Quantitative structure-activity studies of substituted benzyl chrysanthemates

PESTICIDE

BIOCHEMISTRY

AND PHYSIOLOGY

25, 288-294 (1986)

Quantitative Structure-Activity Studies of Substituted Benzyl Chrysanthemates 8. Physicochemical

Properties and Neurophysiological Effects on Membrane Potentials of Crayfish Giant Axon

MASATO OMATSU, KEIICHIRO NISHIMURA,~ AND TOSHIO FUJITA Department

of

Agricultural

Chemistry, Kyoto University, Kyoto 604, Japan

Received February 5, 1985; accepted June 3, 1985 The neurophysiological actions of a set of substituted benzyl (lR)-trans-chrysanthemates and related compounds on the membrane potential of the crayfish giant axon were observed using an intracellular microelectrode. These compounds had one or both of two effects, deceleration of the falling phase of the action potential or elevation of the depolarizing after-potential. The potency of a certain range of compounds to decelerate the rate of the falling phase is mostly determined by the electron-releasing effect of substituents on the aromatic ring. The rate of development of each symptom evaluated in terms of half the time required to reach the maximum response decreased with the hydrophobicity of the compound. o 1986 Academic PXSS. IK. INTRODUCTION

We have examined the effects of pyrethroids, including variously substituted benzyl chrysanthemates, on the action potential emerging in the giant axons of crayfish, using an intracellular procedure (1). We classified the pyrethroids by their effects into one of three types. Type A compounds decelerate the falling phase of the action potential, and type B compounds cause an elevation of the depolarizing after-potential. Type C has both effects. With increasing concentrations, the type C compounds still exhibit both the type A and B effects, although the type A deceleration-phase fuses into the type B depolarization, We determined the potency of type A, B, and C compounds in terms of the concentration required to cause a specific degree of each type of response. We showed that the deceleration of the falling phase of the action potential, which necessarily elevates the after-potential, is closely related to the induction of the repetitive discharges in central nerve cords for the type A activity of type A and C comI To whom correspondence

pounds. The elevation of the depolarizing after-potential observed in type B and C compounds was, however, not directly linked with their repetitive nerve activity although it should be an important factor determining the variations in the activity. Continuing to examine the nature of intracellularly determined neurophysiological activities, we have attempted to quantitatively analyze variations in these activities with the physicochemical properties of the molecule. The variations in the potency of the type A effect have been shown to be governed by an electron-donating effect from the alcoholic moiety in a range of substituted benzyl chrysanthemates. The rate of progress of each symptom has been evaluated in terms of half the time required to reach the maximum response. The lower the hydrophobicity of compounds, the higher is the rate of development of symptoms. MATERIALS

288 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

METHODS

Compounds. The compounds used in this study are the same samples as those used previously (1). Determination of potencies of various ef-

should be addressed.

0048-3575/86 $3.00

AND

PHYSICOCHEMISTRY

fects

on membrane

AND

NEUROPHYSIOLOGY

The cirisolated from

potentials.

cumesophageal connectives both sexes of the crayfish Procambarus clarkii were used as the preparation for the intracellular experiments. The conditions of the neurophysiological tests are the same as those described previously (1). As indicated earlier, pyrethroids had three types of effects on the action potential (Fig. 1). The type A effect on the falling phase of the action potential was observed as the percentage decrease from the maximum rate of fall (-dV/dt) for the control. The rate of fall slowed down gradually leading to a stable value at about 45 min, as shown in Fig. 2a, for the o-F derivative of benzyl chrysanthemate. We evaluated the percentage decrease at the stable phase in the time-response curve for each concentration. The plot of the percentage decrease value against the concentration gave doseresponse curves such as Fig. 2b. To compare the potency, the concentration required to reduce the maximum rate by 10%. termed ICI0 (M), was used for the type A effect of type A and C compounds. The type B effect on the depolarizing afterpotential was expressed as the highest membrane depolarization between 1 and 20 msec after the action potential once fell quickly. The highest depolarizing potential

