Vol. 34, No. 10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1990, P. 1908-1914

0066-4804/90/101908-07$02.00/0 Copyright © 1990, American Society for Microbiology

Synthesis of Active Nitroguaiacol Ether Derivatives of Streptomycin JOSE P. ABAD AND RICARDO AMILS* Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas-Universidad Aut6noma de Madrid, Universidad AutonQma de Madrid, Canto Blanco, 28049 Madrid, Spain Received 29 November 1989/Accepted 6 July 1990

The synthesis, purification, and biological properties of nitroguaiacol ether derivatives of streptomycin and their corresponding radioactive reduced products were examined. These derivatives are biologically active against gram-positive and gram-negative eubacteria and they are also photoreactive because of the presence of the nitroguaiacol group in the molecule. We demonstrated that these derivatives can be used as streptomycin analogs in photoafflinity labeling of the macromolecular structures related to the mode of action of the antibiotic.

Since 1944, when Schatz et al. (16) reported the isolation of streptomycin (Fig. 1) from Streptomyces griseus cultures, this antibiotic has been studied widely. Streptomycin was one of the first antibiotics used clinically; but now, because of its toxic effect on the eighth cranial nerve, causing vertigo and deafness (4), it has been replaced in the treatment of many infectious diseases, although it is still one of the antibiotics of choice in the treatment of tuberculosis (18). In order to find structure-activity relationships that would permit the design of more efficient, less toxic antibiotics which could overcome the resistance of some important clinical strains, different research groups have synthesized several streptomycin derivatives. We now know that the modifications of the antibiotic's guanidine groups affect its biological activity (10, 12). Modifications of the secondary amino group also decrease activity (9). Some investigators have tried to circumvent the resistance produced by the action of antibiotic-modifying enzymes by eliminating the susceptible group affected by the enzyme from the antibiotic structure, as long'as it is not essential for the antibiotic activity. In this way Sano et al. (14) obtained 3'-deoxydihydrostreptomycin, which shows antibiotic activity against susceptible or resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis. The formyl group on streptobiosamine is the functional streptomycin group which has been modified most often, producing derivatives with various degrees of biological activity, ranging from the oxime (3), semicarbazone, and phenylhydrazone (5) of streptomycin, which are inactive, to dihydrostreptomycin, which is as active as streptomycin itself (6). Some research groups have reported that some other streptomycin derivatives, like carbohydrazones (7) and amines (8), are active as well. In this report we describe the synthesis of several carbohydrazone-type streptomycin derivatives that are active against gram-negative and gram-positive bacteria. Because of the photochemical properties of these derivatives, they can be used to map the interaction site of streptomycin with the bacterial ribosome or to study the active transport system of the antibiotic, using photoaffinity labeling techniques. * Corresponding author.

