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Correlations between structure of nitrostyrene derivative fungicides and their reactivity toward low molecular weight thiols
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
39,
1-7 (191)
Correlations between Structure of Nitrostyrene Derivative Fungicides and Their Reactivity toward Low Molecular Weight Thiols G. GULLNER,**' T. Csmu-rArr,t
AND GY. MIKITES
*Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest POB 102, Hungary; Research Institute for Chemistry, H-1525 Budapest POB 17, Hungary; and AEGIS Pharmaceutical H-1475 Budapest POB 100, Hungary
Kentral Works,
Received April 3, 1990; accepted August 27, 1990 Reactivity of 27 fungitoxic R-nitrostyrene derivatives against L-cysteine, glutathione, and 2mercaptoacetic acid was studied for modeling their reactions with essential suhhydryl groups in fungi. A significant linear correlation was found between the reactivity of ring-substituted derivatives and the Hammett u constant of the substituents in the case of each thiol. Principal component analysis and stepwise regression analysis were used to reveal other possible correlations. The reactivity order of nitrostyrene derivatives against the three thiols was very similar. The reactivity was increased the most by nitro and cyan0 substitutions (except substitutions in the ortho position), the presence of halogen atoms influenced to a lesser extent the reactivity of each parent compound. Ortho substitutions generally lessened the reaction rate. Methyl substitution on the a carbon atom of the styryl double bond strongly decreased the reaction rate. The molecular lipophilicity of these nitrostyrene derivatives did not significantly influence the reaction rates. 0 1991 Academic Press, Inc.
product of fenitropan is the unsaturated compound 2-nitro- 1-phenylpropen-3-yl acetate (NPPA) (Fig. 1, II), which also shows antifungal activity (15). The fungitoxicity of NPPA strongly decreased in the presence of glutathione (GSH) (17). NPPA reacts readily with GSH and with other thiols at room temperature. The reaction rate of the first reaction step (a bimolecular nucleophilic addition) is determined by thiol basicity and pH. The supposed product is a thioether conjugate (Fig. 1, III) (14). The chemical interaction of NPPA with thiols was also detected by charge-transfer chromatography (18). The structurally related P-nitrostyrene also reacts with thiols. In this case some thioether products were actually synthesized (12). In the present study the reactivities of l3-nitrostyrene and its 26 derivatives with antifungal activity (among them NPPA and its 21 derivatives) toward L-cysteine, glutathione, and 2-mercaptoacetic acid were investigated as a model for their reaction with essential sulfhydryl groups present in fungi. Preliminary results of this structure-
INTRODUCTION
The biological effect of many xenobiotic compounds appears to be mediated by their binding to cellular nucleophilic groups such as amines and thiols in a Michael-type addition reaction (1,2). The nucleophilicity of the thiolate anion greatly exceeds that of the amino and hydroxyl groups (3). The biological activity of various a&unsaturated nitro compounds has been recognized as due to their reactivity toward the sulfhydryl groups of biomolecules (4-g). Several papers have reported the reaction of these compounds with low molecular weight thi01s (!L14). Fenitropan ((lRS,2RS)-Znitro-l-phenyltrimethylene diacetate) (Fig. 1, I) and its derivatives are effective antifungal agents (15). The antifungal activity of these compounds is determined mainly by the presence (or possible formation) of a double bond in the alkyl chain, suggesting that they exert their antifungal activity via the unsaturated form (16). The first decomposition ’ To whom correspondence should be addressed. 1
004%3575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. AU tights of reproduction in any form reserved.
2
GULLNER,
NO, CH-CH-CH, d A I I
c=o I CH,
NO, CH= C-CH,-0
c-o , tH,
I
CSERHATI,
-C -CH, 6
NO?
I
CH -CH-CH,-O-C-CH,
AND
MIKITE
trostyrene derivatives were plotted versus time. The pseudo-first-order rate constants (kobs) were calculated by the integrated form of Eq. [I]. The second-order rate constants (k) could be evaluated by plotting the log kobs values against the logarithms of the thiol concentrations, according to the equation:
/I
log kobs = log k + log[thiol] III FIG. 1. Structures the suggested first rhiol reaction.
offenitropnn intermediate
(I), NPPA (II), and (III) in the NPPA-
reactivity correlation analysis have been already published (19). The lipophilicities of nitrostyrene derivatives were recently reported (20).