OF CHRYSANTHEMATES

increased gradually with the time of the treatment reaching a maximum. A set of the time-response curves is shown in Fig. 3a for resmethrin. The maximum potential was estimated for each concentration and plotted against the concentration to prepare dose-response curves such as Fig. 3b. The concentration required to elevate the depolarizing after-potential by 5 mV from the resting level, termed EC5 (M), was used to compare the potency for the type B effect of type B and C compounds. The log value of the reciprocal of these equieffective concentrations was used for the indices of type A and B potencies (Table I). Some pyrethroids cause depolarization of the resting potential at high concentrations. Since the depolarizing after-potential was measured from the resting level, such depolarization of the resting potential may perturb the determination of the accurate potency to elevate the depolarizing afterpotential. Therefore, we only used concentrations where the depolarization of the resting potential was insignificant and did not affect the value of EC5. Measurement of the rate of development ofresponses in nerve membranes. The rate

of development of each symptom was calculated in terms of the half-time, tl/z (min), the time required to reach half the max-

cI I

IY- = ,Itl__ Iv-=rIi= ;t4 “II

G!!

bll

289

FIG. 1. Three different effects of pyrethroids on the uction potential: before (a) and 10 min after (d) starting the treatment with 4.74 x lo-’ M o-F benzyl (lR)-trans-chrysanthemate (type A); before (6) and 80 min after (e) starting the treatment with 1.55 x lo-’ M phenothrin (type B); before (c) and I5 min after cf, starting the treatment with 1.00 x 10-j M m-CN benzyl (lR)-trans-chrysanthemate (type C). The lower trace in each subfigure is that for the membrane potential. while the upper is the record of thefirst derivative of the potential with respect to time (dV/dt). The maximum and minimum of the derivative trace represent the maximum rate of the potential change in the rising undfalling phases of the action potential, respectively. Scales for potentiuls 12s time are for the lower trace in each subfigure.

290

OMATSU,

NISHIMURA,

AND FUJITA

7 2 I a 30-” _-----

. 2L .E .__.6.-’--.0 ,‘“l i ! lJ 3 O” I”‘-_ +’ t1/2 30

60

~ime(min)

FIG. 2. (a) Time-response

curves for deceleration of the rate offah ofthe action potenfiai induced M (0) and 1.58 x 10-e M (0) o-F benzyl (lR)-trans-chrysanthemate. The time required to cause half the maximum response in each time-response curve is tl/z. (b) Dose-response curve of o-F benzyl (lR)-tram-chrysanthemate for this deceleration. ZCK, is the concentration required to decrease the rate offal1 by 10%. The integer in the parentheses is the number of determinations. The vertical line shows the standard deviation. The response in each subfigure is expressed by the percentage decrease from that determined before treatment with the compound.

by 4.74

X

1O-6

imum response. The tl/Z value for the decelerating effect on the falling phase of the action potential was measured graphically at each concentration for type A compounds, as shown in Fig. 2a for the o-F benzyl chrysanthemate. The fl/2 for the elevating effect on the after-potential was measured similarly at each concentration for type El and C compounds, as shown in Fig. 3a for resmethrin. The fl/2 values were measured with at least 5 nerve preparations between 1 x 1O-8 and 1O-5 M for each compound. The log of the reciprocal, log (llfti), was used as the index for the rate of development (Table 1). If the rate of devel-

opment obeys ideal first-order kinetics governed by the concentration gradient from the outside solution to the site of action, the log (lltY2) should be constant regardless of the bathing concentrations for each compound. In practice, the values were scattered from one concentration to another. Therefore, the values were averaged over several determinations and used for the analyses. Errors in the measurement of small membrane potential changes can result in large errors in the determination of the fY2 value. Therefore, ti/z was determined only for concentrations inducing more than 10% deceleration in the falling

3. (a) Time-response curves for elevation of the depolarizing IO-‘M (0) and 1.99 x 1Om8 M (0) resmethrin. The time required response in each time-response curve is t%. (b) Dose-response curve the depolarizing after-potential. EC5 is the concentration to elevate the 5 mVfrom the resting potential. The integer in the parentheses is the vertical line shows the standard deviation. FIG.

x

after-potential induced by 1.99 to cause half the maximum of resmethrin for elevation of depolarizing after-potential by number of determinations. The

