAMEWORK FOR THERAPEUTIC USE OF THERAPEUTIC CHARACTERISTICS OF 1-N-NY-AMINO-ALPHA-HYDROXIBUTYRYL SUBSTITUTES FOR CANANYCIN (A ELLER B) -FOERENING - Google Patents

Description

Ι-ιΤί — Ί ΓβΙ rt1xKUU «PUBLICATION ΓΛΑηρ VST * ibj <") UTLÄGGNINGSSKR1FX bbJoo C (4S) Patent granted 10 01 1990 dfirig2, Patent oeddelat V v '(51) Kv.lk. * / Lnt.CI. 07 H 15/22 (21) Puenttlhak «mu $ - Ptt« nUnsöknlng 1988/72 (22) Hakemlipilvl - An «6knlng * dag 12.07 * 72 (Fl) (23) Home - Glltlghetidag 12.07 * 72 (41) Tullut (from outside) · Ktf - Bllvlt © ffentilg 1 ^ .01.73

Patent · Ja rekl * terlhallltu * (44) Date of issue of the publication. -

Patent * och registrstyralMn Antflkan utligd och utl.tkrifMn publlcerad 2o.09 * 79 (32) (33) (31) Requested «tuolkeui — Begird priorltet 13.07 * 71 27.01.72 USA (US) 162315, 221378 (71) Bristol-Myers Company, 3 ^ 5 Park Avenue, New York, NY , USA (72) Hiroshi Kawaguchi, Tokyo, Takayuki Naito, Tokyo, Susumu Nakagawa,

Tokyo, Japan-Japan (JP) (7 * 0 Oy Kolster Ab (5h) Method for the preparation of a new therapeutically useful lNY-amino-ε-hydroxybutyryl-substituted kanamycin (A or B) compound - Förfarande för framställning av en The present invention relates to a process for the preparation of a novel therapeutically useful ly * -amino-of-hydroxybutyryl-substituted kanamycin (A or B) compound of the formula: The present invention relates to a novel therapeutically useful ly * -amino-of-hydroxybutyryl-substituted kanamycin (A or B) compound. is HO -6 'K ch2-nh2 \' ho ^ IT j ^ NH2 (I.) l: tt s ~ '\ V ", ΟΗρΟΗ H ° 0/5" /' —NH-C-CH-CH „- CH0-NH0 \ - * - 0V /, 2 2 2

\ "2" Y / ° H

NH2 '/ HO 1 "^ / 0 3 563ϋό 2 wherein R is an OH or NH2 group, or non-toxic pharmaceutically acceptable acid addition salts thereof.

The process according to the invention is characterized in that the amino group on the 6 'carbon of kanamycin A or kanamycin B is protected with a customary amino-protecting group Y to give a compound of the formula:

HO - H *, CH_-NH-Y

\ 3 '"" °.

V,

HO / \ V

, s 6 "H ° OH OH / --NH_ H ° - / \ 3" ®v / NH2—- / HO \ / 3. ...

wherein R and Y are as defined above, the amino group in the 1-position of the compound of formula II is acylated with an acylating agent of formula:

OH

W - NH - CH 2 - CH 2 - CH - COOH (III) wherein said acylating agent is derived from L- (-) - γ-amino-α-hydroxybutyric acid of formula W, or a functional equivalent thereof, to give a compound with the formula: rv · 6 * 3 583oc

HO --- vl + '. CH 2 NH-Y

Υ \ ^ 5 *

Vl * 2 ^ ° H0 ^ / \\ Χ '(IV) 6 "H0 5 /' 'xW 2 HO_ *" <Χ0Η. / ^ nh-c-ch-ch2-ch2-nh \\ / „// OH w \ —-- 0 / \ 3" 2 "\ / ™ r— / HO \ / 07 3 where R, Y and W denote same as above; and deprotecting W and Y by methods well known in the art to give a 1-NK-amino-X-hydroxybutyryl-substituted kanamycin (A or B) compound of formula I, and optionally converting the resulting product into a pharmaceutically acceptable acid addition salt in a manner known per se. .

Kanamycins are known antibiotics, see Merck Index, B. edition pages 597-59Θ. Kanamycin A is a compound of the formula ° Xa 1.— V — 7 \\ Ia Ησ 0 —BH ^ 1.0 \ /

563ÖC

Kanamycin B is a compound of the formula: HO - \ H2N j Ib I nh2

HO '/ ^ NA

CH 2 OH / ^ NH 2 -? W HO \ / 0 The IUPAC name for “Amikacin” is 0-3-amino-3-deoxy - <* - D-gluco-

O

pyranosyl (1 * 4) -O-Q5-amino-6-deoxy - (* * -D-glucopyranosyl Cl-> 6) DN- (4-amino-L-2-hydroxybutyryl) -2-deoxy-L-streptamine is a compound of formula IV obtained from kanamycin A by the process of the invention. Kanamycin A is called 0-3-amino-3-deoxy - <* - D-glucopyranosyl (1-> 4) -O-Q6-amino-6-deoxy - <* - D-glucopyranosyl Cl-> 6) 3-L-streptamiini.

The phrase "non-toxic, pharmaceutically acceptable acid addition salt" means a mono-, di-, tri- or tetra salt formed by administering 1 mole of Compound IV and 1 to 4 moles of a non-toxic pharmaceutically acceptable acid. These acids include acetic, hydrochloric, sulfuric, almond, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acids and those other acids commonly used in the preparation of amine-containing pharmaceutical salts.