MATERIALS AND METHODS

4-Nitroveratrole, potassium hydroxide, potassium cyanide, silica gel for column chromatography, plates for thinlayer chromatography (TLC), and all the solvents, which were of analytical grade, were purchased from E. Merck AG (Darmstadt, Federal Republic of Germany). Bromoacetonitrile, 5-bromovaleronitrile, 1,8-dibromooctane, and hydrazine were provided by Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Streptomycin sesquisulfate was from Antibi6ticos S.A. (Madrid, Spain). The infrared (IR) spectra were registered on a model 325 apparatus (The Perkin-Elmer Corp., Norwalk, Conn.) by using KBr from Carlo Erba. The 'H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker model WM 360 apparatus (Bruker) by using the solvent indicated in each case. Elemental analysis was performed on a model 240C apparatus (Perkin-Elmer). Elemental analyses of all compounds were in agreement with the expected results (±0.4% for C, H, and N). Synthesis of potassium 4-nitroguaiacoxide. Potassium 4-nitroguaiacoxide' (compound IV) was synthesized 'as described by Pollecoff and Robinson (13). Synthesis of w-(4-nitroguaiacoxy)-alkylcyanides. The syntheses of w-(4-nitroguaiacoxy)-alkylcyanides were performed as described by Jelenc et al. (11). Analytical data for 4-nitroguaiacoxiacetonitrile (compound V), were as follows: IR (KBr) (in cm-') 3,090 (Ar H), 2,930 (al H), 2,840 (CH2-O, CH3-O), 2,260 (-C-N), 1,580, 1,500, 1,460 (Ar-NO2), 1,350 (Ar-NO2), 1,270 to 1,200 (Ar-O--C), 1,200 to 1,000 (Ar-O-C) and 'H NMR (CDC13) (in ppm) 8 4.00 (3H, s, CH3-O-Ar), 4.49 (2H, s, -CH2----Ar), 7.10 (1H, d, Ar 6-H), 7.83 (1H, d, Ar 3-H), 7.93 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 52.10; H, 4.10; N, 13.25. Analytical data for 5-(4-nitroguaiacoxy)pentanenitrile (compound VI) were as follows: IR (KBr) (in cm-l) 3,100 (Ar H), 3,000 to 2,900 (al H), 2,800 (-CH2-O, CH3-O), 2,240 (-C{N), 1,580, 1,500, 1,460 (Ar-NO2), 1,340 (Ar-NO2), 1,280 to 1,200 (Ar---C), 1,200 to 1,000 (Ar-O-C) and 'H NMR (CDCl3) (in ppm) 8 1.94 (2H, m, -CH2--C-CN), 2.06 (2H, m, -CH2-C---O-Ar), 2.51 (2H, t, -CH2--CN), 3.95 (3H, s, CH3-0-Ar), 4.16 (2H, t, -CH2--O-Ar). Elemental analysis was as follows: C, 57.76; H, 5.68; N, 11.20. Synthesis of 8-(4-nitroguaiacoxy)-octyl bromide. For syn1908

SYNTHESIS OF STREPTOMYCIN DERIVATIVES

VOL. 34, 1990

NH -

NH2

HO

O

HNKC

HH

H2N

0

H3C OHp

NH-CH3

HO OH I R=CHO U

R-CH20H

FIG. 1. Chemical structures of streptomycin (compound I) and dihydrostreptomycin (compound II).

thesis of 8-(4-nitroguaiacoxy)-octyl bromide (compound VII), compound IV (67 mmol) and 1,8-dibromooctane (134 mmol) in dimethylformamide (125 ml) were heated at 60 to 80°C until the orange color of the original material disappeared. The cold solution was then added to an ice-water bath, and the mixture was shaken and cooled for several hours. The product was then extracted with CH3Cl and purified by chromatography on silica gel, eluting the column with ethyl acetate-hexane mixtures with increasing polarity. Analytical data were as follows: IR (KBr) (in cm-') 3,090 (Ar H), 2,920 (al H), 2,850 (CH2-O, CH3-O), 1,580, 1,500, 1,465 (Ar-NO2), 1,330 (Ar-NO2), 1,270 to 1,200 (Ar-O-C), 1,200 to 1,000 (Ar----C), 555 (C-Br) and 'H NMR (CDCl3) (in ppm) 8 1.30 to 1.60 [8H, m,

-(C)2-(CCH2)4-(C)2-], 1.91 (4H, m, -CH2-C-Br, -CH2-C---OAr), 3.42 (2H, t, -CH12-Br), 3.95 (3H, s, CH3-{-Ar), 4.10 (2H, t, -CH2-0-Ar), 6.90 (1H, d, Ar 6-H), 7.75 (1H, d, Ar 3-H), 7.9 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 49.89; H, 6.40; N, 3.97. Synthesis of 9-(4-nitroguaiacoxy)-nonanenitrile. For the synthesis of 9-(4-nitroguaiacoxy)-nonanenitrile (compound VIII) the bromoderivative (compound VII) (10 mmol) was treated for 2.5 h with KCN (11 mmol) in 35 ml of ethyleneglycol at 80°C. The reaction mixture was then added to a previously cooled 0.1 M sodium carbonate solution. The suspension was stirred overnight and the solid deposited was filtered and purified by column chromatography on silica gel by using ethyl acetate-hexane mixtures with increasing polarity as eluent. Analytical data were as follows: IR (KBr) (in cm-') 3,100 (Ar H), 2,930 (al H), 2,850 (-CH2--O, CH3-O), 2,240 (-GCN), 1,560, 1,500, 1,460 (Ar-NO2), 1,350 (Ar-NO2), 1,275, 1,260, 1,230 (Ar----C), 1,200 to 1,000 (Ar-0-C) and 1H NMR (CDC13) (in ppm) 8 1.39