H o-F o-SOzMe m-F m-OPr(i)

1 2 3 4 5 6 7 8 9 10 11 12 13

6.15 6.30 5.36 5.85 5.90 5.56 6.60

Obsd

6.26 6.07 5.46 5.86 6.22 5.54 6.40

Calcdb

of’S~~h.stit~tted

6.20 7.34 7.68 7.20 7.53 7.28

6.40 I

logt l/EC)

Actitbiries

8 7 16 I5 IO 8 5

7 IO 6 II 9

No. of Experiments

-0.93 -0.17 --0.38 -1.38 -1.03 -0.58 - 1.20

-0.87 -0.64 -0.61 -0.62 -0.80 I zi 2 2 _t 2 f 2

k ” 2 r t 0.25 0.21 0.50 0.09 0.15 0.25 0.04

0.17 0.23 0.21 0.28 0.09’

Obsd ( ? SD)

log(l/tfi)

TABLE 1 (lR)-trans-Ck~sarIrhetn~tes

Benzyl

-0.72 -0.75 -0.32 - 0.75 -0.86 -0.58 - 0.93 -0.32 - 0.54 - 1.24 - 1.05 -0.53 - 1.20

Calcd’

ce

Compormds

0.00 0.17h 0.69” 0.35 0.04 0.62 -0.12 0.69

und Related

5.49 5.63 3.86 5.63 6.34 4.92 6.05 3.86 4.78 7.57 6.82 4.73 7.44

log P

0.00 0.33 2.78 0.33 3.48 1.22 1.12 2.78 2.70 4.65 4.06 3.24 4.65

AV,,f

(’ Substituent on the benzyl alcohol moiety is shown except for compounds indicated by the common name. b From Eq. [2]. L From Eq. [4]. ClFrom the compilation of 0. Exner; The Hammett equation-the present position. irr “Advances in Linear Free Energy Relationships” Chapman and J. Shorter, Eds.), p, I. Plenum Press, London and New York, 1972. p From Ref. (2) except for compound 11 the value of which is a corrected one from erroneous list in the reference. f From Ref. (5). Value for compounds 9- 13 is the value ( x 0. I) relative for the alcoholic moiety to benzyl alcohol. p From Ref. (I). ‘I The value for the corresponding parer substituents. / Evaluated from type B effect. j Not determined.

Allethrin Phenothrin Resmethrin Tetramethrin Permethrin

m-CN p-Me p-SO,Me

Compound”

No.

lo& l/IC,oJ

Nertr-ophvsioloRic(II

(N. 8.

B

B

B B B

A A A A C C A B

Types

$

2 z ? 5 is

g

8 <

9

3 E

z z r5

%

2

t;

8 z

F:

WJ i;

292

OMATSU,

NISHIMURA,

phase of the action potential at the steady state (Fig. 2a) for type A compounds and more than 3 mV elevation of the depolarizing after-potential at the maximum on the time-response curves (Fig. 3a) for type B and C compounds. Physicochemical parameters. For quantitative analyses, such physicochemical parameters as o”, log P, and OV, were used. u” is an electronic parameter of aromatic substituents similar to the Hammett constant. Log P, an index of the hydrophobicity of the whole molecule, was estimated from P, the I-octanollwater partition coefficient, as described previously (2). AV, is the van der Waals volume (3) relative to that of unsubstituted benzyl chrysanthemate scaled by 0.1 to make it nearly equiscalar with the other parameters. Relevant substituent parameters are listed in Table 1. RESULTS

AND

DISCUSSION

Relationship between neurophysiological ativities and physicochemical properties. Variations in the potency to decelerate

the falling phase of the action potential of type A substituted benzyl chrysanthemates in terms of log (l/I&J were quantitatively analyzed by physicochemical parameters of aromatic substituents of the benzyl chrysanthemates. The activity was correlated with an electronic parameter giving

AND

FUJITA

rentheses are the 95% confidence interval of the regression coefficient and intercept. Since type C compounds also exert the decelerating effect, they were combined in a set to give Eq. [2]. log(l/IC,,)

