Pharmaceutical Chemtstp 3' dourna e' I'o/ 29. \o. I, I995

UDC 615.332.038

REDUCTIVE ALKYLATION OF THE GLYCOPEPTIDE EREMOMYCIN AND ITS DERIVATIVES

ANTIBIOTIC

A. Yu. P a v l o v , 1 T. F. B e r d n i k o v a , 1 E. N. O l s u f ' e v a , t

G. I. Orlova, 1 and M. N. Preobrazhenskaya I

Translated from Khimiko-Farmatsevticheskii Zhumal, Vol. 29, No. 1, pp. 46 -48 , January,, 1995.

Original article submit ted March 1, 1994.

The reaction of formaldehyde and benzaldehyde with the glycopeptide antibiotic eremomyein in the presence of NaBH3CN yielded a number of methyl and benzyl derivatives substituted at the amino groups. Some of the benzyl derivatives have a high antibacterial activity, but it is somewhat less than that of the parent compounds.

Eremomycin (I) is a glycopeptide antibiotic of the van- comycin-ristomycin group [1], of which vancomycin (USA), teicoplanin (Italy) and ristomycin A (Russia) are used in medicine. The antibiotics of the given group are highly active with respect to multiresistant staphylococci, antibiotic-toler- ant streptococci, various species of clostridia, and entero- cocci. They are distinguished by the currently extremely rare appearance of forms of microorganisms resistant to them.

The original domestic glycopeptide antibiotic eremomy- cin was isolated at the Research Institute for Searching for New Antibiotics of the Russian Academy of Medical Sci- ences [2]. Its chemotherapeutic index is 10 times that ofvan- comycin. Currently, eremomycin is being prepared for the first stage of clinical testing.

o~\

ol-I oH I

However, the glycopeptide antibiotics of the vancomy- cin-ristomycin group have shortcomings that restrict their broader application at clinics: namely, a narrow spectrum of

I Research Institute for Searching for New Antibiotics. Russian Academy of Medical Sciences, Moscow, Russia.

51

antibacterial action limited to gram-positive bacteria, side ef- fects (nephro- and ototoxicit-y), and allergic reactions.

Investigations of directed chemical modification of these antibiotics, begun in the middle of the 1980's along with studies of the structure-activity relation were aimed at creat- ing new semisynthetic antibiotics with improved chemotherapeutic properties.

Chemical modification of the glycopeptide antibiotics of the vancomycin-ristomycin group is mainly directed at the C- terminal carboxyl goup and N-terminal amino group of the peptide (aglycone), and also at the amino groups of the ami- nosaccharides. For example, reductivve alkylation of van- comycin yielded N'-benzyl, N'-decyl, and N'-p-butyloxyvan- comycin, substituted at the amino group of the aminosaccha- ride (vancosamine), their in vitro activity being an order of magnitude higher than the activity of the original antibi- otic [3].

Previously, mono-, di-, and triethyl derivatives at the amino groups were obtained on the basis of eremomycin by reductive alkylation [4]. It was found that the first to react is the amino group of the aminosaccharide (eremosamine) of the disaccharide branch (N'), next the N-terminal amino group of the peptide (N), and last of all, the amino group of the eremosamine bound directly to the aglycone (N").

In the present investigation, we continued to study the re- action of reductive alkylation of eremomycin, and the alkyla- tion of the previously obtained N-nitrosoeremomycin (II) [5] and N,N-dimethyleremomycin (III) [6]. We initially planned to prepare the mono-, di-, and trimethyl and benzyl deriva- tives of eremomycin, and the N'-methyl and N' benzyl de- rivatives of compounds II and III. However, unlike reductive alkylation with acetaldehyde [4], when alkylating with for- maldehyde and benzaldehyde we did not succeed in choosing

0091 - 150X/95/2901-0051 �9 1995 Plenum Publishing Corporauon

52 A. Yu. Pavlov et al.

T A B L E I. S t ructure o f E r e m o m y c i n D e r i v a t i v e s I V - IX

C o m p o - und

Hydro lys i s , p r e s e n c e E d m a n ' s Elect ro- o f e r e m o s a m i n e m e t h o d , phoret ic

p r e s e n c e ~H-NMR, mobi l i ty o f u n s u b - n u m b e r o f relat ive

o f s t i tu ted in t roduced to e remo- d i saccha r ide b o u n d to N - t e r m i n a l alk3'l m y c i n in

b r anch a g l y c o n a m i n o - g roups 2 N A c O H g r o u p (pH 2.4,

o f pep t ide 700 V, 3 h)

I + + ~ - l

II + + - - 0.75

III + + - 2 1.02

IV - - 3 0.96

V - - - 3 0.93

VI - - - 2 0.73

V I I - - - 2 0 .70

VIII - - - 2(2)" 0 .99

IX - - - 2(2)" 0.97

*A s ingle t f rom the three me thy l g r o u p s o f the s ta r t ing N N-d ime thy le re - m o m y c i n (111) is p resen t in the H - N M R spec t r a o f these compounds .