[4H, m, -(C)3---(CH2)2(C)3-C],

1.48

[4H, m, -CH2

-(C)2--CN, -CH21-(C)2---Ar], 1.67 (2H, m, -CH2 -C--CN), 1.89 (2H, m, -CH2--C--0--Ar), 2.35 (2H, t, -CH2-CN), 3.95 (3H, s, CH3---Ar), 4.10 (2H, t, -CH2-0-Ar), 6.90 (1H, d, Ar 6-H), 7.75 (1H, d, Ar 3-H), 7.90 (1 H, dd, Ar 5-H). Elemental analysis was as follows: C, 62.49; H, 7.37; N, 9.01. Synthesis of methyl w-(4-nitroguaiacoxy)-alkyl carboxy-

1909

lates. A stream of dry HCl was passed through a solution of the proper nitrile (10 mmol) in boiling absolute methanol (40 ml) containing H20 (10 mmol) until the reaction was completed. The solution was cooled, and the solid product was collected by filtration and recrystallized from ethanol-water. For methyl 4-nitroguaiacoxyacetate (compound IX), analytical data were as follows: IR (KBr) (in cm-') 3,100 (Ar H), 2,950 (al H), 2,850 (CH2-O, CH3-0), 1,755 (C=O), 1,580, 1,520, 1,495 (Ar-NO2), 1,350, 1,330 (Ar-NO2), 1,280 to 1,200 (Ar-O-C), 1,200 to 1,000 (Ar-CO-C), and 1H NMR (CDCl3) (in -ppm) 8 3.83 (3H, s, -CO2-CH3), 3.99 (3H, s, CH3-O-Ar), 4.82 (2H, t, -CH2--O-Ar), 6.82 (1H, d, Ar 6-H), 7.79 (1H, d, Ar 3-H), 7.91 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 49.73; H, 4.70; N, 5.67. For methyl 4-nitroguaiacoxy-pentanecarboxylate (compound X), analytical data were as follows: IR (KBr) (in cm 1) 3,100 (Ar H), 2,960 (al H), 2,850 (-CH2-O, CH3-O), 1,735 (C==O), 1,585, 1,505, 1,470 (Ar-NO2), 1,340 (Ar-NO2), 1,280 to 1,235 (Ar-O-C), 1,200 to 1,000 (Ar-O-C) and 1H NMR (CDCl3) (in ppm) 8 1.86 (2H, m, -CH2--C--C02-), 1.92 (2H, m, -CH2-C--O-Ar), 2.43 (2H, t, -CH2--CO2-C-), 3.68 (3H, s,-CO2-CH3), 3.95 (3H, s, CH3-O--Ar), 4.12 (2H, t, -CH2--O-Ar), 6.90 (1H, d, Ar 6-H), 7.75 (1H, d, Ar 3-H), 7.90 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 55.12; H, 5.84; N, 3.18. For methyl 9-(4-nitroguaiacoxy)-nonanecarboxylate (compound XI), analytical data were as follows: IR (KBr) (in cm'1) 3,100 to 3,020 (Ar H), 2,980, 2,920 (al H), 2,840 (-CH2-O, CH3-O), 1,720 (C=O), 1,575, 1,520, 1,500, 1,490 (Ar-NO2), 1,360, 1,330 (Ar-NO2), 1,290 to 1,200 (Ar---C), 1,200 to 1,000 (Ar-O--C) and 'H NMR (CDCl3) (in ppm) 8 1.35 [6H, m, -(C)3-(CH2)3-(QC)2 -C02-CH3], 1.46 [2H, m, -CH2--(C)2---Ar], 1.63 (2H, m,-CH2-C--O2-CH3), 1.88 (2H, m, -CH2 -C--O--Ar), 2.31 (2H, t, -CH2-CO2CH3), 3.67 (3H, s, -CO2CH3), 3.95 (3H, s, Ar-OCH3), 4.10 (2H, t, -CH2--}-Ar), 6.90 (1H, d, Ar 6-H), 7.75 (1H, d, Ar 3-H), 7.9 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 60.46; H, 7.58; N, 3.93. Synthesis of w-(4-nitroguaiacoxy)-acyl-hydrazides. The suitable methyl ester (10 mmol) was boiled with a 5 to 10 times molar excess of hydrazine hydrate in absolute ethanol (33 ml) until the reaction was completed. The desired product crystallized on cooling and was further purified by recrystallization in ethanol. For 4-nitroguaiacoxy-acetyl-hydrazine, (compound XII), analytical data were as follows: IR (KBr) (in cm-') 3,330, 3,260 (N-H), 3,100 (Ar H), 2,990 (al H), 2,840 (-CH2-O, CH3-O), 1,670, 1,620 (C=O), 1,585, 1,515 (Ar-NO2), 1,350, 1,340 (Ar-NO2), 1,280 to 1,230 (Ar-O-C), 1,200 to 1,000 (Ar- C) and 'H NMR (CDC13) (in ppm) 8 1.25 (1H, s, N-H), 3.98 (3H, s, CH3A-O-Ar), 4.68 (2H, t, -CH2--0-Ar), 6.90 (1H, d, Ar 6-H), 7.79 (1H, d, Ar 3-H), 7.91 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 45.12; H, 4.84; N, 17.12. For 5-(4-nitroguaiacoxy)-pentanoyl-hydrazide (compound XIII), analytical data were as follows: IR (KBr) (in cm-') 3,330 (N-H), 3,080 (Ar H), 2,960 to 2,900 (al H), 2,865 (-CH2-O, CH3-O), 1,640, 1,625 (C-O), 1,580, 1,500, 1,455, 1,330 (Ar-NO2), 1,270, 1,230, 1,215 (Ar-O---C), 1,200 to 1,000 (Ar---C) and 1H NMR (CDC13) (in ppm) 8 1.92 [4H, m, Ar-O-C-(CH2)2-C-CONHNH2], 2.33 (2H, t, --CH2-CONHNH2), 3.93 (2H, -NH2), 4.00 (3H, s, CH3-0-Ar), 4.15 (2H, t, -CCH2--O--Ar), 6.90 (1H, d, Ar