[31
The rate constants given in Table 1 are the mean values of three independent experiments. The standard deviations varied between 5 and 8% from the mean values. Mathematical
Calculations
The electronic effects of the various benzene ring substituents were studied for NPPA and its derivatives (compounds 6-27 MATERIALS AND METHODS in Table 1) by applying the Hammett equaThe nitrostyrene derivatives were pretion. The Hammett u constants were taken pared according to Ref. (15). Their chemifrom Ref. (21). cal structures are given in Table 1. All other To assess the similarities in the reactivity chemicals were reagent grade and used as of the 3 thiols toward the 27 nitrostyrene supplied. derivatives and to compare the reactivity of Kinetic Studies [According to Ref. (14)I the nitrostyrene derivatives simultaneously In all cases the solvent used was a 0.2 M with each thiol, principal component analysis (PCA) was applied (22). The 3 x 27 rate sodium acetate buffer (pH 4.00tethanol constants formed the data matrix (with 3 (8:2, v/v) mixture. The transformations of variables and 27 observations). The limit of the nitrostyrene derivatives upon addition the explained variance was set to 99%. The of L-cysteine, glutathione, or 2-mercaptotwo-dimensional plot of the principal comacetic acid were followed spectrophotometponent loadings and variables was also calrically at the maximum absorbance of the cuiated. The thiols showing similar reactivstarting nitro compound at 26°C. The initial ity form distinct groups on the plot of the concentration of each nitrostyrene derivaPC loadings and the nitrostyrene derivative was 9 x IO-’ M. Thiols were added in tives, exhibiting similar reactivity against large excesses with concentrations varying between 1.8 x IOK and 8.1 x 10m3 M. On thiols, form clusters on the map of PC varithe basis of previous results (14), the fol- ables . To elucidate the role of the various molowing equations were used for calculating lecular substructures and the lipophilicity the rate constants: of the nitrostyrene derivatives in their in-d[nitrostyrene]ldt = k,,,[nitrostyrene], teractions with thiols, a stepwise regression [II analysis was applied (23). The lipophilicity value [taken from Ref. (20)] and the 23 varwhere, because of the thiol excess, ious structural characteristics of these nik obs = k[thiol]. 121 trostyrene derivatives (the value of these The logarithms of the concentration of ni- variables can be 0 or 1, showing that a sub-
REACTIONS
OF
NITROSTYRENES TABLE
The A,,
WITH
THIOLS
3
1
Values of the Nitrostyrene Derivatives and the Rate Constants for Their Reactions with L-Cysteine, Glutathione, and 2-Mercaptoacetic Acid
CR,, R,,R,,R4,R,,andR,= H unless otherwise indicated) k (M-’ Compound number
A,,,
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
320 326 340 345 322 310 305 326 302 317 321 322 315 303 330 325 330 358 320 356 373 320
23 24 25
325 362 350
26
320
27
318
1
Substituents
R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R,
L-Cysteine
R 1-6 --H R, = Cl R, = CH, = CH, R,, R,, R, = OCH, = CH,-O-CO-C,H, = CH,-O-CO-CH, = CH,-O-CO-CH, R, = F = CH,-O-CO-CH, R, = Cl = CH,-O-CO-CH, R, = Cl = CH,-O-CO-CH, R4 = CI = CH,-O-CO-CH, R4 = Br = CH,-0-CO-CH, R, = NO, = CH,-0-CO-CH, R, = NO, = CH,-O-CO-CH, R4 = NO, = CH,-O-CO-CH, R, = CH, = CH#-CO-CH, R, = CH, = CH,-O-COCH, R, = CH, = CH,-O-CO-CH, R, = OCH, = CH,-CWO-CH, R, = OCH, = CH#-CO-CH, R, = OCH, = CH,-O-CO-CH, R,, R, = OCH, = CH,-O-CO-CH, R, = OCH, R, = O-CO-CH, = CH,-O-CO-CH, R, = OC,H, = CH,-O-CO-CH, R4 = OC,H, = CH,-O-CO-CH, = O-CH&H, = CHIC-CO-CH, R, = NO, R, = CH, R, = CH,-0-CO-CH, R, = CN
stituent is absent or present in the molecule, respectively) were the independent variables. As the impact of lipophilicity may deviate from linearity, the logarithmic and quadratic forms of the lipophilicity values were also included as independent variables. The calculations were carried out three times: the reaction rates of each thiol were taken as dependent variables. The number of accepted variables was not limited, the partial F value was set to F = 2.
mini’)
Glutathione
2-Mercaptoacetic acid
45 163 9 6 107 115 156 78 222 164 208 42 335 411 65 108 88 57 129 78 105
39 93 9 5 64 74 101 52 126 97 108 36 190 259 26 58 47 27 64 47 49
70 266 12 11 229 186 300 170 320 288 303 84 674 787 105 175 152 108 222 141 177
173 128 71
85 66 31
294 207 133
72
42
140
284 432
152 232
618 664
RESULTS
AND
DISCUSSION
AH P-nitrostyrene derivatives reacted readily with the three thiols at 26°C. The second-order rate constants together with the X,, values of the nitrostyrene derivatives are compiled in Table I. A significant linear relationship was found between the Hammett u constants for the ring substituent(s) of compounds 627 and the logarithm of the rate constants
4
GULLNER,
A 01 .-
CSERHATI,
.2
01
-0.1 .j
-0.11
1
FIG. 2. Two-dimensional map of PC loadings. 1, 2mercaptoacetic acid; 2, cysteine; 3, glutathione.
for these compounds
with the three thiols:
log k = 2.067 + 0.669 CT r = 0.961 (L-cysteine) log k = 1.794 + 0.715 u r = 0.954 (glutathione) log k = 2.299 + 0.695 u r = 0.%3 (Zmercaptoacetic acid) where r is the correlation coefficient. The positive slopes of these equations refer to nucleophilic addition reactions. The highest rates were observed for nitro and
AND
MIKITE
cyano ring substituents (with each thiol). These electron withdrawing groups facilitate nucleophilic attack of the thiolate anion onto the carbon atom adjacent to the benzene ring. In the PCA the first principal component explained the overwhelming majority of variance (eigenvalue 2.%, sum of variance explained 98.72%). This result indicates that the reactivity order of the thiols is highly similar, that is, no specific interaction was observed between the nitrostyrene derivatives and the thiols. The map of PC loadings is shown in Fig. 2. Each thiol differs considerably from the others. As the high eigenvalue of the first principal component showed that their reactivity order is similar, the difference in the map may be due to the differences in the absolute value of the reaction rates. The map of PC variables (Fig. 3) shows that compounds with NO, or CN substituents (except o-NO,) form a distinct group (cluster I). These substitutions considerably increase the reactivity. The presence of halogen substituents (these compounds form cluster II) influences the reaction rates to a lesser extent. Cluster II also indicates that the position (R2, R,, or R4) or the type (F, Cl, or Br) of halogen substitution is of secondary impor-
.. 2,s I
c _--f 27 .