conditions for the selectivity of the reaction. Alkylation oc- curred simultaneously at all the amino groups. Treatment of the relevant starting compound with formaldehyde or benzal- dehyde in the presence of cyanoborohydride in an aqueous-

methanol medium yielded N,N',N"-.trimethyleremomycin

(IV), N,N',N"-tribenzyleremomycin (V), N',N"-dimethyl-N-

nitrosoeremomycin (VI), N',N"-dibenzyl-N-nitrosoere-

momycin (VII), N,N,N',N"-tetramethyleremomycin (VIII),

N',N"-dibenzyl-N,N-dimethylerem omycin (IX).

gnz . . . . M-m~ZR"

HOOC~_._t-R : nooc-~ H~O-M~:gr 'I(I:I) , 20"C~ 8h

1, II. III IV - IX

R = N(CH3) _, (I) R ' = N ( C H 3 ) =. R" = H (IV)

R = N ( C H 3 ) N O (II) R ' = N ( C H 3 ) C H : C 6 H s. R" = C~H s (V)

R = N§ - (lII) R ' = N ( C H 3 ) N O . R" = H (VI)

R'= N ( C H 3 ) N O , R " = C6H.~ (VII)

R ' = N*(CH3)31- , R" = H (VIII)

R ' = N*(CH3)3 I-. R" = C6H s (IX)

The structure of the obtained compounds I V - I X was proved on the basis of the 1H-NMR spectra, Edman's method, electrophoresis, and acid hydrolysis.

In the IH-NMR spectra of all the synthesized derivatives, we identified all the signals from protons of the correspond- ing eremomycin molecules, and also the protons from the in-

troduced alkyl groups, namely: at 6 3.21 and 3.04 p p m - the

methyl groups; and in the region of 8 7.60 - 7.30 ppm - the aromatic protons of the benzyl groups. Moreover. in the 1H-

NMR spectra of compounds VIII and IX at 8 3.15 ppm, we see a nine-proton singlet from the three methyl groups at the

nitrogen atom of the residue of N,N,N-trimethylleucine of the starting N,N-dimethyleremomycin.

Analysis (as described by Olsuf'eva, et al., [4]) of the products of acid hydrolysis, under the conditions for which (0.2 N HC1, 100~ 10 min) only one residue of the ami- nosaccharide of the disaccharide branch is detached, revealed that in all the obtained compounds I V - I X , this aminosac- charide differs from eremosamine. In hydrolysis under more rigorous conditions (1 N HCI, 100~ 30 min), when the glu- cose and aminosaccharide bound to the aglycone are de- tached, this aminosaccharide was also found to differ from eremozamine, and glucose was found to be present in all the synthesized compounds IV - IX.

The use of Edman's method as described by Berdnikova, et at., [7] showed that in compounds I I - I X the N-terminal amino group of the peptide is substituted. The presence of a substituted N-terminal amino group of the peptide in deriva- tives VI and VII, confirmed by of electrophoresis data, points to the stability of the N-nitroso group under conditions of re- ductive alkylation.

All the data on the structure of the obtained compounds are presented in Table 1.

CHEMICAL EXPERIMENTAL PART

We used samples of eremomycin sulfate obtained =)*/,

from the Institute's testing facility: ;~-m~ 280 nm, '--1r 40,

[a]~ ~ - 100 ~ (c 1, water), Rf 0.08 (AI), 0.56 (A2), 0.30 (A3), vancomycin from the Eli Lilly company (USA). The deriva- tives were analyzed by thin layer chromatography on plates with silica gel (Merck 60F254) (Germany) in the systems: ethyl acetate-n-propanol-25% aqueous ammonia 2 : 2 : 1 (A1), 2 : 2 : 5 (A2), 3 : 3 : 4 (A3), and also by electrophoresis on paper in 2 N acetic acid (700 V, 3 h). We used an ultra- chemiscope to detect the antibiotic and its derivatives on the chromatograms, and Pauly's reagent to visualize these sub- stances on the chromatograms and electrophoregrams.

We studied the optical rotation of the samples on a Per- kin-Elmer-241 polarimeter (Sweden). We obtained the UV spectra in 0.01 N HCI on a Pye-Unicam SP-8000 spectro- photometer (Great Britain), and recorded the IH-NMR spec- tra on a Varian VXP-400 spectrometer (Switzerland).

Preparation of N,N',N"-trimethyleremomyein (IV). To a solution of 165 mg (0.1 mmole)oferemomycin sulfate in 8 ml of a mixture of H20 and CH3OH (1 : 1), we added 0.075 ml (1 mmole) of 37% aqueous CH_~O (formalin) and 32 mg (0.5 mmole) of NaCNBH3. We mixed it for four hours at 20~ and added 32 mg of NaCNBH3, and after two hours. 32 more mg of NaCNBH3. We mixed the solution for two hours, added 100 ml of H:O, acidified it with 6 N H:SQ up to pH 2, and passed it through a column with the sulfocation-exchange resin SDV-3 (H') (0.58 x 8 cm) for four hours. We rinsed the column with water, and desorbed compound IV with 0.25 N NH4OH. We evaporated the eluate in a vacuum to 2 ml. acidified it with 6 N H_,SO4 to pH 6. and precipitated the tar- get compound with 100 ml of acetone. The yield of IV was

Reductive Alkylation of the Glycopeptide Antibiotic Eremomyein and its Derivatives 53

102 mg (60%). Rf 0.62 (A2), 0.39 (A3). '-~cm~l~176 39.

