Relation between chemical structure and biological activity of anticholinesterases

Gen. Pharmac., 1978, I/ol. 9, pp. 49 to 53. Pergamon Press. Printed in Great Britain

RELATION BETWEEN CHEMICAL STRUCTURE AND BIOLOGICAL ACTIVITY OF ANTICHOLINESTERASES ANGEL M. RELIMPIO* Department of Biochemistry, Faculty of Sciences, University of Madrid, Madrid, Spain (Received 30 June 1977)

Abstract--1. A series of ethyl- mono- and disubstituted phenyl-methylphosphonates have been synthesized and their biological activities in vitro and in vivo studied. 2. The treatment p-cr-n of biological activity in vivo leads to the conclusion that the electronic effects of the para substituents play a preponderant role on the activity, whereas the role of the meta substituents is moreover conditioned by the steric factors. 3. The study of the effects that the solubility exerts on the activity has been shown to be too small to represent an appreciable factor. 4. The results obtained have been compared with their diethyl-phosphate analogues.

INTRODUCTION

have on in vivo activity was made, using the approach of rationalisation of biological response in terms of electronic and solubility factors as developed by Hansch et al. (1964) and Fujita (1964, 1966, 1967) and expressed in general terms as follows:

The toxicity of organophosphorous compounds depends to a large degree on their physicochemical properties, and their action on certain biochemical processes is mainly centered on inhibition of the cholinesterase enzyme system, although other enzymes may be affected. Many studies on these compounds were carried out with a view either to finding out their anticholinesterase activity in vitro, as in Fukuto's experiment (1956), or to the study the cholinesterase activities due to the acyl group of the different p-nitrophenol derivates (Fukuto & Metaff, 1959; Fukuto et al., 1959, 1961). In our previous papers several phosphorous compounds were studied with the object of relating the anticholinesterase activities in vitro and in vivo to structural factors such as electronic and steric effects of the several substituents (Corral, 1966; Gonzalez, 1967). In this paper several series of m-p-disubstituted phenyl-ethyl-methylphosphonates have been studied from the same point of view, observing the analogies and differences to the previously cited organophosphorous compounds. The series studied were phenyl, methyl-phenyl and ethyl-phenyl with all these substituents in the meta position. The idea of changing the meta substituents is to observe the change in activity due to the molecular complementarity in the anionic center of the enzyme. The change at the para position was made in such a way as to cover an ample range of a values, so that the transference capacity of the anionic group could be studied step by step. As the biolbgical activity of any compound in vivo depends not only on its capacity to inhibit the enzyme, but also on other factors such as the penetrability and the possibility of suffering any kind of metabolic process before acting on the enzyme, a study of the effects that meta and para substituents

log 1/c = kTr2 + k'n + p a + k" Where c is the molar concentration of a compound of a series which provokes an equal biological response, a is the Hammet constant of a given compound, and n is a parameter obtained by comparing the partition coefficient of a substituted compound to a non-substituted analogue (Fujita et al., 1964; Hansch et al,, 1975). For the present work the values of a and n which are used are the same as those used in our previous papers for the considerations taken into account therein (Gonzalez, 1967). A study of the in vitro activities will be carried out with the phenyl series and several compounds of the others to observe whether they behave in the same way as the phosphates and to compare their activities. MATERIALS AND METHODS The required compounds were synthetized by the method described by Relimpio (1977). Inhibition in vitro

Determination of Iso values for the inhibition of flybrain cholinesterase was carried out by the Warburg standard manometric technique (Aldridge et al., 1952; Metcalf, 1949). Houseflies were decapitated by the Morefield (1957) procedure. A homogenous mixture containing a sufficient number of fly heads per milliliter to give an enzymatic activity of 6ml CO2/min after centrifuging for 1 h, was used. The plso were obtained graphically, plotting negative logarithms of molar concentration against percentages of inhibition. Toxicity test in vivo

Determinations of toxicity by contact were carried out by the Metcalf method (1953). Musca domestica L. were fed a standard diet at 22°C and 60% relative humidity

* Present address: Departamento de Bioquimica, Facultad de Ciencias, Universidad de Sevilla, Sevilla, Spain. 49 G.P. 9 I

II

50

ANGEL M. RELIMPIO

for 3 or 4 days. The LDs0 determination were carried out by Probit analysis (Finney, 1959). From the data fed into an IBM ll30-M2B computer the corresponding equations were obtained. Structural parameters used in the regression analysis were obtained from the literature on the subject (Gonzalez et al., 1967).