---.,;\, f-
I ;16 -!-
FIG. 3. Two-dimensional Table 1.
,’
/’
/
/ 1.
.I 3, ’ c
/’
map of PC variables. Numbers indicate the nitrostyrene derivatives in
REACTIONS
OF NITROSTYRENES
WITH
5
THIOLS
TABLE 2 Effect of Various Substituents on the Reactivity (R) of Nitrostyrene Derivatives toward L-Cysteine: Results of Stepwise Regression Analysis (n = 27) F = 30.2 Independent variable
R = a + b,x, + . . . + b,g6 a = 102.3 Position
? = 0.8709 b
XI
Rl
x2
R3
-9
R4
Cl Br
x4
R3
NO2
-94.8 119.7 105.7 207.2 308.7 329.7
CH,
x5
NO2
x6
CN
tance. The compounds with electron donating ring substituents form the tightest group (cluster III) with the greatest number of data points. These substituents lessen the reactivity of the parent compound NPPA (compound 6), which is situated between clusters II and III. The ortho-Cl-substituted compound 8 can also be found in cluster III. Substitutions in the ortho position (R2) generally decreased the reaction rate (except compound 2). Three or&o-substituted compounds and also l3-nitrostyrene are situated in the proximity of cluster III. Methyl substitution in the R, position resulted in the lowest reactivities observed (cluster IV). The results of stepwise regression analysis (Tables 2-4) correspond to those of PCA
b’%
sd
29.7 40.9 40.9 29.7 40.9 40.9
10.25 9.33 8.24 22.40 24.07 25.71
and support the conclusions drawn from it. The lipophilicity of the nitrostyrene derivatives does not significantly influence their reaction rates, that is their reactivities did not depend on the molecular lipophilicity. The fitness of equations selected by the stepwise regression analysis was in each case over the 99.9% significance level (the calculated F values are higher than thqcorresponding tabulated ones). The substituents selected (4-g out of the 25 independent variables) explained about 87-90% of the total variance (see 4 values). The methyl substitution at the R, position significantly decreased the reaction rate (see b values). Halogen substitution increased the reaction rate. In the case of glutathione (Table 3) this effect increased with increasing atomic ra-
TABLE 3 Effect of Various Substituents on the Reactivity (R) of Nitrostyrene Derivatives toward Glutathione: of Stepwise Regression Analysis (n = 27) R = a + b,x, + . . . + bGs a = 52.9
F = 32.4 Independent variable
Position
Substituent
Xl
Rl
CH,
x2
4
x3
R3
F Cl Cl Br
x4
R4
X5
%
x6
R3
X7 -%
NO2 NO2
CN
Results
rJ = 0.9065 b -45.9 48.1 73.1 44.1 55.1 118.1 206.1 179.1
sd
14.5 20.0 20.0 20.0 20.0 14.5 20.0 20.0
b’% 7.65 5.77 8.77 5.29 6.61 19.66 24.74 21.50
6
GULLNER,
CSERHATI,
TABLE Effect
of Various
Substituents Acid:
Independent variable
MIKITE
4
on the Reactivity (R) of Nitrostyrene Derivatives toward Results of Stepwise Regression Analysis (n = 27) R = a f b,x, a = 193.8
F = 42.9
AND
+
2-Mercaptoacetic
+ b,x,
? = 0.8864
Position
Substituent
b
sd
X1
R,
CH,
x2
R3
NO,
x3
R4
NO,
X4
R4
- 182.3 452.2 593.2 470.2
55.1 55.1 76.3 16.3
CN
dii: Br > Cl > F at the R, and R4 positions. The effect of a Cl substitution is higher at R, than at the R, position. The nitro and cyano groups also enhance the reactivities of the parent molecules. However, opposite to the Cl substitution, the effect of a nitro substitution is less at R, than at the R, position. These findings can be explained by the fact that the nitro group and the Cl atom cause opposite tautomer effects (electron withdrawing and electron donating effects, respectively) in the benzene ring. The path coefficients (b’% values) show that the impact of methyl substitution is similar to those for the halogens. The nitro and cyano groups have a significantly higher influence on the reaction rate. Further studies are necessary to reveal the possible correlations between the reactivities of nitrostyrene derivatives with thiol compounds and their actual antifungal activities. However, many other factors can also influence the fungicidal activity. Furthermore, the inactivation of these nitrostyrene derivatives may be connected with their blocking by nonessential thiol compounds in fungi. REFERENCES
1. A. Dal Pozzo, M. Acquasaliente, G. Donzelli, P. DeMaria, and M. C. Nicoli, 3-Carbonylacrylic derivatives as potential antimicrobial agents. Correlations between activity and reactivity towards cysteine, J. Med. Chem. 30, 1674 (1987). 2. W. H. Schaefer, T. M. Harris, and F. P. Guengerich, Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide, a Michael acceptor with extended con-
3.