The process according to the invention is described by the following reaction scheme: ΙΙ3Θ6

l) Kanamycin A N- (benzoyloxycarbonyloxy) Q

(Ia) or -ΓΤ ": -: - ^ n succinimide H0 CHo-NH-C-O-CHo-C.H_

Kanamysuni B ^ ^ d 0 ->

, lb> KL

0 V nh2 (6'-N-monobenzyloxydicarbonylcannarysin A or B) \ ^ \ / *> 1 H2B Λ - yvA / ho A /

563GC

6 2) The compound obtained in l) L - (-) - / - benzyloxycarbonylamino-o (-hydroxy) N-hydroxysuccinimide ester of butyric acid_ ^ 0 HO-K CH2m-i- ° -CH2-C6B3 | WH2

-NH

H0 / f = °

CHpOH / S ° jE

*> - vJU— \ / Γ2 2 \ / CHo-NH-C-O-CHo-C.H [- - | J / 2 2 O? h2n ^^ ho ^

HP / Pd / C CH NH

3) the compound ΓΧ / HO-i___ ^ 0 obtained in 2) ^ HO- ^ 2 n2

'VI

H0 - ^ ----- \ CH OH 7 \ \ HO -i V \\

\ __ \ // NH

NH- X I

2 HO ^ C = 0

'✓ HO-CH

0 I

CH? i 2 CH "I 2"; nh Λ '' * - M; '

7 563ÖG

Useful acylating agents include those which provide labile amine protecting groups and are commonly used in peptide synthesis. It must be possible to easily remove labile protecting groups by methods well known in the art. Examples of such labile protecting groups and their removal can be found in the article by A. Kapoor, J. Pharm. Sciences 59, pp. 1-27 (1970). Suitable acylating agents for primary amine groups include the following: carboxylic acid chlorides and bromides and anhydrides, including anhydride mixtures, and especially those anhydride mixtures prepared from stronger acids such as carbonic acid and alkyl and aryl sulfonic acids, aliphatic acids such as In addition, an acid azide or an active ester or thioester (e.g. with p-nitrophenol, 2,4-dinitrophenol, thiophenol or thioacetic acid) may be used, or the free acid itself may be coupled with the kanamycin derivative (II) after said free acid has been reacted With Ν, Ν'-dimethylchloroformium chloride (cf. British Patent 1,008,170 and Novak and Weichet, Experientia XXI / 6, 360 (1965)) or using enzymes or Ν, Ν-carbonyldiimidazole or Ν, Ν'-carbonylditriazole (cf. Sheehan and Hess, 3rd Amer. Chem. Soc., 77, 1067, (1955) or an alkynylamine reagent (cf.

R. Buijile and H.G. Viehe, Angew. Chem., International Edition 3, 582 (1964)) or keteneimine reagent (cf. CL Stevens and NE Monk, J. Amer. Chem. Soc. 80, 4065 (1958)) or isoxazolium salt reagent (cf. RB Woodward, RA Olofson and H. Mayers, 3. Amer. Chem. Soc., 83, 1010 (1961)). Another substance corresponding to the acid chloride is the corresponding azolide, i. an amide of the corresponding acid, the amide nitrogen of which is a member of a semi-aromatic five-membered ring containing at least two nitrogen atoms, i. imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and substituted derivatives thereof. As an example of a general process for the preparation of an azolid, N, N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or the like in an inert solvent imideolide Diimidazolides are obtained from dicarboxylic acids. The by-product imidazole precipitates and can be separated and the imidazolide isolated, but this is not necessary. These reactions are well known in the art (cf. U.S. Patent Nos. 3,079,314, 3,117,126 and 3,129,224 and English Patent Nos. 932,644, 957,570 and 959,054).

When preparing 6'-protected aminocanamycin, it is recommended to adjust the molar ratios of the reagents so that the acylating agent is used in a ratio of 1 mole or less per mole of 6'-protected aminocanamycin. Increasing the amount of acylating agent θ 56386 results in lower yields of the desired intermediate due to the increase in competing side reactions that produce polyacyl derivatives as impurities.

Temperature is not very critical. However, the use of significantly higher temperatures (above about 5 ° C) is not recommended, as the proportion of side reactions then becomes significant.

The compound 1-N- (L - (-) - X'-amino - '* - hydroxybutyryl) -kanamycin A: 11a prepared according to the invention has excellent bactericidal properties which prove to be superior to kanamycin A: 11a itself.

Below are two tables showing the lower inhibitory concentrations (MICs) of kanamycin A and the compound of the present invention against various gram-positive and gram-negative bacteria obtained by the Steers agar dilution method (Table 1) and two-fold by the dilution method (Table 2). Mueller-Hinton agar medium was used in the study of Table 1 and Heart bath solution was used in the study of Table 2.

5 6 3 o l

Table 1 9

Minimum inhibitory concentration of kanamycin A and a compound of the invention

Lowest effective concentration (mg / mlj

The organism Kanamycin A 1-N- (L - (-) - V "-amino-o (-

hydroxybutyryl-kanamycin A

lot no. 4 1. Alk. faecalis A-9423 16 8 2. ”" A-2064B> 125> 125 3. Ent. cloacae A-9656 4 4 4. "species A-20364> 125 2 5." hafniae 1 A-20674 1 1 6. E coli A-0636 2 1 7. "A-20664 16 4 8." A-20665> 125 1 9. "A-20507 32 2 10." A-20520> 125 4 11. "A-20365> 125 1 12. "A-20684 2 2 13." A-20682> 125 2 14. "Α-206Θ3> 125 8 15." Α-206Θ1> 125 2 16. "A — 15119 4 4 17. K. pneumoniae Α- 9Θ67 4 4 18. ”species A-20328) 125 2 19." "A-20330 32 32 20." "A-20634> 125 4 21." pneunnoniae A-20680> 125 4 22. "" A-9977 1 1 23. Pr. mirabilis A-9900 2 2 24. Pr. morganii A-15153 2 2 25. "vulgaris A-9555 2 1 26. '' rettgeri A-9636 0,25 0,25 27. 'mirabilis A-20645 4 4 28.' mirabilis A-20454 2 2 29. Providence stuartii A-20615 2 1 30. ”alkalifaciens A-20676 1 1 563υϋ 10

Table 1 (continued)