Table 1. Biological activity in vitro of ethyl 3-4-disubstituted phenyl-methylphosphonates

~/0 --CH2--CH3

CH~Po \~ X R

RESULTS AND DISCUSSION In general the methylphosphonate studied have been found to be powerful inhibitors of acetylcholinesterase. Table 1 shows the pIs0 of the phenyl series. Almost all of them present an activity ranging from 10 -6 to 10 9 M. If the values are compared with those obtained for their diethylphosphate analogues as taken from the literature, it can be observed that methylphosphonates are 10-fold more powerful. Just as in the phosphate series the pIs0 increases with the electron acceptor capacity of the para substituent, there being a good relation between both values. This relation can be expressed by the equation pls0 = a ~ r + b . The degree of approximation in the series for this equation is superior to the phosphate series in accordance with the statistic data (Fig. 1). The 3.16 value indicates the sensitivity of the inhibiting reaction to electronic effects and this is greater in this series than in the phosphate one (2.96). When the in vitro biological activities are studied, it might be expected that the methylphosphonate series would be less active than the phosphate, since activity is related to the transferring power of the acyl group to stereactic point of the enzyme, which at the same time is related with a low electron density on the P atom. In accordance with this idea the substitution of one ethoxy group (electron withdrawing) by a methyl-group (electron donor) would diminish the electrophylic capacity of the P, producing a lesser activity. Nevertheless, the explanation of the increase of activity, must be attributed to the presence of resonance effects due to an oxygen atom which just like any other atom with unshared electron pairs, exerts a + M effect when it is united to an organic

X

R

pI~0

H --CH3 C1 Br CH30-C2H50-~CN CH3-S-CH3-SO2-CH3 SO2---COOCH 3 C1 ~N CI ~N

H H H H H H H H H H H CH a CH 3 C2H5 C2H5

4.24 4.16 5.43 5.58 3.88 3.97 7.46 5.89 7.07 8.02 7.22 6.6 7.6 7.0 7.8

Experimental error is +0.05 for the 4-substituted series and +0.20 for other compounds. group. The ethoxyde group can conjugate the unshared electron pair of the oxygen atom with the P ~ O bond (RO - P = O - R O = P - O - ) (Gould, 1967; Rivers, 1973). This effect is superior to the + I effect of the methyl group, which is very small, probably only due to a hyperconjugating process (AlvarezOssorio, 1960). Thus, the presence of two ethoxyde groups reduces the electrophylic power of the P atom and so the products show a less inhibitory effect. Although the presence of a steric effect should be taken into account, as is postulated by Fukuto & Metcalf (1959) and confirmed by Hansch (1970), its contribution is negligible. The effect of the substituents in meta position is very similar to that found in the study of phosphates for the pI~o increases, with an increase in the volume

.J j

-

o 6

SOz CH3

COOC~ .CN Jeso CH3

-

--S-CH3

B ~

o

-c.3 v : 0 . 0 6 4

I

02

I

0

I

02

I

04 0

I

06

J

08

f

hO

Fig. 1. Plot of pIso of the series ethyl-4-substituted phenyl-methylphosphonates vs a values of the substituents.

Biological activity of anticholinesterase of the substituent, and the difference between the m-ethyl substituted and methyl-substituted series is less than the corresponding one between m-methyl substituted and non-substituted series, as can be deduced from Table 1. The results show that the effects producing this difference must be basically the same in the phosphate and phosphonate series. The study of in vivo biological activity of these potential insecticides indicates a high biological response, as was to be expected from the study made in vitro. In Table 2 the LDso values are shown. Logically, the compounds which present a high inhibition of acetyl cholinesterase are those which have a higher in vivo biological response, but if the meta substituent is taken into account, an increment in the van der Waals radius of the substituent produces a disminution of the activity. The most likely explanation for this fact, can be found from the studies carried out by several workers. According to Gonzalez et al. (1966) an increment of the lipophylic character of the molecule is not accompanied by an increment in the activity, due to a variation in the conditions of its union to the enzyme. Another explanation was that, the meta isomers have different modes of inhibition to the para substituents. Hansch (1966, 1970) observed in his study with diethyl-phenyl-phosphates, that for meta substituents the steric factor were more important than the electronic ones and so this effect should be reflected in their modes of action upon the enzyme. Fukuto & Metcalf (1965) have shown evidence that the meta substituents can join at a place on the enzyme, and that this union Table 2. Biological activity in vivo of ethyl 3-4-disubstituted phenyl-methylphosphonates xa