4. 5.
6.
7.
b’%
13.02 32.26 30.52 24.19
jugation, Arch. Biochem. Biophys. 257, 186 (1987). M. Friedman, J. F. Cavins, and J. S. Wall, Relative nucleophilic reactivities of amino groups and mercaptide ions in addition reactions with a$-unsaturated compounds, J. Amer. Chem. Sot. 87, 3672 (1965). T. Zsolnai, Die Untersuchung der insekticiden Wirkung von Thiol-Reagentien, Acta Phytopathol. Acad. Sci. Hung. 9, 143 (1974). R. T. Louis-Ferdinand and G. C. Fuller, The mechanism of the biologic activity of a series of nitroolefins, Toxicol. Appl. Pharmacol. 16, 668 (1970). W. Billard and E. Peets, Sulfhydryl reactivity: Mechanism of action of several antiviral compounds-Selenocystine, 4-(2-propinyloxy)-Bnitrostyrene and acetylaranotin, Antimicrob. Agents Chemother. 5, 19 (1974). E. Sturdik, L. Drobnica, and S. Bat&z, Interactions of 2-furylethylenes with thiol enzymes, Collect.
Czech.
Chem.
Commun.
48,
327
(1983). 8. E. Sturdik, L. Drobnica, and S. BalBz, Reactions of 2-furylethylenes with thiols in vivo, Collect. Czech. Chem. Commun. 48, 336 (1983). 9. L. F. Cason and C. C. Wanser, The preparation of some aryl aminoalkyl sulfides and sulfones, J. Amer. Chem. Sot. 73, 142 (1951). 10. W. C. McCarthy and B.-T. Ho, 2-Mercapto2-phenylethylamine, J. Org. Chem. 26, 4110 (1961). 11. C. E. Lough, D. J. Currie, and H. L. Holmes, Rates of reaction of n-butanethiol with some conjugated heteroenoid compounds, Canad. J. Chem. 46, 771 (1968). 12. W. Winter, G. Heusel, H. Fouad, and G. Jung, Rontgen-Strukturanalyse, absolute Konfiguration und Circulardichroismus von B-Nitrostyrol-Addukten des Cysteins, Chem. Ber. 112, 3171 (1979). 13. S. BalBz, E. Sturdik, and L. Drobnica, Biochemically important reactions of 2-furylethylenes. Characterization of the reactivity towards thi-
REACTIONS
14.
IS.
16.
17.
18.
OF NITROSTYRENES
ols, Collect. Czech. Chem. Commun. 41, 1659 (1982). G. Gullner, Gy. Josepovits, and Gy. Mikite, Kinetics of the reaction of the fust metabolite of fenitropan with some low molecular weight thiols, Pestic. Sci. 26, 253 (1989). Gy. Mikite, E. Jakucs, A. Kis-Tamas, F. Darvas, and A. Lopata, Synthesis and antifungal activity of new nitro-alcohol derivatives, Pestic. Sci. 13, 557 (1982). A. Lopata, F. Darvas, K. Valk6, Gy. Mikite, E. Jakucs, and A. Kis-Tam&, Structure-activity relationships in a series of new antifungal nitroalcohol derivatives, Pestic. Sci. 14, 513 (1983). Gy. Josepovits, M. Gasztonyi, A. Kis-Tamas, Gy. Mikite, and G. Gullner, Chemical and microbiological transformations of the fungicide fenitropan, Tag.-Ber. Acad. Landwirtsch.-Wiss. DDR, Berlin 222, 225 (1984). G. Gullner, T. Cserhati, A. Kis-Tamas, and Gy. Mikite, Interaction of amino acids and glutathione with the fungicide I-phenyl-2-nitro-3-
19.
20.
21.
22. 23.
WITH THIOLS
7
acetoxyprop-1-ene studied by charge-transfer chromatography, J. Chromatogr. 355, 211 (1986). A. Lopata, F. Darvas, Gy. Mikite, A. Kis-Tamas, G. Gullner, and Gy. Josepovits, On the mechanism of antifungal action of nitroalcohol derivatives, in “QSAR and Strategies in the Design of Bioactive Compounds” (J. K. Seydel, Ed.), p. 424, VCH Verlagsgesellschaft, Weinheim, 1985. T. Cserhati, A. Kis-Tamas, and Gy. Mikite, Lipophilicity determination of some nitrostyrene derivatives on RP-2, RE-8 and RP-18 layers. Chromatographia 25, 82 (1988). C. Hansch, A. Leo, S. H. Unger, K. H. Kim, D. Nikaitani, and E. J. Lien, Aromatic substituent constants for structure-activity correlations, .I. Med. Chem. 16, 1207 (1973). K. V. Mardia, J. T. Kent, and J. M. Bibby, “Multivariate Analysis,” Academic Press, London, 1979. H. Mayer, “Modeme Regressionsanalyse,” Salle, Sauerlander, Frankfurt am Main, 1982.