1910

ABAD AND AMILS

6-H), 7.51 (1H, s, -CO-NH-N-), 7.75 (1H, d, Ar 3-H), 7.90 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 50.80; H, 6.20; N, 14.72. For 9-(4-nitroguaiacoxy)-nonanoyl-hydrazide (compound XIV), analytical data were as follows: IR (KBr) (in cm-') 3,300 (N-H), 2,915 (al H), 2,840 (-CH2-O, CH3-O), 1,670, 1,620 (C=O), 1,585, 1,500, 1,460 (Ar-NO2), 1,335 (Ar-NO2), 1,270 to 1,230 (Ar-CO-C), 1,200 to 1,000 (ArC-O-C) and 1H NMR (CDC13) (in ppm) 8 1.35 [6H, m, -(C)3-(CH2)3-CONHNH2], 1.45 [2H, m, -CH2-(C)2 -0--Ar], 1.63 (2H, m, -CH2-C-CONHNH2), 1.88 (2H,- m, -CH2-C-C---Ar), 2.15 (2H, t, -CH2-CO-NH -NH), 3.91 (1H, -N-NH2), 3.95 (3H, s, Ar-OCH3), 4.10 (2H, t, -CH2-O-Ar), 6.70 (1H, -CO-NH-N-), 6.90 (1H, d, Ar 6-H), 7.75 (1H, d, Ar 3-H), 7.90 (1H, dd, Ar 5-H). Elemental analysis was as follows: C, 56.92; H, 7.63; N, 12.09. Synthesis of &-(4-nitroguaiacoxy)-acyl-hydrazones of streptomycin. For the synthesis of w-(4-nitroguaiacoxy)-acylhydrazones of streptomycin (compounds XV, XVI, and XVII), an aqueous solution of streptomycin sesquisulfate (111 mmol) was mixed with a solution of the proper hydrazide (167 mmol) in dimethyl sulfoxide. The solution was kept at 50°C for 1 h and cooled, and ethanol (5 volumes) was added. The solution was kept at -20°C for several hours and then centrifuged to collect the precipitate. The streptomycin derivatives were purified by ion-exchange chromatography on carboxymethyl cellulose. To purify the derivative obtained from 162 mg of streptomycin, a column of 1.5 by 20 cm was used. The resin was swollen with 0.01 M ammonium formate buffer (pH 4). The precipitate was dissolved in the same buffer, and a few microliters of [3H]dihydrostreptomycin was added as a marker for streptomycin. The- column was eluted with a formate gradient from 0.01 to 1 M with constant pH (pH 4). The collected fractions were analyzed for their radioactivities, optical densities at 344 nm, and guanidine group concentrations. The fractions that were not radioactive, that had guanidine groups, and that exhibited an A344 contained the pure streptomycin derivative. Those fractions were pooled and lyophilized in order to remove the volatile ammonium formate. The purity of the compounds obtained was checked by TLC by using a mixture of pyridine-water-ethyl acetateacetic acid (5:2:2:1) as the eluent and was developed by heating a plate that was sprayed with 50% H2SO4. Elemental analysis of compound XV was as follows: C, 45.39; H, 5.80; N, 15.96. Elemental analysis of compound XVI was as follows: C, 47.20; H, 6.20; N, 15.15. Elemental analysis of compound XVII was as follows; C, 49.50; H, 6.70; N, 14.40. Reduction of hydrazones of streptomycin. The corresponding streptomycin derivatives (compounds XV to XVII) (85 mmol) were dissolved in water and the pH was adjusted to 8 with NaOH. The solution was cooled (0°C) and sodium borohydride (1.7 mol) was then added. The mixture was stirred for 30 min at 0°C and 1 h at room temperature and then lyophilized and purified as indicated above for the hydrazone. To obtain reduced streptomycin derivatives which were radioactive, we used tritiated sodium borohydride with a high specific activity (5 to 10 Ci/mmol). We usually added 100 mCi to a solution of the derivative in phosphate buffer, pH 8 (20 times cxcess of NaBH4). The reaction was performed as described above, but prior to lyophilization in aliquots, the solution was evaporated two to three times under an N2 stream to release any radioactivity that was not incorporated in the derivative. The resultant product was not

ANTIMICROB. AGENTS ICHEMOTHER.