Ra

LDso 7/g

H --CH3 C1 Br CH30

--NO 2 H Cl Br

H H H H H H H H H H H H ---CHa --CH3 ----CH3 ---CH3 ---CH3 ---CH3 --CH3 -42H 3 ---C2H5 --C2Hs "--C2H 5 ---C2H5

--CN

---C2 H 5

CH3-S-CHa-SO-CH3-SO2---NO2

-'-C2Hs -'--C2Hs --"C2H~ ---C2H5

586 1239 95 63 260 2458 1.83 1.44 1.62 1.63 1.08 1006 3210 1690 162 340 6.90 3.43 5.41 7.31 5.49 740 256 192 11 6.11 15.90 19.40 11.80

C2H50

--CN CHa-S-CH3-SO-CH3-SO2---NO2 H ~H

3

---OCH3 C1 Br ---CN CH3-S-CH3-SO-CH3-SO 2 -

° X and R mean the same as in Table 1.

51

is determined by the size of the substituent. If this explanation is correct, it should be expected that either the enzyme has a different conformation when in vivo and in vitro experiments are carried out, or that there is some physiological barrier of special characteristics which would be in accordance with the first hypothesis. The study of the effects of the substituents on biological activity can be related by the Hansch equation, and ideas may thus be obtained as to the contribution of the electronic and lipophylic effects to biological activity. LDs0 values were related to electronic and solubility factors, first independently and then all together. The application of this relation to the phenyl series give rise to the equations (1-3) pLDso = - 0 . 8 2 n + 1.18

n 11

V r 1.2431 0.2587 (1)

pLDs0 = 1.68a + 0.37

11 0.5996 0.6858 (2)

pLDs0 = - 0 . 5 n + 1.64a + 0.52

11 0.4775 0.7009. (3)

The inspection of the above set of equations reveals that solubility properties exert little influence on the biological activity as can be deduced from the variance (v) and correlation coefficient (r) of equation (1). If the electronic factors present a better correlation, it is much lower than that presented in the study of in vitro inhibition. The equation obtained using both kinds of factors does not present a more favorable correlation, since equation (2) can only explain 45~o of the variance. The difference found between the correlation of the regression analysis for LDso and plso can be explained if we remember that for the compound to exert a biological response it must suffer biological interactions which are completely different in the two cases, since it may undergo diverse transformations before reaching the enzyme target. If we represented the pLDso/a values by plotting a graph (Fig. 2), a high correlation is observed in the compounds studied except in the cases of methyl-thio and methyl-sulphynil compounds. Observing these results, it is logical to think that just as in the phosphate series, the compounds with a thioether linkage undergo in vivo oxidation processes, which lead to a biological behaviour like a sulphone compound. The same should occur with sulphynil products. If this phenomenon took place, it should become apparent on replacing the first a values of -S-CH3 group (0.00) by those corresponding to the -SO2-CH3 group (1.05). Thus, equations (4) and (5) are obtained and statistic data show that these equations represent a model better fitted to biological activity, which this possible transformation should always take into account. n

v

pLDs0 = 1.94a + 0.1

11 0.1291 0.9419 (4)

pLD~0 = 1.90a + 0.06

11 0.1072 0.9603. (5)

The introduction of the solubility factors 7~2 and does not increase significantly the correlation coefficient of equations (4) and (5), thus confirming the small contribution of these effects.

