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The influence of lipophilic character on the biological activity of oligosaccharide antibiotics in Escherichia coli
Pharmacological Research Communications, Vol. 2, No.. 2, 1970
121
THE INFLUENCE OF LIPOPHILIC CHARACTER ON THE BIOLOGICAL ACTIVITY OF 0LIGOSACCHARIDE ANTIBIOTICS IN ESCHERICHIA COLI.
Gian Luigi Biagi, Anna Maria Barbaro, Maria Clelia Guerra Istituto di Farmacologia e Farmacognosia dell'Universit~ di Bologna Received 26 May 1970 In a previous paper a relationship was shown between lipophilic character, as expressed by means of the chromatographic Rm value, and antibacterial activity of cephalosporins and penicillins in Escherichia coli, Staphylococcus aureus and Treponema pallidum (Biagi et al., 1970). The compounds most active against the Gram-negative E. coli were found to be more hydrophilic than those most active against the Gra~-positive Staph. aureus. This result, which was in agreement with analogous findings of Hansch et al. (1963, 1964 a, 1964 b, 1965) and Lien et al. (1968), suggested the present work dealing with the Rm values and the antibacterial activity in E. coli of six oligosaccharide antibiotics.
MATERIALS AND METHODS
The lipophilic character of the streptomycin-
like antibiotics given in Table I was expressed by means of the chromatographic Rm value , calculated from the formula Rm = log ( 1 - 1). The Rf values Rf were determined by a thin-layer chromatographic method, the details of which have already been described (Biagi et al., 1969 b). The non-polar stationary phase was represented by silicon oil, which impregnated a Silica Gel G layer. The polar mobile phase was an aqueous buffer (sodium acetate-Veronal buffer at pH 7.4), which provoked a suitable migration of all the tested compounds. The antibiotics were assayed against E. coli 0-25 by means of the cylinderplate method as previously described (Biagi et al., 1969 a). The biological activity was expressed as log ( I ) where C is the molar (mH x 10-2 ) C concentration of each antibiotic which gives an inhibition diameter of 20 mm. The molecular weights used in the calculations are reported in
Pharmaco~gicalResearch Commun~ations, VoL 2, No. 2,1970
122
Table I. Kanamycin and neomycin were assumed to be represented by the sulphate salt of respectively pure kanamycin A and neomycin B.
Table I - Activity and lipophilic character of oligosaccharide antibiotics.
Compound
M.w.
log
I
C
Rm value
Neomycin B sulphate
7]2.67
3. 113
O. 184
Paramomycin sulphate
713 968
3 9068
-0.029
Aminosidine sulphate
896.80
3.198
-0.037
Kanamycin A sulphate
582.59
3. 060
-0.227
Dihydro st rept omycin sulphate
1459.41
3.594
-O. 322
Streptomycin sulphate
1457.40
3.438
-0.485
RESULT AND DISCUSSION
The Rm values and the observed log
I
data of C oligosaccharide antibiotics are reported in Table I. Higher and/or positive
Rm values indicate compounds more lipophilic than those characterized by lower aud/or negative Rm values. The data show that reversed-phase TLC is a suitable technique for the determination of partition data of oligosacharide antibiotics. The ranking of the compounds according to their lipophilic character, as shown by their Rm values in Table I, is in fair agreement with the results of Ito et al. (1964), who carried out the chromatography of neomycin, paromycin, kana~ycin A, dihydrostreptomycin and streptomycin on cellulose with propanol-pyridine-acetic acid-water (15 : 10 : 3 : 12). The only important difference being that they found tha same Rf value for both streptomycin and dihydrostreptomycin. In the case of penicillins and
Pharmaco~gicalResearchCommun~adons,
VoL 2, No. 2,1970
123
cephalosporins there was a linear or parabolic relationship between Rm and log (-~-~) values. In particular the quadratic dependence of log ( 1 ) on C C the Rm values was in agreement with the postulate of the parabolic relationship between the penetration rate of compounds through~biological membranes and their lipophilic character (Penniston et al., 1969). The presence, in the case of peni~illins in E. coli, of a linear relationship was explained on the basis of the lack of more hydrophilic compounds. On the contrary the regression analysis showed no significant relationship between the Rm values I and the l o g ~ v a l u e s of the oligosaccharide antibiotics in E. coli. This C could be due to the fact that the few compounds at present available all possess a similar degree of activity against E. coll.
It is possible that
the availability of more hydrophilic and respectively more lipophilic compounds would cause a parabolic relationship between Rm and log ( ~ ) values. At any rate, the R m v a l u e s of the oligosaccha~ideantibiotics, ranging from 0.13 to -0.48, practically overlap those of the penicillins most active against E. coli, i.e. ampicillin, methylenampicillin and carbenecill~ the Rm values of which range from 0.08 to -0.46 (Biagi et al., 1969 b). I On the other hand the log values observed in Table I indicate a degree C of activity in E. coli close to that of ampicillin, methylenampicillin I and carbenecillin. In fact, while the log values of oligosaccharide C antibiotics vary from a minimum of 3.06 to a maximum of 3.59, those of the three mentioned penicillins range from 2.94 to 3.85. The present data seem therefore to support the previous finding that Gr~m-negative organisms are likely to be most sensitive to hydrophilic compounds (Lien etal.,
1968; Biagi et al. 1969). An interpretation of
this point has been suggested (Lien et al., 1968). The hydrophilic compounds may be the most active against Gram-negative organisms, as the high lipid content of the cell wall in these organisms would not retain hydrophilic molecules. Anand e t a l .