purified any further. The specific activity obtained in derivatives XVIII and XIX was between 1,000 and 2,000 cpm/ pmol, depending on the reductive reactiop. In the cage of derivative XX, the. specific activity was between 500 and 1,000 cpm/pmol. Elemental analysis of compound XVIII wAs as follows: C, 45.30; H, 6.02; N, 16.05. Elemental analysis of compound XIX was as follows: C, 47.12; H, 6.39; N, 15.47. Elemental analysis of compound XX was as follows: C, 49.21; H, 7.10; N, 14.49. Bacterial strains and culture media. Escherichia coli MRE 600 and Bacillus subtilis, ATCC 6633 were grown in medium containing 5 g of yeast extract per liter, 10 g of tryptone (Difco Laboratories, Detroit, Mich.) per liter, 5 g of NaCl per liter, and 0.2% glucose at 37°C. The petri plates were prepared with the same6medium including 1%. of agarose. Plates (diameter, 10 cm) were prepared with a base layer of 5 ml of the medium and a. covering layer containing the bacteria at 0.005 optical idehsity units at 660 nm. The antibiotics were put on 3MM paper disks (diameter, 5 mm). The plates were incubated for 12 to 14 h at 37°C in a stove. Concentration of guanidiine groups. Guanidine group concentrations were determined by the method of Tomlinson and Viswanatha (17). Preparation of ribosomal particles. For the preparation of ribosomal particles, the meth}ods described by Amils et al. (2) were used. Irradiation experiments. Ribosomal particles (final concentration, 1 ,uM) were irradiatea with the different streptomycin derivatives in a 1:2 molar ratio except when indicated. A mercury medium-pressure lamp was used, and the samples were placed in glass tubes at 5 cm from the light source in a container and refrigerated at 4°C. Samples were taken at appropriate times. Controls were incubated under the same conditions but wrapped in aluminum foil to prevent photolysis. RESULTS Three carbohydrazones of s,treptomycin and their related reduced compounds were synthesized. The p-nitroguaiacoxide group on these derivatives was separated from the streptomycin moiety by spacers of different lengths. These derivatives were obtained by the synthesis pathway outlined in Fig. 2. As shown above (Fig. 2), carbohydrazones with one, four, or eight methylene grotps between theit aromatic and carbonyl groups were synthesized. Commercial 4-nitroveratrole (compound III) was transformed into its potassium salt (compound IV) by heating it in a basic medium. The following step was to bind the aromatic ring to the methylene chain by means of an ether bond. By using suitable alkyl bromides, the nitroguaiacoxide group was attached to the alkylene spacers by a bromine substitution. Bromoalkyl nitriles were us.ed for the synthesis of compounds V and VI. Compound VIII was prepared by treating the appropriate bromode-rivative (compound VII) with potassium cyanide. Methanosysis of the nitriles catalyzed by dry gaseous hydrogen chloride gave the corresponding esters (compounds IX, X, and XI). The hydrazides were obtained by reaction of the esters with hydrazine hydrate. Esters were chosen over acids because the former react better with hydrazine-releasing monohydrazides (compounds XII, XIII, and XIV) (15), while the acids give mainly dihydrazide derivatives. The yields of synthesis of all the intermediates were quantitative. When necessary, the intermediate products were purified by chromatography and recrystallization. The carbohydrazides were condensed with