BIOCHEMISTRY
AND
PHYSIOLOGY
39,
1-7 (191)
Correlations between Structure of Nitrostyrene Derivative Fungicides and Their Reactivity toward Low Molecular Weight Thiols G. GULLNER,**' T. Csmu-rArr,t
AND GY. MIKITES
*Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest POB 102, Hungary; Research Institute for Chemistry, H-1525 Budapest POB 17, Hungary; and AEGIS Pharmaceutical H-1475 Budapest POB 100, Hungary
Kentral Works,
Received April 3, 1990; accepted August 27, 1990 Reactivity of 27 fungitoxic R-nitrostyrene derivatives against L-cysteine, glutathione, and 2mercaptoacetic acid was studied for modeling their reactions with essential suhhydryl groups in fungi. A significant linear correlation was found between the reactivity of ring-substituted derivatives and the Hammett u constant of the substituents in the case of each thiol. Principal component analysis and stepwise regression analysis were used to reveal other possible correlations. The reactivity order of nitrostyrene derivatives against the three thiols was very similar. The reactivity was increased the most by nitro and cyan0 substitutions (except substitutions in the ortho position), the presence of halogen atoms influenced to a lesser extent the reactivity of each parent compound. Ortho substitutions generally lessened the reaction rate. Methyl substitution on the a carbon atom of the styryl double bond strongly decreased the reaction rate. The molecular lipophilicity of these nitrostyrene derivatives did not significantly influence the reaction rates. 0 1991 Academic Press, Inc.
product of fenitropan is the unsaturated compound 2-nitro- 1-phenylpropen-3-yl acetate (NPPA) (Fig. 1, II), which also shows antifungal activity (15). The fungitoxicity of NPPA strongly decreased in the presence of glutathione (GSH) (17). NPPA reacts readily with GSH and with other thiols at room temperature. The reaction rate of the first reaction step (a bimolecular nucleophilic addition) is determined by thiol basicity and pH. The supposed product is a thioether conjugate (Fig. 1, III) (14). The chemical interaction of NPPA with thiols was also detected by charge-transfer chromatography (18). The structurally related P-nitrostyrene also reacts with thiols. In this case some thioether products were actually synthesized (12). In the present study the reactivities of l3-nitrostyrene and its 26 derivatives with antifungal activity (among them NPPA and its 21 derivatives) toward L-cysteine, glutathione, and 2-mercaptoacetic acid were investigated as a model for their reaction with essential sulfhydryl groups present in fungi. Preliminary results of this structure-
INTRODUCTION
The biological effect of many xenobiotic compounds appears to be mediated by their binding to cellular nucleophilic groups such as amines and thiols in a Michael-type addition reaction (1,2). The nucleophilicity of the thiolate anion greatly exceeds that of the amino and hydroxyl groups (3). The biological activity of various a&unsaturated nitro compounds has been recognized as due to their reactivity toward the sulfhydryl groups of biomolecules (4-g). Several papers have reported the reaction of these compounds with low molecular weight thi01s (!L14). Fenitropan ((lRS,2RS)-Znitro-l-phenyltrimethylene diacetate) (Fig. 1, I) and its derivatives are effective antifungal agents (15). The antifungal activity of these compounds is determined mainly by the presence (or possible formation) of a double bond in the alkyl chain, suggesting that they exert their antifungal activity via the unsaturated form (16). The first decomposition ’ To whom correspondence should be addressed. 1
004%3575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. AU tights of reproduction in any form reserved.
2
GULLNER,
NO, CH-CH-CH, d A I I
c=o I CH,
NO, CH= C-CH,-0
c-o , tH,
I
CSERHATI,
-C -CH, 6
NO?
I
CH -CH-CH,-O-C-CH,
AND
MIKITE
trostyrene derivatives were plotted versus time. The pseudo-first-order rate constants (kobs) were calculated by the integrated form of Eq. [I]. The second-order rate constants (k) could be evaluated by plotting the log kobs values against the logarithms of the thiol concentrations, according to the equation:
/I
log kobs = log k + log[thiol] III FIG. 1. Structures the suggested first rhiol reaction.
offenitropnn intermediate
(I), NPPA (II), and (III) in the NPPA-
reactivity correlation analysis have been already published (19). The lipophilicities of nitrostyrene derivatives were recently reported (20).