The organism Kanamycin A 1-N- (L - (-) - γ-amino-c <-

hydroxybutyryl-kanamycin A

31. See aeruginosa A-20229 32 2 32. "" A-9843A 125 16 33. "" A-20653) 125 32 34. "species A-20601 125, 63 16 35." "A-20621> 125> 125 36. "maltophilia A-20620 32> 125 37. Sal. enteritidis A-9531 1 0,5 38. "Derby A-20087> 125 1 39. Ser. marcescens A-20019 2 4 40." "A-9933 4 8 41." "A-20460> 125 4 2 42. "" A-20459 4 16 43. Shig. Flexneri Α-9Θ64 4 4 44. Aeromonas sp. A-20670 2 2 45. Arizona sp. A-20671 2 1 46. Sitrobacter sp. A-20673 4 4 47. Edwardsiella sp. A-20678 4 4 48. Staph, aureus A-9606 1 1 49. "" A-4749 0.5 1 50. "" A-9537 2 1 51. "" A-20610> 125 2 52. " "A-20240> 125 8 53." "A-15197 1 2

Mueller-Hinton medium + 4% sheep blood 54. Str. Faecalis A-9854 63 63 55. "" A-9575 125> 125 56. "pyogenes A-20200 32, 16 32 57." "A-9604 125 125 58 . "" A-15040 125 125 59. "" A-20065 125 125 60. D-pneumoniae A-9585 63, 32 63 61. "" A-20159> 125> 125

Table 2 563ο o 11

Minimum effective concentration (mg / ml)

Organism Kanamycin A 1 -N-CL-C-3'-amino-t * -

hydroxy ibutyryl-kanamycin A

1. D. pneumoniae + 5% blood whey Α-95Θ5 63 63 2. Str. Pyrogenes + 5% blood whey A-9604 125 125 3. Staph, aureus Smith A-9537 0.5 0.5 4. Staph, aureus A- 9497 0.5 0.5 5. Staph, aureus A-20239> 125 4 6. Staph, aureus A-20240> 125 4 7. Enter, cloacae A-0656 2 2 8. Enter, species A-20364> 125 2 9. K. pneumoniae Α-9Θ67 2 4 10. E. coli K.-12 ML1410 A-20361 2 4 11. E. coli K-12 ML1630 A-20363> 125 2 12. E. coli K-12 A- 9632 2 1 13. E. coli A-20664 32 Θ 14. E. coli A-20665> 125 8 15. Pr. mirabilis A-9900 2 16 16. Pr. morganii A-15153 4 16 17. Pr. vulgaris A-9436 1 2 18. Ps aeruginosa A-20227 4 1 19. Ps species A-20499 63 4 20. Ps aeruginosa A-20653> 125 4 21. Ps species A-20621> 125> 125 22 Ser. marcescens A-20019 2 4 23. Ser. marcescens A-20141 16 16

12,563iiL

The above experimental results show that 1-N-CL-C -) - / '- amino- *. -hydroxybutyryl) -kanamycin A is clearly superior to kanamycin A, especially against kanamycin A tolerant organisms.

The MIC results are also in good agreement with the in vivo results in all three organisms against which kanamycin A and 1-N-CL-C - (- (') - amino- (*-hydroxybutyryl) -kanamycin A were tested.

1 —N— CL— (CD- / "- amino - ** - hydroxybutyryl) - kanamycin A and kanamycin A were equally effective against infections caused by kanamycin A-susceptible E. coli A 15119 and Staph, aureus A 9537 mice. Although CD4 values (a healing dose for 50% of lethal infected mice) appear to indicate that 1-N-CL-C-D - / '- amino - * (hydroxybutyryl) -kanamycin A is slightly less active than kanamycin A, this small difference is presumably not significant because the dose concentrations were far apart (5 x dilutions).

Kanamycin was not expected to be effective in vivo against kanamycin-tolerant E. coli A 20520, whereas 1-N- (L - (-) - V "-amino-o <-hydroxybutyryl) -kanamycin A showed a significant protective effect, being approximately 10-fold more active against this E. coli species when administered as a four-course regimen rather than a two-course regimen.

Table 3

Comparison of in vitro and in vivo activities of a compound of the invention and kanamycin A.

Compounds Experimental Staphylococcus Escherichia coli Escherichia coli _number aureus A 9537 A 15119_A 20520_ MICaiCD50b) MIC CD5QMIC C05Q 1-N-CL-C -) - Γ-amino-1 1 2; 0 x 2 22x2 2 66 x 2 -hydroxybutyryl-2 -c) - - -5x4

kanamycin A

Kanamycin A 1 2 0.5 x 2 44x2 125 200 x 2 2 - - - - - 200 x4 (a) MIC = minimum effective concentration (ug / ml). The experiments were performed as described by Chrisholm et al. (Antimicrob. Agents and Chemotherapy - 1969, p. 244, 1970) using Mueller-Hinten agar as the test medium.

13 5 6 3 b it b) CD ^ q = Healing dose, 50% (mg / kg / treated x number of treatments). Mice were treated subcutaneously 1 and 4 hours post-infection with 2 treatments and 0.2, 4, and 6 hours post-infection with 4 treatments. The other parts of the experiment were performed as described by Price et al. (0. of Antibiotics 22: 1 1969).

c) = not tested

The novel compounds prepared by the process of the invention are valuable bactericidal agents as a dietary supplement in animal feed, as therapeutic agents for poultry and other animals, including man, and are particularly valuable in the treatment of infectious diseases caused by Grarr-positive and Gram-negative bacteria.

The new compounds are useful as oral adjuvants in preoperative intestinal sterilization. Both aerobic and anaerobic organisms that are sensitive to these drugs are reduced in the colon. When they are used with sufficient mechanical cleaning, they will be useful in preparation for colon surgery.