SYNTHESIS OF STREPTOMYCIN DERIVATIVES

VOL. 34, 1990

O,N-~OCH,3

1911

0O2N~O

KO

E E

HIBrCt,,r

OCH3

O

KOH

).15 Z

Br-(CH2)8-Br E

z

OCH3

Br- (CH2 )n6 CN

1.10 °

CY z rn

Li

0X

n=1,4

0

KCN

1.05

> 0

Xr CL

40 60

80

100 120 140 160 180 200 220 240 260 280 FRACTION NUMBER

FIG. 3. Purification by ion-exchange chromatography of streptomycin derivative XVII. 0, Streptomycin group concentrations determined by the method of Tomlinson and Viswanatha (16); 0, optical density at 344 nm; A, radioactivity.

12N300 nm), (ii) higher yields of the photoreaction that only takes place with nucleophilic groups, thus preventing dissipation of the photolabel with the solvent, (iii) short lifetime of the excited state, which should favor the specificity of photolabeling, and (iv) haptenic properties of the photoproducts that allow the use of specific antibodies for their analysis. The photoreactive group was attached to the antibiotic through the carbonyl group because (i) previous experience has shown that there are a number of streptomycin derivatives on this group that retain biological activity and (ii) this group is unique, easily modifiable, and one of the most reactive in the molecule. The photoreactive ring was bound to methylene chains of different lengths in order to produce derivatives which kept the photoreactive group at different distances from the antibiotic. This was extremely useful for testing the topographical consistency of the photolabeling. The methylene chain was functionalized to obtain a hydrazide group that reacted easily with the aldehyde group of streptomycin. The condensation between the carbonyl and the hydrazide groups gave a double C=N bond that could be reduced allowing the introduction of tritium into the molecule, facilitating the analysis of the photoreaction and its products. Purification of the streptomycin derivatives was done by ion-exchange chromatography at low pH, which maintained positive charges on the streptomycin guanidine and amino groups. The peaks of the streptomycin derivatives were well separated from that of streptomycin, and the ammonium formate used to elute the column was easily eliminated by lyophilization, leaving the derivatives as formate salts. The purity of the synthesized compounds was checked by TLC. Developing conditions which distinguished between streptomycin (Rf = 0), the hydrazides (Rf = 1), and the derivatives (Rf 0.7) were used. All the streptomycin derivatives obtained were free of streptomycin or hydrazide contamination (Fig. 4). Elemental analyses of the compounds and their 'H NMR spectra (Fig. 5) were in agreement with the expected structures. Once the purity and structure of the streptomycin derivatives were checked, their biological activities were tested. As shown in Table 1, all the nonreduced (compounds XV, XVI, and XVII) and reduced (compounds XVIII, XIX, and XX) streptomycin derivatives interfered with the growth of gram-negative and gram-positive eubacteria, although at TABLE 1. Activities of streptomycin derivatives on E. coli and B. subtilis growth CI50 (p.g/ml)b Derivativea