= 6.264 - 1.166~” (+0.274)(+0.716) n = 7, s = 0.214, r = 0.882

PI

The situation is depicted in Fig. 4. The negative of u” term in these equations indicates that the electron-donating substituents are favorable to the deceleration of the falling phase of the action potential in type A and C benzyl chrysanthemates. The fact that the regression coefficient value, p, was not varied much by including type C compounds indicates that the deceleration of the falling phase by type C compounds is brought about by a molecular mechanism common with that of type A compounds, even though type C compounds simultaneously elevate the depolarizing after-potential. It seems peculiar for this kind of biological effect to be determined solely by the electronic effect of substituents. Because the data are collected after a steady biological response was attained (Fig. 2a), the potency analyzed here may be independent of transport processes that could be controlled by hydrophobicity. The aromatic

Eq. [Il. = 6.360 - 1.4148 [II ( _t 0.297)( k 0.830) n = 5, s = 0.166, r = 0.953 u” is the “normal” electronic substituent constant that is supposed to contain no fraction of through-resonance effect on the functional side chain. For ortho substituents, the value of corresponding para substituents was used (4). Substitution by or addition of other parameters did not improve the correlation. In this and the following equations, II is the number of compounds, s, the standard deviation, r, the correlation coefficient. The figures in palog(l/IC,,)

FIG. 4. Relationship between the decelerating activity (log ZIZC,O)of type A (0) and C (A) compounds on the rate of fall of the action potential and o” values.

PHYSICOCHEMISTRY

AND

NEUROPHYSIOLOGY

ring as well as the benzyloxylic part of the alcoholic moiety were made electron-rich by electron-donating substituents and could interact with an electron-deficient critical site of action located on the nerve membrane electronically. In our previous analyses of repetitive nerve activity using a wider range of substituted benzyl chrysanthemates, aromatic substituent effects were shown to be highly specific to position (5). In general, steric bulkiness of substituents, represented by the van der Waals volume, is of major importance in determining variations in the activity in each series of positional isomers. In ortho substituted isomers (n = 14), however, the electron-donating effect of substituents was also important. The participation of the electronic effect in the repetitive activity seemed to conform with Eq. [2]. We have recently found that the great majority of ortho substituted derivatives (13 out of 14) are either type A or C in terms of their effect on the action potential (61.

For type B and C compounds, we have attempted to analyze variations in their activity to elevate the depolarizing after-potential in terms of log(l/EQ. Unfortunately, for compounds other than substituted benzyl chrysanthemates, the electronic parameter of “substituents” is hard to estimate. Moreover, no correlation was found using such parameters for hydrophobicity of the molecule and steric bulkiness of the alcoholic moiety as log P and AV,. Relationship between rate of development of neurophysiological symptoms and physicochemicat properties. The rate of de-

velopment of the elevation of the depolarizing after-potential of type B and C compounds in terms of log (lltfi) was quantitatively analyzed to give Eq. [3]. log(l/t’h)

= 0.951 - 0.294 log P [3] ( 2 0.439)( ? 0.072) n = 7, s = 0.102, r = 0.978

The addition of or the replacement

by such

OF CHRYSANTHEMATES

293

parameters as AV, in Eq. [3] did not improve the correlation. After some trials, the rate of the development of the deceleration of the falling phase of the action potential for type A compounds was found to be analyzable together with that for the type B effect to yield Eq. [4]. log(l/tl/z)

= 0.642 - 0.248 log P [4] ( t 0.481)( r 0.083) n = 12, s = 0.153, r = 0.903

The good correlation indicates that the development of each symptom in terms of the potential change at the axonal membrane is basically due to penetration. The negative log P term in Eq. [4] means that the lower the hydrophobicity of the compound, the easier the penetration through a number of barriers in the membrane to the target sites. For a series of compounds with log P values covering a sufficiently wide range, the penetration rate has been shown to vary parabolically with log P (7). Since the higher hydrophobicity makes the penetration more difficult, the hydrophobicity would be supraoptimal for the compounds tested here. The optimal log P of the rate of development of these potential changes should be lower than that of the least hydrophobic compound among those used (3.86 for the p-SO,Me derivative). Since the log (W/i) values of type A, B, and C compounds were correlated with the log P value by a single equation, penetration processes to the target sites of action for two kinds of effects, A and B, may be very similar to each other hydrophobically, although our recent voltage clamp experiments suggest that the receptors for type A and B effects are independent from each other. For type C compounds, only the rate of development of the type B effect was determined, since that of the type A effect was not accurately measured due to their potency generally being lower than that of type A compounds. The rate of the development of these symptoms was also evaluated according to a first-order kinetic model. Thus, we as-