[31
The rate constants given in Table 1 are the mean values of three independent experiments. The standard deviations varied between 5 and 8% from the mean values. Mathematical
Calculations
The electronic effects of the various benzene ring substituents were studied for NPPA and its derivatives (compounds 6-27 MATERIALS AND METHODS in Table 1) by applying the Hammett equaThe nitrostyrene derivatives were pretion. The Hammett u constants were taken pared according to Ref. (15). Their chemifrom Ref. (21). cal structures are given in Table 1. All other To assess the similarities in the reactivity chemicals were reagent grade and used as of the 3 thiols toward the 27 nitrostyrene supplied. derivatives and to compare the reactivity of Kinetic Studies [According to Ref. (14)I the nitrostyrene derivatives simultaneously In all cases the solvent used was a 0.2 M with each thiol, principal component analysis (PCA) was applied (22). The 3 x 27 rate sodium acetate buffer (pH 4.00tethanol constants formed the data matrix (with 3 (8:2, v/v) mixture. The transformations of variables and 27 observations). The limit of the nitrostyrene derivatives upon addition the explained variance was set to 99%. The of L-cysteine, glutathione, or 2-mercaptotwo-dimensional plot of the principal comacetic acid were followed spectrophotometponent loadings and variables was also calrically at the maximum absorbance of the cuiated. The thiols showing similar reactivstarting nitro compound at 26°C. The initial ity form distinct groups on the plot of the concentration of each nitrostyrene derivaPC loadings and the nitrostyrene derivative was 9 x IO-’ M. Thiols were added in tives, exhibiting similar reactivity against large excesses with concentrations varying between 1.8 x IOK and 8.1 x 10m3 M. On thiols, form clusters on the map of PC varithe basis of previous results (14), the fol- ables . To elucidate the role of the various molowing equations were used for calculating lecular substructures and the lipophilicity the rate constants: of the nitrostyrene derivatives in their in-d[nitrostyrene]ldt = k,,,[nitrostyrene], teractions with thiols, a stepwise regression [II analysis was applied (23). The lipophilicity value [taken from Ref. (20)] and the 23 varwhere, because of the thiol excess, ious structural characteristics of these nik obs = k[thiol]. 121 trostyrene derivatives (the value of these The logarithms of the concentration of ni- variables can be 0 or 1, showing that a sub-
REACTIONS
OF
NITROSTYRENES TABLE
The A,,
WITH
THIOLS
3
1
Values of the Nitrostyrene Derivatives and the Rate Constants for Their Reactions with L-Cysteine, Glutathione, and 2-Mercaptoacetic Acid
CR,, R,,R,,R4,R,,andR,= H unless otherwise indicated) k (M-’ Compound number
A,,,
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
320 326 340 345 322 310 305 326 302 317 321 322 315 303 330 325 330 358 320 356 373 320
23 24 25
325 362 350
26
320
27
318
1
Substituents
R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R,
L-Cysteine
R 1-6 --H R, = Cl R, = CH, = CH, R,, R,, R, = OCH, = CH,-O-CO-C,H, = CH,-O-CO-CH, = CH,-O-CO-CH, R, = F = CH,-O-CO-CH, R, = Cl = CH,-O-CO-CH, R, = Cl = CH,-O-CO-CH, R4 = CI = CH,-O-CO-CH, R4 = Br = CH,-0-CO-CH, R, = NO, = CH,-0-CO-CH, R, = NO, = CH,-O-CO-CH, R4 = NO, = CH,-O-CO-CH, R, = CH, = CH#-CO-CH, R, = CH, = CH,-O-COCH, R, = CH, = CH,-O-CO-CH, R, = OCH, = CH,-CWO-CH, R, = OCH, = CH#-CO-CH, R, = OCH, = CH,-O-CO-CH, R,, R, = OCH, = CH,-O-CO-CH, R, = OCH, R, = O-CO-CH, = CH,-O-CO-CH, R, = OC,H, = CH,-O-CO-CH, R4 = OC,H, = CH,-O-CO-CH, = O-CH&H, = CHIC-CO-CH, R, = NO, R, = CH, R, = CH,-0-CO-CH, R, = CN
stituent is absent or present in the molecule, respectively) were the independent variables. As the impact of lipophilicity may deviate from linearity, the logarithmic and quadratic forms of the lipophilicity values were also included as independent variables. The calculations were carried out three times: the reaction rates of each thiol were taken as dependent variables. The number of accepted variables was not limited, the partial F value was set to F = 2.
mini’)
Glutathione
2-Mercaptoacetic acid
45 163 9 6 107 115 156 78 222 164 208 42 335 411 65 108 88 57 129 78 105
39 93 9 5 64 74 101 52 126 97 108 36 190 259 26 58 47 27 64 47 49
70 266 12 11 229 186 300 170 320 288 303 84 674 787 105 175 152 108 222 141 177
173 128 71
85 66 31
294 207 133
72
42
140
284 432
152 232
618 664
RESULTS
AND
DISCUSSION
AH P-nitrostyrene derivatives reacted readily with the three thiols at 26°C. The second-order rate constants together with the X,, values of the nitrostyrene derivatives are compiled in Table I. A significant linear relationship was found between the Hammett u constants for the ring substituent(s) of compounds 627 and the logarithm of the rate constants
4
GULLNER,
A 01 .-
CSERHATI,
.2
01
-0.1 .j
-0.11
1
FIG. 2. Two-dimensional map of PC loadings. 1, 2mercaptoacetic acid; 2, cysteine; 3, glutathione.
for these compounds
with the three thiols:
log k = 2.067 + 0.669 CT r = 0.961 (L-cysteine) log k = 1.794 + 0.715 u r = 0.954 (glutathione) log k = 2.299 + 0.695 u r = 0.%3 (Zmercaptoacetic acid) where r is the correlation coefficient. The positive slopes of these equations refer to nucleophilic addition reactions. The highest rates were observed for nitro and
AND
MIKITE
cyano ring substituents (with each thiol). These electron withdrawing groups facilitate nucleophilic attack of the thiolate anion onto the carbon atom adjacent to the benzene ring. In the PCA the first principal component explained the overwhelming majority of variance (eigenvalue 2.%, sum of variance explained 98.72%). This result indicates that the reactivity order of the thiols is highly similar, that is, no specific interaction was observed between the nitrostyrene derivatives and the thiols. The map of PC loadings is shown in Fig. 2. Each thiol differs considerably from the others. As the high eigenvalue of the first principal component showed that their reactivity order is similar, the difference in the map may be due to the differences in the absolute value of the reaction rates. The map of PC variables (Fig. 3) shows that compounds with NO, or CN substituents (except o-NO,) form a distinct group (cluster I). These substitutions considerably increase the reactivity. The presence of halogen substituents (these compounds form cluster II) influences the reaction rates to a lesser extent. Cluster II also indicates that the position (R2, R,, or R4) or the type (F, Cl, or Br) of halogen substitution is of secondary impor-
.. 2,s I
c _--f 27 .