The new compounds are effective in the treatment of bacterial infections in the body when administered from a parenteral dosage level of about 250 to about 3000 mg per day in divided doses three or four times a day. In general, these compounds are effective when administered at a dose of about 5.0 to 7.5 mg / kg of body weight every 12 hours.

The compound prepared according to the invention, 1-N-1H-f-1- (4-amino- (N-hydroxybutyryl) -kanamycin B, has excellent bactericidal properties. Table 4 below shows the lower inhibitory concentrations of kanamycin A and said compound against various grarr positive and gram-negative bacteria (Steers agar dilution method with nutrient agar medium KM = kanamycin A and GM = gentamicin).

1 ^ r c ^ · 'r 563υ t

Table ^

Comparison of a compound of the invention and kanamycin A.

Minimum effective concentration (mg / ml)

Organism Substance for which 1- (L - (-) - N-amino-Kanamycin A

resistance oL-hydroxyhutyryl)

able -kanamycin B

E. coli N1HJ 0, U 0.8 "Juhl A-15119 1.6 0.8" A-15169 1.6 1.6 "A-20363 KM 0.8 50" A-98UU 0.8 0.8 "A-20365 KM 0, U> 50

"K-12 0.8 0, U

"K-12 A-20664 KM 0.8 3.1" K-12 A-20665 KM 0.8 50 K. pneumoniae Dl 1 0.2 0.2 "A-9678 0.8 0.8 S. marcescens A-20019 1.6 1.6

Ps. Aeruginosa D15 3.1 12.5 "D113 KM 50> 50" A-9923 1.6 25 "A-9930 0.8 6.3" A-15150 6.3 25 "A-1519 ^ 1.6 12.5 "A-20U79 KM 1.6 50" A-20616 KM 3.1 25 "A-20653 KM 25 50" A-98U3 1.6 12.5

Pseudomonas sp. A-20355 1.6 6.3 "A-20358 KM 3.1 12.5" A-2O368 GM 50 25 "A-20598 GM 6.3 25" A-2O6OO GM 3.1 12.5 (continued) ) 15 5630 (,

Table U (continued)

Minimum effective concentration (mg / ml)

Organism Substance for which 1- (L - (-) - ^ -amino- Kanamycin A

resistance-αC-hydroxybutyryl) able -kanamycin B

Pseudomonas sp. A-20603 GM 6.3> 50 "A-20618 GM 50 50

PR. vulgaris A-9U36 1.6 0.8 "A-9526 0.8 0.8

PR. mirabilis A ~ 955 ^ 1.6 1.6 A-99OO 1.6 1.6

PR. morganii A-9553 1.6 1.6 "A-20031 6.3 3.1

PR. rettger A-15167 0.8. S. aureus 209P

"Smith 0.8 0, U

"209P 1.6 0.8" A-20239 KM 1.6 25 S. lutea PCI-1001 1.6 3.1 M. flavus 0, U 0.8 B. subtilis PCl-219 0.1 0.1

St. pyogenes S-23 25 25 "Dick 1, 6 1.6" Digonnet A-960k 12.5 25 "A-20065 25 25 D. pneumoniae 50 25 itycobacterium 607 6.3 0.8" 607 D105 KM> 100 > 100 "607 D107 KM> 100> 100" phlei 1.6 1.6 "Ranae 6.3 1.6

"H37RV (by tube dilution method)

The above results show that the activity of the new compound is clearly better than that of kanamycin A, especially kanamycin A-resistant organisms.

16

5638C

Evaluation of the in vivo activity of the new compound was performed against both kanamycin B-sensitive and tolerant E. coli and Ps. Aeruginosa species.

Evaluation of the in vivo activity of the new compound was performed against both kanamycin B-sensitive and tolerant E. coli and Ps. Aeruginosa species. The organisms are named as follows:

Code No Kanamycin susceptibility E. coli A 15119 sensitive E. coli ML-163O A 20363 resistant (phosphorylation)

Ps. Aeruginosa D 15 - quite durable

Ps. Aeruginosa D 113 - highly resistant (phosphorylation) 1- (L - (-) - / '- amino - (N-hydroxyhutyryl) -kanamycin B and kanamycin B were equally effective against E. coli Juhl A 15119 ~ mice at a dose of 6.3 mg / kg. The new compound was most effective against kanamycin B-resistant E. coli ML-1630 at a dose of 6.3 mg / kg compared to a dose of 100 to 100 mg / kg of kanamycin B.

The activity of 1- (L - (-) -? The new compound proved to be significantly better in activity against the highly resistant Ps. Aeruginosa D-113 species than kanamycin B (see Table 5) ·

17 S 6 3 8 O

Table 5

Comparison of new compound and kanamycin B E. coli Juhl A 15119 E. coli ML-1630

Dose (s) 1- (L - (-) - / -amino- Kanamy- 1- (L - (-) - 1H-amino- Kanamy-

-hydroxybutyryl) busine »A -hydroxybutyryl) busine B

-cananycin B B -kanamycin B

1 + 00 mg / kg - - 2/5 100 5/5 5/5 5/5 2/5 25 5/5 5/5 5/5 0/5 6.3 5/5 5/5 5/5 0/5 1.6 0/5 0/5 2/5 CD ^ q (mg / kg) 3.1 3.1 1.8 ca 300

Ps. Aeruginosa D15 Ps. Aeruginosa D-113

Dose (se) 1- (L - (-) - / '- amino-Kanamy- Dose 1 - (L - (-) - / "- amino-Chicken- (<-> hydroxybutyryl) sine (se) -hydroxybutyryl )

-kanamycin B B -kanamycin B B

100 mg / kg 5/5 5/5 1 + 00 mg / kg - 0/5 25 1/5 0/5 200 3/5 6.3 1/5 0/5 50 1/5 1.6 0/5 0/5 12 , 5 0/5 3.1 0/5 CD (mg / kg) 38 50 CD50 (mg / kg) 11 + 5> 1 + 00

The entries 0/5, 1/5, 5/5, etc. indicate the number of surviving animals per five exposed animals \ for example, 5/5 indicates five animals out of five lethal doses of the exposed organism.