294

OMATSU,

NISHIMURA,

sumed that the maximum or the steady state in Figs. 2a and 3a are the states where the concentration gradient disappears between the outside immersing solution and the site of action (8). With the rate constant derived according to this model, we formulated a correlation (not shown) similar to but slightly poorer than Eq. [4]. Previously, we estimated the “penetration” rate constant, k, of a number of pyrethroids from the rate of development of the knockdown symptom induced in house flies using a first-order kinetic model (9). The rate constant (log k) was correlated with the log P value of the molecule. Reanalysis of the correlation including the p-SO,Me derivative (k = 2.32 min-I) yielded Eq. [5]. log k = 3.536 - 0.259 tog P (-+ 0.334)( Itr 0.055) n = 23, s = 0.128, r = ~0.907

PI

In this analysis, we used log P values for resmethrin (log P = 6.82) and for NRDC 134 (log P = 6.69) corrected from those listed previously (2). Eq. [5] indicates that the lower the hydrophobicity of pyrethroids, the faster the development of the symptom. The coefficient of the log P term in Eq. [5] is very close to that in Eq. [4]. This fact may indicate that the rate of development of knockdown symptoms of house flies is mainly determined by the rate process occurring in the axonal membrane in spite of the difference in the animal species. The indication observed here should be further examined using larger insect species such as cockroaches. ACKNOWLEDGMENTS

This investigation was supported in part by a Grantin-Aid for Scientific Research and for Special Project

AND FUJITA

Research from the Ministry of Education, Science, and Culture of Japan and by a fund from Nippon UCLAF K.K. The calculations were performed on a FACOM-382 computer at the data processing center of this university.

REFERENCES

1. M. Omatsu, K. Nishimura, and T. Fujita, Quantitative structure-activity studies of substituted benzyl chrysanthemates. 7. Relationship between induction of repetitive discharges and effects on the action potential, Pestic. Biochem. Physiol.,

24, 192 (1985).

2. S. Nakagawa, N. Okajima, T. Kitahaba, K. Nishimura, T. Fujita, and M. Nakajima, Quantitative structure-activity studies of substituted benzyl chrysanthemates. 1. Correlations between symptomatic and neurophysiological activities against American cockroaches, Pestic. Biochem. Physiol. 17, 243 (1982). 3. A. Bondi, van der Waals volumes and radii. J. Phys. Chem. 68,441 (1964). 4. T. Fujita and T. Nishioka, The analysis of the ortho effect, Prog. Phys. Org. Chem. 12, 49 (1976). 5. S. Nakagawa, N. Okajima, K. Nishimura. T. Fujita, and M. Nakajima, Quantitative structureactivity studies of substituted benzyl chrysanthemates. 2. Physicochemical substituent effects and neurophysiological and symptomatic activities against American cockroaches, Pestic. Biochem. Physiol. 17, 239 (1982). 6. M. Omatsu, K. Nishimura, and T. Fujita, unpublished data. 7. J. T. Penniston, D. L. Bentley, and C. Hansch, Passive permeation of organic compounds through biological tissues: A non-steady-state theory, Mol. Pharmaco/. 5, 333 (1969). 8. G. L. Atkins, “Multicompartment Models for Biological Systems,” p. 19, Methuen, London, 1969. 9. K. Nishimura, N. Okajima, T. Fujita, and M. Nakajima, Quantitative structure-activity studies of substituted benzyl chrysanthemates. 4. Physicochemical properties and the rate of progress of the knockdown symptom induced in house files, Pestic. Biochem. Physiol. 18, 341 (1982).