(1960) showed that in Eo coli streptomycin is
initially accumulated by the cell wall, which swells and becomes porous.
124
PharmacologicalResearch Communications, YoL Z No. ~ 1970
Streptomycin then enters the bacterial cell more easily and reaches its site of action on the ribosomes. The penetration of the oligosaccharide antibiotics into the bacterial cell, after having been accumulated in the cell wall, could actually be due to their hydrophilic character. The differences in the diffusion rates of the antibiotics in the gel were assumed not to influence the results in a qualitative way. According to Schlesinger (1954) the diffusion constant of the antibiotics may be disregarded. Ericsson et al. (1959) seemed to accept this opinion, when considering substances characterized, as in the present cases, by relatively small variations in molecular weight, as the diffusion constant mainly depends on the molecular weight of the Substance.
REFERENCES Anand N., Davis B.D. and Armitage A.K., (1960), Nature, Lond., 18~, 23. Biagi G.L., Barbaro A.M., Gamba M.F. and Guerra M.C., (1969), .J.Chromato~. 41, 371. Biagi G.L., Guerra M.C., Barbaro A.M. and Gamba M.F., (1970) J. Med. Chem. accepted for pubblicati0n. Ericsson H. and Svartz-Malmberg G., (1959), Antibiotica et Chemiotherapia, 6, 41. Hansch C., Muir R.M., Fujita T., Maloney P.P., Geiger F. and Streich M., (1963), J. Am. Chem. Soc., 85, 2817. Hansch C. and FUjita T., (1964 a), J. Am. Chem. Soc., 86, 1616. Hansch C. and Steward A.E., (1964 b), J. Med. Chem., 7, 691. Hansch C. and Deutsch E.W., (1965), J. Med. Chem., 8, 705. Ito Y., Namba M., Nagahama N., Yamaguchi T. and Okuda T., (1964), J. Antibiotics - (Tokyo) Ser. A, 17, 218. Lien E.J., Hansch C. and Anderson S.M. (1968), J. Med. Chem., 11, 430. Penniston J.T., Beckett L., Bentley D.L. and Hansch C., (1969), Mol. Pharmacol., 5, 333. Schlesinger F., (1954), Acts med. scand., 148 , 357.
121
THE INFLUENCE OF LIPOPHILIC CHARACTER ON THE BIOLOGICAL ACTIVITY OF 0LIGOSACCHARIDE ANTIBIOTICS IN ESCHERICHIA COLI.
Gian Luigi Biagi, Anna Maria Barbaro, Maria Clelia Guerra Istituto di Farmacologia e Farmacognosia dell'Universit~ di Bologna Received 26 May 1970 In a previous paper a relationship was shown between lipophilic character, as expressed by means of the chromatographic Rm value, and antibacterial activity of cephalosporins and penicillins in Escherichia coli, Staphylococcus aureus and Treponema pallidum (Biagi et al., 1970). The compounds most active against the Gram-negative E. coli were found to be more hydrophilic than those most active against the Gra~-positive Staph. aureus. This result, which was in agreement with analogous findings of Hansch et al. (1963, 1964 a, 1964 b, 1965) and Lien et al. (1968), suggested the present work dealing with the Rm values and the antibacterial activity in E. coli of six oligosaccharide antibiotics.
MATERIALS AND METHODS
The lipophilic character of the streptomycin-
like antibiotics given in Table I was expressed by means of the chromatographic Rm value , calculated from the formula Rm = log ( 1 - 1). The Rf values Rf were determined by a thin-layer chromatographic method, the details of which have already been described (Biagi et al., 1969 b). The non-polar stationary phase was represented by silicon oil, which impregnated a Silica Gel G layer. The polar mobile phase was an aqueous buffer (sodium acetate-Veronal buffer at pH 7.4), which provoked a suitable migration of all the tested compounds. The antibiotics were assayed against E. coli 0-25 by means of the cylinderplate method as previously described (Biagi et al., 1969 a). The biological activity was expressed as log ( I ) where C is the molar (mH x 10-2 ) C concentration of each antibiotic which gives an inhibition diameter of 20 mm. The molecular weights used in the calculations are reported in
Pharmaco~gicalResearch Commun~ations, VoL 2, No. 2,1970
122
Table I. Kanamycin and neomycin were assumed to be represented by the sulphate salt of respectively pure kanamycin A and neomycin B.
Table I - Activity and lipophilic character of oligosaccharide antibiotics.