S 6 3 8 € 18 Preparation of starting materials A. Preparation of L - (-) - N-benzyloxycarbonylamino-cC-hydroxybutyric acid L - (-) - V "-amino - <> <- hydroxybutyric acid (T> ^ g, 0.062 mol) was added To a solution of 5.2 g (0.13 mol) of sodium hydroxide in 50 ml of water was added dropwise to the stirred solution at 0-5 ° C over 0.5 hour, 11.7 g (0.068 mol) of benzoxycarboxochloride, and stirring was continued. The reaction mixture was washed with 50 ml of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four 80 ml portions of ether.The ether 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 was crystallized from benzene to give 11.6 g (7U%) of colorless plates, m.p. 78.5-79.5 ° C, (· ^) D = * + »5 (c = 2, CH 3 OH). Infrared (IR) (KBr): IR (KBr) λmax, 1690 cm -1. Nuclear Magnetic Resonance (NMR) (acetone-dg) δ (ppm from TM5) 2.0 (2H, m), 3.29 (2H, dd, J = 6.7 and 12 Hz), H, 16 ( 1H, dd, J = U, 5 and 8 Hz), U, 99 (2H, s), 6.2 (2H, broad), 721 (5H, s).

Analysis: Calculated from the formula C 18 H 18 NO 2: C, 56.91 ° H, 5.97 L N, 5.53 Experimental: C, 36.66 °, H, 5. 979 N, 5.1 + 7 · B L - (-) -), '- Benzyloxycarbonylamino-o-hydroxybutyric acid N-hydroxysuccinimide ester

A solution of 10.6 g (0.01 + 2 moles) of the compound obtained in A and U.8 g (0.0U2 moles) of N-hydroxysuccinimide (GW Anderson et al., J.Am.Chem.Soc., 86.1839 (196U)) in 200 ml of ethyl acetate, cooled to 0 ° C, after which 8.6 g (0.0U2 moles) of dicyclohexylcarbodiimide were added. The mixture was kept. overnight in the refrigerator. The separated dicyclohexylurea was filtered off and the filtrate was concentrated to about 50 ml under reduced pressure and filtered to give 6.1 g of the title product as colorless crystals, m.p. 121 to 122.5 ° C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 ml of a mixture of benzene and n-hexane to give further said product. The total yield was 13 + g (92 *). (<0D 1.5 ° (c = 2, OH) IR (KBr)) λ = 1810, 1755, 17?, 1680 cm-1. NMR (acetone-d 6) δ (ppm from TMS) 2.0 (2H, m), 2.83 (1 + H, s), 3.37 (2H, dd, J = 6.5 and 12, 5 Hz), 1 +, 56 (1H, m), U, 99 (2H, s), 6.3 (2H, broad), 7.23 (5H, s).

Analysis: Calculated from the formula C 18 H 18 N 2 N 2: C, 5 * +, 85 ", H, 5.18", N, 8.00 experimentally C, 5I1.79 *. 51 * .70; H, 5.21, 5.20 ", N, 8.1, 8.12.

19 S6388 C. Preparation of N- (benzyloxycarbonyloxy) succinimide N-hydroxysuccinimide (GW Anderson et al., J.Am.Chem.Soc., 86.1839 (196M (23 g, 0.2 mol)) was dissolved in a solution of 9 g (0.22 mol) of sodium hydroxide in 200 ml of water To the stirred solution was added dropwise 3 * + g (0.2 mol) of benzoxycarboxochloride under cooling with water, and then the mixture was stirred at room temperature overnight to separate the benzoxycarboxy derivative which was collected by filtration. washed with water and air dried to give U1.1 g (82%) Recrystallization from a mixture of benzene and n-hexane (10: 1) gave colorless prisms melting at 78-79 ° C.

D. Preparation of 6, -N-carbobenzoxycanamycin A.

A solution of U2.5 g (90 mmol) of kanamycin A free base in U50 ml of water and 500 ml of dimethylformamide (DMF) was cooled to below 0 ° C and stirred vigorously. A solution of 22 μg (90 mmol) of N-benzyloxycarbonyloxy-succinimide in 500 ml of DMF was added dropwise to the solution over about two hours. The mixture was stirred at -10 to 0 ° C overnight and then at room temperature for one day. The reaction mixture was evaporated under reduced pressure below about 50 ° C. The oily residue was dissolved in a mixture of 500 ml of water and 500 ml of butanol, and the mixture was filtered to remove insoluble matter, and separated into two layers. The butanol and aqueous solutions were treated with butanol-saturated water (2 x 500 mL) and water-saturated butanol (2 x 500 mL), respectively, using a countercurrent partitioning technique. All three aqueous layers were combined and evaporated to dryness under reduced pressure to give an oily residue, some of which crystallized on standing at room temperature. To the residue containing crystals was added about 100 ml of methanol, which dissolved the oil and separated it from the crystals. After about 300 ml of ethanol was added, the mixture was kept at room temperature overnight to give a crystalline mass which was collected by filtration. It weighed UU g. The product contained a small amount of kanamycin A as indicated by thin layer chromatography using a solvent system of an aqueous mixture of n-propanol-pyridine acetic acid (15: 10: 3: 12) and ninhydrin as a spray reagent.

The crude product was dissolved in 300 ml of water and chromatographed on a column (diameter 30 mm) with CG-50 ion exchange resin (NH 4+ type, 500 ml). The column was immersed in 0.1 N ammonia and the eluate was collected in 10 ml fractions. The desired product was included in tubes 10-100, while kanamycin A was recovered from the slower moving fractions and the positional isomer (s) of the product appeared to be included in the faster moving fractions. Fractions 10-110 were combined and evaporated to dryness under reduced pressure to give 2h, 6 g (i + 5%) of a colorless 6'-carbobenzoxycanamycin A product (1'-Cbz-kanamycin A) which began to melt and color at 20 ° C and decomposed at 212 ° C producing gas. U) + = 106 ° (c = 2 *, HgO).