Streptomycin (Sm-CHO) (I) Dihydrostreptomycin (II)

E. coli

1.43 0.95 9.29 Sm-CH=NNH-C0-CH2-O--Ar (XV) 6.43 Sm-CH=NNH-CO-(CH2)4--Ar (XVI) 3.33 Sm-CH=NNH-CO-(CH2)8--Ar (XVII) Sm-CH2-NHNH-CO-CH2--Ar (XVIII) >20.00 Sm-CH2-NHNH-C0-(CH2)4--O-Ar (XIX) 12.62 4.17 Sm-CH2-NHNH--CO-(CH2)8--Ar (XX) a Ar, C6H3 (p-NO2,o-OCH3). b CT50, Concentration of derivative that caused

growth.

B. subtilis

0.09 0.15 2.09 1.41 0.88 9.56 1.38 0.76

50%o inhibition of bacterial

VOL. 34, 1990

a

z CD

4.

0

CD CD

00:. 0 3

U

0 0-

-o

0 CD 0.

0

CD

10 0 C,

0CD

X

0co

C. CD

_. CK

Z

00D 0 D

a

C.0.

3

0: 0

CA

I 4

.0

I I

4

~I

SYNTHESIS OF STREPTOMYCIN DERIVATIVES

1913

0

0

3*-.

S

0*

Q.

3

s

1914

ANTIMICROB. AGENTS CHEMOTHER.

ABAD AND AMILS

streptomycin, the structures related to its ribosomal binding site, and its active transport system. ACKNOWLEDGMENTS This work was supported by grants from the Comisi6n Asesora de Investigaci6n, Fundaci6n Ram6n Areces, and institutional grants from the Fondo de Investigaciones Sanitarias. One of us (J.P.A.) was a predoctoral fellow from the Fondo de Investigaciones Sanitarias de la Seguridad Social. We thank 0. Nieto and M. Bernabe for critical reading of the manuscript and J. P. G. Ballesta and D. Vazquez for many valuable discussions.

c0

0

X (nm)

FIG. 6. Kinetics of the photolysis of streptomycin derivative XIX irradiated in the presence of equimolar concentration of 70S ribosomes. O.D., Optical density.

lower efficiencies than unmodified streptomycin and with slight differences between them. In order to use these streptomycin analogs in the study of the antibiotic ribosomal binding site, it is necessary to ascertain whether they bind to the same site. As demonstrated previously, this is, in fact, the case because all the derivatives inhibit protein synthesis and compete efficiently with dyhydrostreptomycin for the same ribosomal binding site (1). The photochemical performances of the derivatives were tested in two types of experiments. In the first experiment, nonradioactive derivatives were irradiated in the presence of E. coli ribosomes, and the corresponding UV spectra were obtained after different irradiation times under the conditions described above. There was a concomitant decrease in the A340 when ribosomes were irradiated for increasing periods of time (Fig. 6), indicating that photosubstitution took place. To demonstrate that the photoreaction actually promoted the binding of the derivative to the ribosomal components, irradiation was performed with radioactive derivatives. Figure 7 shows that increasing amounts of radioactive derivative appeared to be covalently bound to the trichloroacetic acid-precipitable ribosomal components when ribosomes or 30S ribosomal subunits were irradiated for different periods of time. After 2 h of irradiation the photoincorporation leveled off. The addition of cold streptomycin to the irradiation mixture reduced the photoincorporation drastically, suggesting that specific photolabeling of the streptomycin binding site was achieved. These results allow us to postulate that these derivatives are suitable for characterization of the mode of action of V

° .04

a

-

E

a .03 2 x .02 x

2

.01

1,0l 1

-zlwl

1

L.