Compound
M.w.
log
I
C
Rm value
Neomycin B sulphate
7]2.67
3. 113
O. 184
Paramomycin sulphate
713 968
3 9068
-0.029
Aminosidine sulphate
896.80
3.198
-0.037
Kanamycin A sulphate
582.59
3. 060
-0.227
Dihydro st rept omycin sulphate
1459.41
3.594
-O. 322
Streptomycin sulphate
1457.40
3.438
-0.485
RESULT AND DISCUSSION
The Rm values and the observed log
I
data of C oligosaccharide antibiotics are reported in Table I. Higher and/or positive
Rm values indicate compounds more lipophilic than those characterized by lower aud/or negative Rm values. The data show that reversed-phase TLC is a suitable technique for the determination of partition data of oligosacharide antibiotics. The ranking of the compounds according to their lipophilic character, as shown by their Rm values in Table I, is in fair agreement with the results of Ito et al. (1964), who carried out the chromatography of neomycin, paromycin, kana~ycin A, dihydrostreptomycin and streptomycin on cellulose with propanol-pyridine-acetic acid-water (15 : 10 : 3 : 12). The only important difference being that they found tha same Rf value for both streptomycin and dihydrostreptomycin. In the case of penicillins and
Pharmaco~gicalResearchCommun~adons,
VoL 2, No. 2,1970
123
cephalosporins there was a linear or parabolic relationship between Rm and log (-~-~) values. In particular the quadratic dependence of log ( 1 ) on C C the Rm values was in agreement with the postulate of the parabolic relationship between the penetration rate of compounds through~biological membranes and their lipophilic character (Penniston et al., 1969). The presence, in the case of peni~illins in E. coli, of a linear relationship was explained on the basis of the lack of more hydrophilic compounds. On the contrary the regression analysis showed no significant relationship between the Rm values I and the l o g ~ v a l u e s of the oligosaccharide antibiotics in E. coli. This C could be due to the fact that the few compounds at present available all possess a similar degree of activity against E. coll.
It is possible that
the availability of more hydrophilic and respectively more lipophilic compounds would cause a parabolic relationship between Rm and log ( ~ ) values. At any rate, the R m v a l u e s of the oligosaccha~ideantibiotics, ranging from 0.13 to -0.48, practically overlap those of the penicillins most active against E. coli, i.e. ampicillin, methylenampicillin and carbenecill~ the Rm values of which range from 0.08 to -0.46 (Biagi et al., 1969 b). I On the other hand the log values observed in Table I indicate a degree C of activity in E. coli close to that of ampicillin, methylenampicillin I and carbenecillin. In fact, while the log values of oligosaccharide C antibiotics vary from a minimum of 3.06 to a maximum of 3.59, those of the three mentioned penicillins range from 2.94 to 3.85. The present data seem therefore to support the previous finding that Gr~m-negative organisms are likely to be most sensitive to hydrophilic compounds (Lien etal.,
1968; Biagi et al. 1969). An interpretation of
this point has been suggested (Lien et al., 1968). The hydrophilic compounds may be the most active against Gram-negative organisms, as the high lipid content of the cell wall in these organisms would not retain hydrophilic molecules. Anand e t a l .
(1960) showed that in Eo coli streptomycin is
initially accumulated by the cell wall, which swells and becomes porous.
124
PharmacologicalResearch Communications, YoL Z No. ~ 1970
Streptomycin then enters the bacterial cell more easily and reaches its site of action on the ribosomes. The penetration of the oligosaccharide antibiotics into the bacterial cell, after having been accumulated in the cell wall, could actually be due to their hydrophilic character. The differences in the diffusion rates of the antibiotics in the gel were assumed not to influence the results in a qualitative way. According to Schlesinger (1954) the diffusion constant of the antibiotics may be disregarded. Ericsson et al. (1959) seemed to accept this opinion, when considering substances characterized, as in the present cases, by relatively small variations in molecular weight, as the diffusion constant mainly depends on the molecular weight of the Substance.
REFERENCES Anand N., Davis B.D. and Armitage A.K., (1960), Nature, Lond., 18~, 23. Biagi G.L., Barbaro A.M., Gamba M.F. and Guerra M.C., (1969), .J.Chromato~. 41, 371. Biagi G.L., Guerra M.C., Barbaro A.M. and Gamba M.F., (1970) J. Med. Chem. accepted for pubblicati0n. Ericsson H. and Svartz-Malmberg G., (1959), Antibiotica et Chemiotherapia, 6, 41. Hansch C., Muir R.M., Fujita T., Maloney P.P., Geiger F. and Streich M., (1963), J. Am. Chem. Soc., 85, 2817. Hansch C. and FUjita T., (1964 a), J. Am. Chem. Soc., 86, 1616. Hansch C. and Steward A.E., (1964 b), J. Med. Chem., 7, 691. Hansch C. and Deutsch E.W., (1965), J. Med. Chem., 8, 705. Ito Y., Namba M., Nagahama N., Yamaguchi T. and Okuda T., (1964), J. Antibiotics - (Tokyo) Ser. A, 17, 218. Lien E.J., Hansch C. and Anderson S.M. (1968), J. Med. Chem., 11, 430. Penniston J.T., Beckett L., Bentley D.L. and Hansch C., (1969), Mol. Pharmacol., 5, 333. Schlesinger F., (1954), Acts med. scand., 148 , 357.