TLC (silica gel F, Rf value)

Solvent system 6'-N-carbobenzoxy-Kanamycin A

kanamycin A

n-PrOH-pyridine-AcOH-HgO 0, * + 2 (0.33 ^ 0.15 / 0.0.1+ (15: 10: 3: 12) (head) smaller

Acetone-AcOH-HgO 0.2 * t 0.1 * + (20: 6: 7 * +) CHCl 3 -MeOH-c.NH 2 O-OH-HgO 0.76 0.50 (1: 1+: 2: 1)

AcOMe-n-ProOH-c.NH.OH 0.22 * 0.0U * (1 + 5: 105: 60) * Observed with anthrone-sulfuric acid.

In one of the solvent systems tested, two smaller components were found to be associated with the final product by TLC analysis. However, the final product was used without further purification to prepare 1-N- (L - (-) - / '- amino-o-hydroxybutyryl) -canamycin A.

E. Preparation of 6'-carbobenzoxycanamycin B To a cooled solution of 8.1 g (0.0168 moles) of kanamycin B in 120 ml of water and 80 ml of 1,2-dimethoxyethane was added dropwise with stirring. , containing 1+, 2 g (0.0168 moles) of N-benzyloxycarbonyloxy) succinimide in 1 + 0 ml of 1,2-dimethoxyethane. The reaction mixture was stirred overnight and evaporated to dryness under reduced pressure. The residue was dissolved in 100 ml of water and shaken twice with 50 ml of water-saturated n-butanol. The aqueous layer was separated and adsorbed onto a column of 100 ml of CG-50 resin (NH 4 type). The column was washed with 200 ml of water and eluted with 0.05 N ammonia. The eluate was collected in 10 ml fractions. Fractions 121-180 were collected, evaporated and lyophilized to give 1.58 g (15%) of the desired product. Fractions 1-120 were evaporated and rechromatographed on CG-50 resin (NH 4+) to give 1.21 g (12%) of the title compound. product. Sp. 151-152 ° C (decomposes) (qf) ^ + 10Ho (c = 2.5, H2 ° ^^ c = 0 ^ 10 <™ _1 · Analysis, calculated from the formula ^ 26¾3 ^ 1 + ^ 12: 50 * 56 ; H, 7.02; N, 11.3 * + experimentally: C, 50.71; H, 7.38; N, 11, * + 8.

TLC (silica gel) »Rf 0.03 in n-PrOH-AcOH-H 2 O (15: 10: 3: 12);

Rf 0.16 in acetone-AcOH-HgO (20: 6: 7 * +) · F. 1- (L- (-) - Ί ^ βηΐ syloxycarbonylamino-4-hydroxybutyryl) -6'-carbobenzoxycanamycin B (III b) manufacture

To a stirred solution of 1.85 g (3.0 mmol) of 6'-carbobenzoxy-kanamycin B * in 0 ml of HgO and 50 ml of 1,2-dimethoxyethane was added in one portion at 5 ° C. in 1.1 g (3.1 mmol) of N-IL-T-carbobenzoxyamino-cis-hydroxy-butyryloxy) succinimide. The reaction mixture was stirred overnight at room temperature and subjected to hydrogenolysis without isolating the carbobenzoxy derivative (III b). TLC (silica gel F 25 * +), Rf 0.06 (starting material), 0.1 * +1, 0.57 (n-PrOH-pyridine-AcOH-H 2 O = 15: 10: 3: 12). Rf 0.11 (starting material) 0.21, 03 * +, 0 * + 6 (acetone-AcOH-H 2 O = 20: 6: 7 * +) ·: ·> ;! '·' 21

S6 3 b G

F. Preparation of L - (-) - / - eunino - "<- hydroxybutyric acid from DL -« <- hydroxy-phthalimidobutyric acid (a) Dehydroabietylammonium L-<< -hydroxy- /? - phthalimidobutyrate: containing 25 g (0.1 mol) of 2-hydroxy-N-phthalimidobutyric acid (Y. Saito et al., Tetrahedron Letters, 1970, UO63) in 200 ml of ethanol, a solution of 29 g (0.1 mol) of dehydroabietylamine in 130 ml of ethanol The solution was stirred vigorously for one minute and allowed to stand at room temperature for five hours during which time fine needles crystallized from solution.

The crystals were collected by filtration, washed with 50 ml of ethanol and air-dried to give 30.1 g (56%) of the diastereomer of the dehydroabietylamine salt. Sp. ^ 93 ~ 9 ° C. (α) + 15 ° (c = 2.5, methanol). Recrystallization from 300 ml of ethanol gave 23.2 g (k3%) of pure product. Sp. ^ 9 ~ 95 ° C.

(>) ^ + 10.8 (c = 2.5 MeOH). Recrystallization did not change the melting point or the specific rotation.

Analysis calculated by the formula Ο ^ Η ^ Ν ^ Ο ^ .Η ^ Ο: C, 69.5 ° H, 8.02 ', N, 5.07 Experimental: C, 69.58 *, H, 8.88 ', N, 5.07 (b) L - (-) - Ϋ'-amino-hydroxybutyryl acid: To a solution of 1.5 g (0.014 mol) of sodium carbonate in U0 ml of water was added 5.3 g (0 .01 moles) of dehydroabietylammonium L-N-hydroxy-N-phthalimidobutyrate and 60 ml of ether. The mixture was shaken vigorously until all the solid had dissolved. The Eet blade layer was separated. The aqueous layer was washed twice with 20 ml portions of ether and evaporated to 15 ml under reduced pressure. To the concentrate was added 10 ml of concentrated hydrochloric acid, and the mixture was refluxed for ten hours. After cooling, the separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The remaining syrup was dissolved in 10 ml of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was absorbed onto an IR-120 column (H +, 1 cm x 35 cm), the column was washed with 300 ml of water and eluted with 1 N ammonia. The eluate was collected in 15 ml fractions. Ninhydrin-positive fractions 10-16 were combined and evaporated under reduced pressure to give 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 - (-) - / - - amino -? - hydroxybutyric acid. Mp 206-207 ° C (< c = 2.5, HgO) The IR spectrum was identical to a reliable sample obtained from ambutyrosine.