---.,;\, f-
I ;16 -!-
FIG. 3. Two-dimensional Table 1.
,’
/’
/
/ 1.
.I 3, ’ c
/’
map of PC variables. Numbers indicate the nitrostyrene derivatives in
REACTIONS
OF NITROSTYRENES
WITH
5
THIOLS
TABLE 2 Effect of Various Substituents on the Reactivity (R) of Nitrostyrene Derivatives toward L-Cysteine: Results of Stepwise Regression Analysis (n = 27) F = 30.2 Independent variable
R = a + b,x, + . . . + b,g6 a = 102.3 Position
? = 0.8709 b
XI
Rl
x2
R3
-9
R4
Cl Br
x4
R3
NO2
-94.8 119.7 105.7 207.2 308.7 329.7
CH,
x5
NO2
x6
CN
tance. The compounds with electron donating ring substituents form the tightest group (cluster III) with the greatest number of data points. These substituents lessen the reactivity of the parent compound NPPA (compound 6), which is situated between clusters II and III. The ortho-Cl-substituted compound 8 can also be found in cluster III. Substitutions in the ortho position (R2) generally decreased the reaction rate (except compound 2). Three or&o-substituted compounds and also l3-nitrostyrene are situated in the proximity of cluster III. Methyl substitution in the R, position resulted in the lowest reactivities observed (cluster IV). The results of stepwise regression analysis (Tables 2-4) correspond to those of PCA
b’%
sd
29.7 40.9 40.9 29.7 40.9 40.9
10.25 9.33 8.24 22.40 24.07 25.71
and support the conclusions drawn from it. The lipophilicity of the nitrostyrene derivatives does not significantly influence their reaction rates, that is their reactivities did not depend on the molecular lipophilicity. The fitness of equations selected by the stepwise regression analysis was in each case over the 99.9% significance level (the calculated F values are higher than thqcorresponding tabulated ones). The substituents selected (4-g out of the 25 independent variables) explained about 87-90% of the total variance (see 4 values). The methyl substitution at the R, position significantly decreased the reaction rate (see b values). Halogen substitution increased the reaction rate. In the case of glutathione (Table 3) this effect increased with increasing atomic ra-
TABLE 3 Effect of Various Substituents on the Reactivity (R) of Nitrostyrene Derivatives toward Glutathione: of Stepwise Regression Analysis (n = 27) R = a + b,x, + . . . + bGs a = 52.9
F = 32.4 Independent variable
Position
Substituent
Xl
Rl
CH,
x2
4
x3
R3
F Cl Cl Br
x4
R4
X5
%
x6
R3
X7 -%
NO2 NO2
CN
Results
rJ = 0.9065 b -45.9 48.1 73.1 44.1 55.1 118.1 206.1 179.1
sd
14.5 20.0 20.0 20.0 20.0 14.5 20.0 20.0
b’% 7.65 5.77 8.77 5.29 6.61 19.66 24.74 21.50
6
GULLNER,
CSERHATI,
TABLE Effect
of Various
Substituents Acid:
Independent variable
MIKITE
4
on the Reactivity (R) of Nitrostyrene Derivatives toward Results of Stepwise Regression Analysis (n = 27) R = a f b,x, a = 193.8
F = 42.9
AND
+
2-Mercaptoacetic
+ b,x,
? = 0.8864
Position
Substituent
b
sd
X1
R,
CH,
x2
R3
NO,
x3
R4
NO,
X4
R4
- 182.3 452.2 593.2 470.2
55.1 55.1 76.3 16.3
CN
dii: Br > Cl > F at the R, and R4 positions. The effect of a Cl substitution is higher at R, than at the R, position. The nitro and cyano groups also enhance the reactivities of the parent molecules. However, opposite to the Cl substitution, the effect of a nitro substitution is less at R, than at the R, position. These findings can be explained by the fact that the nitro group and the Cl atom cause opposite tautomer effects (electron withdrawing and electron donating effects, respectively) in the benzene ring. The path coefficients (b’% values) show that the impact of methyl substitution is similar to those for the halogens. The nitro and cyano groups have a significantly higher influence on the reaction rate. Further studies are necessary to reveal the possible correlations between the reactivities of nitrostyrene derivatives with thiol compounds and their actual antifungal activities. However, many other factors can also influence the fungicidal activity. Furthermore, the inactivation of these nitrostyrene derivatives may be connected with their blocking by nonessential thiol compounds in fungi. REFERENCES
1. A. Dal Pozzo, M. Acquasaliente, G. Donzelli, P. DeMaria, and M. C. Nicoli, 3-Carbonylacrylic derivatives as potential antimicrobial agents. Correlations between activity and reactivity towards cysteine, J. Med. Chem. 30, 1674 (1987). 2. W. H. Schaefer, T. M. Harris, and F. P. Guengerich, Reaction of the model thiol 2-mercaptoethanol and glutathione with methylvinylmaleimide, a Michael acceptor with extended con-
3.