[ct]~ ~ - 96 ~ (c 1, water).

Preparation of N,N',N"-tribenzyleremony- tin (V). To a solution of 165 mg (0.1 mmole) of eremomycin sulfate in 8 ml of a mixture of H20 and CH3OH (1 : 1), we added 0.051 ml (0.5 mmol) of C6HsCHO and 32 mg (0.5 mmole) of NaCNBH3. We mixed it for four hours at 20~ added 8 ml of CH3OH, 0.051 ml of C6HsCHO, and 32 mg (0.5 mmole) of NaCNBH3; and after two hours, we added 32 more mg of NaCNBH3. We stirred the solution for two hours, added 100 ml of H20, acidified it with 6 N H2SO4 up to pH 5, and extracted three times with n-butanol in 50 ml portions. We combined the or- ganic layers, evaporated them to 2 m[, and precipitated the target compound V with 80 ml of ethyl acetate. The yield of

-, 1% V was 106 mg (55%). Rf 0.30 (A1), 0.51 (A,), El~m 40.

[Ot]~ ~ - 89 ~ (c 1, methanol).

Preparation of N',N"-dimethyl-N-nitrosoeremomyein (VI). The procedure for preparing compound VI is similar to the procedure for preparing compound IV. From 163 mg of II, we prepared 96 mg (58%) of VI. Rf 0.26 (A1), 0.66 (A2),

0.48 (A3). EI~, 40. [ct]~ ~ - 93 ~ (c 1, water).

Preparation of N',N"-dibenzyl-N-nitrosoeremomycin (VII). The procedure for preparing compound VII is similar to the procedure for preparing compound V. From 163 mg of II, we prepared 102 mg (56%) of VII. Rf 0.38 (AI), 0.55 (A3). El% lcm 39. [Ct]~ ~ - 90 ~ (c 1, methanol).

Preparation of N,N,N",N"-tetramethyleremomycin (VIII). The procedure for preparing compound VIII is similar to the procedure for preparing compound IV. From 168 mg of III, we prepared 98 mg (57%) of VIII. Rf 0.52 (A2), 0.27

~1 ~ "~ o [Ct.]~O -- 93 ~ (c 1, water). (A3). eqc,n .~o.

Preparation of N',N"-dibenzyl-N,N-dimethyleremo- mycin (IX). The procedure for preparing compound IX is similar to the procedure for preparing compound V. From 168 mg of III, we prepared 106 mg (57%) of IX. Rf 0.23 (A1),

~ 1 % 0.45 (A3). mlcm 39. [Ct]~ ~ -- 96 ~ (C 1, methanol).

B I O L O G I C A L E X P E R I M E N T A L PART

The antibacterial activity of the prepared compounds with respect to Baci l lus subti les ATCC 6633 was determined by serial dilutions on a medium of the following composi- tion: Hottinger's hydrolyzate containing 28 rag% of amine ni t rogen- 1000 ml, a g a r - 30 g, peptone- 5 g, N a C I - 5 g, CaCO3 - 2.5 g, pH after sterilization 7.2 - 7.4. The activityS

TABLE 2 Antibacterial Activity m wtro of Deri,,atives IV - IX in Comparison with 1. [l. 11I. and Vancomycin

V a n c o m Compound I 11 III IV V VI VII VIII IX ycin

B. subtilis MIC, p.~ml 0.08 0.16 0.16 3.20 4.00 1.60 0.I6 1.60 0.32 0.16 S. aureus MtCs0, B~ml 0.50 2.00 2.00 16 32 16 4.00 16 2.00 2.00

with respect to 25 methicillin-resistant strains of Staphylo-

coccus aureus was studied by serial dilutions in a dense nutri- ent medium (AGV); the microbial load was 107 microbe bod- ies per ml. The bacterial suspensions were deposited onto the surface of the medium with a replicator. The data on the an- tibacterial activity in vitro of the prepared compounds in comparison with the starting compounds I, II, III, and van- comycin are presented in Table 2. A glance at the latter re- veals that the introduction into the eremomycin molecule of alkyl substituents at all three amino groups (compounds IV, V), and also methylation in compounds II and III of the amino groups of the saccharides (compounds VI, VIII) sub- stantially lowers the antibacterial activity. At the same time, the dibenzyl derivatives of compounds II and III at the amino groups of saccharides (compounds VII, IX), have either the same or half the activity of the starting compounds and of the antibiotic vancomycin used at clinics. These derivatives are two to eight times less active than eremomycin. The data are quite consistent with earlier results on the more favorable ef- fect on the activity from substituents at the amine groups of saccharides having increased lipophilic properties or aro- matic in nature [3].

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