22 56388

Example 1 Preparation of 1-N- (L - (-) - / '- amino-.4-hydroxybutyryl) -kanamycin A

A solution of 1.6 g (U, 6 mmol) of N - hydroxysuccinimide ester of L - (-) - N, N-benzyloxycarbonylamino-N-hydroxybutyric acid in 10 ml of ethylene glycol dimethyl ether (DME) was added dropwise to a stirred solution of 2, 6 g (H, 2 mmol) of 6'-monobenzyloxycarbonylcananyl Aita i + 0 in 50 ml of an aqueous solution of 50-ethylene glycol dimethyl ether and the mixture was stirred overnight. The reaction mixture was evaporated under reduced pressure to give a brown residue of 1-N- (L - (-) - N * -) benzyloxycarbonylamino - * <- hydroxybutyryl-β-N-carbobenzoxycanamycin A, which was used in the next reaction without further purification.

The obtained crude product was dissolved in U0 ml of 50 ¾ aqueous dioxane solution, and a small amount of insoluble matter was removed by filtration. To the filtrate were added 0.8 ml of glacial acetic acid and 1 g of 10-¾ palladium-carbon catalyst, and the mixture was hydrogenated at room temperature for 2 hours in a Parr hydrogenator. The reaction mixture was filtered to remove the palladium catalyst and the filtrate was evaporated to dryness in vacuo. The residue was dissolved in 30 ml of water and chromatographed on a column of CG-50 ion exchange resin (NH 4 type, 50 cm x 1.8 cm). The column was washed with 200 ml of water and then eluted with 800 ml of 0.1 N NH 4 OH, 500 ml of 0.2 N NH 4 OH and finally with 500 ml of 0.5 N NH 4 OH. Ten ml fractions were collected and fractions 1U6-15U contained 552 mg of the title compound, as a crude product, m.p. 18 ° C (decomposes). Relative potency B, against subtilis species (agar plate) = 560 mcg / mg (standard: Kanamycin A free base).

A solution of 250 mg of the crude product obtained in 10 ml of water was chromatographed on a column of CG 50 resin (NH 4 -type, 30 cm x 0.9 cm).

The column was washed with 50 ml of water and then eluted with 0.2 N ammonia. Ten ml fractions were collected. Fractions 50-63 were combined and evaporated to dryness under reduced pressure to give 98 mg of pure product ", m.p. 19 U ° C (dec).

(c <) ^ 1 = + 85 ° (c = 2. H ^ O). Relative potency against B. subtilis (agar plate) = 960 μg / mg (standard kanamycin A free base).

Analysis: Calculating C 18 H 18 N 2 O 3 SH 9 CO 3: C 10 I 6 62 ", H, 6.68", N, 9.87 Experimental: C 10 H 21, 39.79 *, H, 6.96 *, 6.87 *, N, 9.37, 9, fc9-2366388

Example 2 Preparation of 1-N- (L - (-) - / "- amino-N-hydroxybutyryl) -canamycin B

To a stirred solution of 1.85 g (3.0 mmol) of 6'-carbobenzoxy-kanamycin B in 1 + 0 ml of H 2 O and 50 ml of 1,2-dimethoxyethane was added in one portion 5 1.1 ° (3.1 mmol) of N- (N- (N-carbobenzoxyamino) -4-hydroxy-butyryloxy) succinimide at 0 ° C. The reaction mixture was stirred overnight at room temperature and subjected to hydrogenolysis without isolating the 1-N- (L - (-) - / ”- benzylcarbonylamino-hydroxybutyryl) -6'-N-carbobenzoxycanamycin B formed without isolation. TLC (silica gel F 25 * +), Rf 0.06 (starting material), 0.1 + 1 and 0.57 (n.PrOH-pyridine-AcOH-HgO 15: 10: 3: 12). Rf 0.11 (starting material), 021, 0.3 * + and 0.1 + 6 (acetone-AcOH-HgO 20: 6: 71+).

To the resulting solution was added 0.2 g of 10% palladium on carbon catalyst. After the mixture was hydrogenated under normal pressure for 5 hours, an additional 0.1 g of 10% palladium-on-carbon catalyst and 10 ml of water were added. Hydrogenation was continued overnight. The reaction mixture was filtered, the filtrate was evaporated under reduced pressure, the residue was dissolved in 50 ml of water and chromatographed on a CG-50 column (NH 4, 1.2 cm x 50 cm). The column was washed with 200 ml of water and then eluted with 500 ml of 0.1 N ammonia, 500 ml of 0.2 N ammonia, 900 ml of 0.5 N ammonia and 500 ml of 1 N ammonia. ammonia. The effluent was collected in 10 ml fractions. Kanamycin B was recovered from fractions 60-76 in 32% yield (1 + 59 mg). Fractions 128-138 were collected, evaporated under reduced pressure and lyophilized to give 318 mg (17% based on carbobenzoxyananine B) of the title compound ', m.p. 186-187 ° C (decomposes). (*) ^ 9 = + 78 ° (c = 1.15, Ho0) V ^ 16U0 cm -1.

u d 9 c = 0

Analysis: Calculated from the formula C22H1 + 1 + W6 ° 12'H2CO3: C, 1 + 2.72 ', H, 7.17', N, 13.00 Experimental: C, 1 + 2.23 *, H, 7, 19 ', N, 12.37 TLC (silica gel F 2 N, ninhydrin), R f 0.11 in acetone-AcOH-H 2 O (20: 6: 71+)', R f 0.19 CHCl 3 -MeOH conc. In NH 4 OH- 2 O (1: 1+: 2: 1).