4. 5.
6.
7.
b’%
13.02 32.26 30.52 24.19
jugation, Arch. Biochem. Biophys. 257, 186 (1987). M. Friedman, J. F. Cavins, and J. S. Wall, Relative nucleophilic reactivities of amino groups and mercaptide ions in addition reactions with a$-unsaturated compounds, J. Amer. Chem. Sot. 87, 3672 (1965). T. Zsolnai, Die Untersuchung der insekticiden Wirkung von Thiol-Reagentien, Acta Phytopathol. Acad. Sci. Hung. 9, 143 (1974). R. T. Louis-Ferdinand and G. C. Fuller, The mechanism of the biologic activity of a series of nitroolefins, Toxicol. Appl. Pharmacol. 16, 668 (1970). W. Billard and E. Peets, Sulfhydryl reactivity: Mechanism of action of several antiviral compounds-Selenocystine, 4-(2-propinyloxy)-Bnitrostyrene and acetylaranotin, Antimicrob. Agents Chemother. 5, 19 (1974). E. Sturdik, L. Drobnica, and S. Bat&z, Interactions of 2-furylethylenes with thiol enzymes, Collect.
Czech.
Chem.
Commun.
48,
327
(1983). 8. E. Sturdik, L. Drobnica, and S. BalBz, Reactions of 2-furylethylenes with thiols in vivo, Collect. Czech. Chem. Commun. 48, 336 (1983). 9. L. F. Cason and C. C. Wanser, The preparation of some aryl aminoalkyl sulfides and sulfones, J. Amer. Chem. Sot. 73, 142 (1951). 10. W. C. McCarthy and B.-T. Ho, 2-Mercapto2-phenylethylamine, J. Org. Chem. 26, 4110 (1961). 11. C. E. Lough, D. J. Currie, and H. L. Holmes, Rates of reaction of n-butanethiol with some conjugated heteroenoid compounds, Canad. J. Chem. 46, 771 (1968). 12. W. Winter, G. Heusel, H. Fouad, and G. Jung, Rontgen-Strukturanalyse, absolute Konfiguration und Circulardichroismus von B-Nitrostyrol-Addukten des Cysteins, Chem. Ber. 112, 3171 (1979). 13. S. BalBz, E. Sturdik, and L. Drobnica, Biochemically important reactions of 2-furylethylenes. Characterization of the reactivity towards thi-
REACTIONS
14.
IS.
16.
17.
18.
OF NITROSTYRENES
ols, Collect. Czech. Chem. Commun. 41, 1659 (1982). G. Gullner, Gy. Josepovits, and Gy. Mikite, Kinetics of the reaction of the fust metabolite of fenitropan with some low molecular weight thiols, Pestic. Sci. 26, 253 (1989). Gy. Mikite, E. Jakucs, A. Kis-Tamas, F. Darvas, and A. Lopata, Synthesis and antifungal activity of new nitro-alcohol derivatives, Pestic. Sci. 13, 557 (1982). A. Lopata, F. Darvas, K. Valk6, Gy. Mikite, E. Jakucs, and A. Kis-Tam&, Structure-activity relationships in a series of new antifungal nitroalcohol derivatives, Pestic. Sci. 14, 513 (1983). Gy. Josepovits, M. Gasztonyi, A. Kis-Tamas, Gy. Mikite, and G. Gullner, Chemical and microbiological transformations of the fungicide fenitropan, Tag.-Ber. Acad. Landwirtsch.-Wiss. DDR, Berlin 222, 225 (1984). G. Gullner, T. Cserhati, A. Kis-Tamas, and Gy. Mikite, Interaction of amino acids and glutathione with the fungicide I-phenyl-2-nitro-3-
19.
20.
21.
22. 23.
WITH THIOLS
7
acetoxyprop-1-ene studied by charge-transfer chromatography, J. Chromatogr. 355, 211 (1986). A. Lopata, F. Darvas, Gy. Mikite, A. Kis-Tamas, G. Gullner, and Gy. Josepovits, On the mechanism of antifungal action of nitroalcohol derivatives, in “QSAR and Strategies in the Design of Bioactive Compounds” (J. K. Seydel, Ed.), p. 424, VCH Verlagsgesellschaft, Weinheim, 1985. T. Cserhati, A. Kis-Tamas, and Gy. Mikite, Lipophilicity determination of some nitrostyrene derivatives on RP-2, RE-8 and RP-18 layers. Chromatographia 25, 82 (1988). C. Hansch, A. Leo, S. H. Unger, K. H. Kim, D. Nikaitani, and E. J. Lien, Aromatic substituent constants for structure-activity correlations, .I. Med. Chem. 16, 1207 (1973). K. V. Mardia, J. T. Kent, and J. M. Bibby, “Multivariate Analysis,” Academic Press, London, 1979. H. Mayer, “Modeme Regressionsanalyse,” Salle, Sauerlander, Frankfurt am Main, 1982.