I

E

1

2

3

4

5

IRRADIATION TIME (h)

FIG. 7. Kinetics of photoincorporation of radioactive streptomycin derivative XIX to 70S ribosomes (A) and 30S ribosomes (0). A, Competition when irradiation of 70S ribosomes was performed in the presence of a 10 times molar excess of cold streptomycin.

LITERATURE CITED 1. Abad, J. P., G. Le6n, and R. Amils. 1987. Biological activity of nitroguaiacol ether derivatives of streptomycin. J. Antibiot.

40:685-691. 2. Amils, R., E. A. Matthews, and C. R. Cantor. 1979. Reconstitution of 50S ribosomal subunits from E. coli. Methods Enzymol. 59:449-461. 3. Brink, N. G., F. A. Kuehl, Jr., and K. Folkers. 1945. Streptomyces antibiotics. III. Degradation of streptomycin to streptobiosamine derivatives. Science 102:506-507. 4. Caldwell, J. R., and L. E. Chuff. 1974. Adverse reactions to antimicrobial agents. J. Am. Med. Assoc. 230:77-80. 5. Donovick, R., G. Rake, and J. Fried. 1946. The influence of certain substances on the antibiotic activity of streptomycin in vitro. II. The action of some carbonyl reagents on streptomycin. J. Biol. Chem. 164:173-181. 6. Edison, A. O., B. M. Frost, 0. E. Graessle, J. E. Hawkins, Jr., S. Kuna, C. W. Mushett, R. H. Silber, and M. Solotorovsky. 1948. An experimental evaluation of dihydrostreptomycin. Annu. Rev. Tuberc. 58:487-493. 7. Hall, J., J. P. Davis, and C. R. Cantor. 1977. Interaction of a fluorescent streptomycin derivative with E. coli ribosomes. Arch. Biochem. Biophys. 179:121-130. 8. Heding, H., and A. Diedrichsen. 1975. Streptomycylamines: difference in activity and mode of action between short-chain and long-chain derivatives. J. Antibiot. 28:312-316. 9. Heding, H., G. J. Fredericks, and 0. Lutzen. 1972. New active streptomycin derivatives. Acta Chem. Scand. 26:3251-3256. 10. Heding, H., and 0. Lutzen. 1972. Streptomycin: correlation between in vivo and in vitro activity of ten representative derivatives. J. Antibiot. 25:287-291. 11. Jelenc, P. C., C. R. Cantor, and S. R. Simon. 1978. High yield photoreagents for protein crosslinking and affinity labelling. Proc. Natl. Acad. Sci. USA 75:3564-3568. 12. Munro, M. H. G., R. M. Stroshane, and K. L. Rinehart, Jr. 1982. Location of guanidino and ureido groups in bluensomycin from '3C-NMR spectra of streptomycin and related compounds. J. Antibiot. 35:1331-1337. 13. Poliecoff, F., and R. Robinson. 1918. Nitro-derivatives of guaiacol. J. Chem. Soc. 113:645-656. 14. Sano, H., T. Tsuchiya, S. Kobayashi, M. Hamada, S. Umezawa, and H. Umezawa. 1976. Synthesis of 3' deoxydihydrostreptomycin active against resistant bacteria. J. Antibiot. 29:978-980. 15. Satchell, D. P. N., and R. S. Satchell. 1969. Substitution in the groups COOH and COOR, p. 375-452. In S. Patai (ed.), The chemistry of functional groups. The chemistry of carboxilic acids and esters. John Wiley & Sons, Inc., N.Y. 16. Schatz, A., E. Bugie, and S. A. Waksman. 1944. Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria. Proc. Soc. Exp. Biol. Med. 55:6669. 17. Tomlinson, G., and T. Viswanatha. 1974. Determination of the arginine content of proteins by the Sakaguchi procedure. Anal. Biochem. 60:15-24. 18. Trnka, L., P. Mison, K. Bartmann, and H. Otten. 1988. Experimental evaluation of efficacy, p. 31-91. In K. Bartmann (ed.), Handbook of experimental pharmacology, vol. 84. SpringerVerlag, New York.