Example 3 Preparation of 1-N- (L - (-) - [? - amino-β-hydroxybutyryl) kanamycin A or B monosulfate salt

One mole of 1-N- (L - (-) - N-amino-hydroxybutyryl) kanamycin A or B was dissolved in 2 L of water. The solution was filtered to remove insoluble solids. To the cooled and stirred solution was added 1 mole of sulfuric acid dissolved in 500 ml of water. The mixture was allowed to stir for 30 min, after which cold ethanol was added to the mixture until precipitation for 2k 56306 occurred. The solids were collected by filtration and found to be the desired monosulfate salt.

Example k Preparation of the disulfate salt of 1N-tL-N-amino-N-hydroxybutyryl-kanamycin A 35 g of 1-N- (L - (-) - N-amino- * 4-hydroxybutyryl) kanamycin A (monobicarbonate trihydrate) was dissolved in 125 ml of deionized water. The pH was found to be about 9.0.pH was lowered to 7 ~ 7.5 with 50- $ (v / v) sulfuric acid.

8.5 g of activated carbon ("Darco G-βθ") was added and the mixture was shaken at ambient temperature for 0.5 hours. The carbon was removed by filtration and washed with 1 + 0 ml of water. Wash water was added to the filtrate.

The pH of the above combined filtrate-washing solution was adjusted to 2.0-2.6 with 50- $ (v / v) sulfuric acid. A large amount of carbon dioxide evolved. The solution was allowed to stand for another 20 minutes under reduced pressure to remove residual carbon dioxide. 8.5 g of activated carbon ("Darco G-60") was added to the degassed solution. The mixture was shaken for 0.5 hours at room temperature. The carbon was removed by filtration and washed with 35 ml of deionized water. Water was added to the filtrate.

The combined filtrate and washings were adjusted to pH 1-1.3 with 50- $ (v / v) sulfuric acid. This solution was added with rapid stirring over 10 minutes to 600-800 ml of methanol (3 parts by volume of methanol). The mixture was stirred for 5 min. At pH 1-1.3, passed through a 100 mesh screen, stirred for 2 min. and allowed to settle for 5 min. Most of the supernatant was decanted. The remaining slurry was suitably filtered, washed with 200 ml of methanol and dried in vacuo at 50 ° C for 2k hours. The yield of the title product was 32-3 * + g ", (^) p H 2 O = + 7 ^, 75, decomposition at 220-230 ° C.

Elemental analysis (dry weight) *

Experimentally In Theory $ 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 not at all ----- x Karl Fosher water content: 2.33, 1.79, $ 2.87 (in theory, the monohydrate contains $ 2.25 water). This salt is hygroscopic but not hydrated. After a small portion was stored in air at room temperature for 18 hours, the water content rose to $ 9.55, $ 9.89 (in theory, the pentahydrate contains $ 10.33 in water).

Claims (1)

563B8 Claim: A process for the preparation of a novel therapeutically useful 1-N-N-amino-N-hydroxybutyryl-substituted kanamycin (A or B) compound of the formula HO_ 4 '6' yV. ch2-nh2 \ 3 '0 ho ^ ΑΛ m 1,3 4 NHj HO \ CM / ^ SNS \ ^ \ 1 0 ^ / XSSss \ n H0 4 »/ CH2OH / ^^ NH-C-CH-CH2-CH2- NH2 g / OH νη2Λ ^ - ^ \ / ho xry 3 in which R is an OI or 1H-H22 group, or non-toxic pharmaceutically acceptable acid addition salts thereof, characterized in that 6 'of kanamycin A or kanamycin B is protected. carbon group with a standard amino protecting group Y to give a compound of formula: HO __4 'CH2-NH-Y HO - \ 3/1 »RI 4 NH2 (II) 6” \, HO _5 / Η2 ° Η Wherein the R and Y have the same meaning as above, the amino group in the 1-position of the compound of formula II is acylated with an acylating agent of the formula: □ HW - NH - CH2 - CH2 - CH - COOH (III) said acylating agent being derived from a 1- (-) - N -amino-N-hydroxybutyric acid of the formula wherein W is a common amino-protecting group, or a functional equivalent thereof, to give a compound having the formula: 6 'H0 4' CH2-NH-Y HO '° \ «VI r3 / nh2 hct 1 \ o H0 4 "W20H / \ Lnh-c-ch-ch2-ch2-nh _o, / 0H w \ 3” 2 ”\ / NH2 —'-- 7 ^ A / Hu rj / where R , Y and W are as defined above, and the protecting groups W and Y are removed by generally known methods to give a 1-NK-amino-β-hydroxybutyryl-substituted kanamycin (A or B) compound of formula I and, if desired, the product in a manner known per se as a pharmaceutically acceptable acid addition salt. 4 / 563ϋε • Patent: For use in the preparation of therapeutically active compounds 1-N-V ”-amino - hydroxybutyryl-substituted with kanamycin (A or B) -formation with the form HO 4 '6' Vs. / CH2-NH2 1 '(I) R ^ 4 NH2 ΗΟ ^ δ ^ - ^ \ 6 ”/ \\ 1 0 PH ΓΊΗ / \\'» »H0 4„ / 2 n / NH-C-CH-CH2- CH2-NH2 \ 3 "7" \ / NH2 .— / HO / 1 \ / 0 3 of R is an OH- or NH2-group, and is toxic, a pharmaceutical product containing a total of 6 groups of amino acids, -staining with kanamycin A or B blockers with a conventional amino acid group Y for the formulation of the form HO -J * '/ CH2-NH-Y HO TvA R3 ^^ 4 NHZ (ID o - \ 2 6 "HD 5 ^ , HO_NH2 nh2 —7 ^ \ / HO 1 „\ / 0