52

ANGEL M. RELIMPIO

2.0

I;SCH3

1.6

-20CH~_c:SOz~~, ~

~

fiN02

1.2 o o

0.4-o-

J

eCt __ pLDso:l.94o-+O.i r: 0.940 v=O.150

-OEi* /

el/

eH

I" ~

_0.4~f 1

0I

I 0.4 cr

I 0.8

t 1.2

Fig, 2. Plot of pLDso of the series ethyl-4-substituted phenyt-methylphosphonates vs ~r values of the substituents. n

v

r

pLDso = -0.13r~ + 1.88a+0.10

ll

0.1126 0.9611

pLDso = - 1.15 r~z - 1.16 ~ + 1.92a + 0.13

11

0.1122 0.9675 (7)

(6)

O n studying the series with substitutions in the meta and para positions, we have to take into account the values of the parameters corresponding to b o t h substituents. In these cases, the a and ~ values will be the sum in b o t h cases of the a values and the ~ values, respectively. In accordance with this idea, equations (8-11) were derived n v r pLDso = 1.46lEa - 0.19 9 0.2748 0.7218 (8) pLDso = - 1 . 1 1 Z n + 1.34

9

0.4192 0.6024

pLDso = -0.35 Zn 2 + 1.43 Ya + 0.48

9

0.2915 0.7336 (10)

pLDso = -5.18 lEn2 + 10.20 Z(r + 0.85 lea - 3.63

(9)

9

9

0.2675 0.8221. (11)

9

0.0772 0.9836 (13)

0.0812 0.9849. (14)

The ethyl-phenyl series gives similar results. For this series the following equations were derived. /1

s

r

pLDso = 0.92 Ea + 0.35

8

0.3853 0.5790 (15)

pLDs0 = -0.88 Y~rc+ 1.93

8

0.4351 0.4510 (16)

pLDs0 = -0.58 lEn + 0.74lEa + 1.18

8

0.3235 0.6437 (17)

pLDs0 = -3.21 Ym2 + 8.81 left + 0.58 Y,a - 4.99

8

0.2829 0.6984. (18)

If we adjust this set of equations, using the corrected values for thio-derivate, the results are the following equations /1

As can be observed, the introduction of a substituent in meta position, gives rise to a set of equations in which there is a correlation of coefficients similar to that found in the former case, but with greater variance. This fact indicates that the solubility factors do not play an important role in the activity of these compounds. O n studying the electronic effects the same p h e n o m e n o n as already cited is observed, viz. when there is a replacement of methyl-thio and methyl-sulphynil values by those corresponding to methylsulphone, a substantial increase of correlation coefficients is observed, and at the same time the variance diminishes. F r o m the observance of the equations in which both factors participate, the different role of the factors may be confirmed. n S r pLDs0 = 1.92lEa + 0.28 9 0.0714 0.9776 (12) pLDs0 = -0.002 Zn + 1.92lEa -- 0.28

pLDso = 0.89 lElt2 + 1.80 Y4t + 2.04Za + 0.38

s

r

pLDs0 = 1.55 Za - 0.21

8

0.0786 0.9658 (19)

pLD5o --- 0.16 En + 1.63 2cr - 0.47

8

0.080

0.9685 (20)

pLDs0 = -0.78 £n 2 2.08 Z~ + 1.70 Zcr + 0.96 8 0.0889 0.9703. (21) Throughout this study the preponderancy of the electronic effects on the biological activity is clear, whereas solubility does not play any appreciable role. The difference found in the in vivo biological activity among the series must be originated by a cause not related with solubility since it is very difficult to explain that the difference in activity is due to a factor which has demonstrated so small an influence. So we must come to the conclusion that these changes in activity may be originated by steric factors due to meta substituents which may produce a change in the mode of action of these compounds, in accordance with Hansch's conclusions. The difference with the p h o s p h a t e series, in which a little, but appreciable, influence of the solubility fac-

Biological activity of anticholinesterase tors was found, may be due to the difference in solubility of b o t h kinds of compounds, which although slight, may be of value at biological level. Acknowledgements--The author would like to thank Dr Antonio Quijada from Centro de C/tlculo Escuela T6cnica Superior de Ingenieros Industriales for his aid in the statistical work and Miss Teresa Silva for technical assistance.

REFERENCES

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53

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