Structure-Activity Relationship Studies with Mosquito Repellent Amides M. V. S. SURYANARAYANA~, K. S. PANDEY,SHRIPRAKASH, C. D. RAGHUVEERAN, R. S. DANGI, R. v. SWAMY, AND K. M. RAO Received November 6, 1989, from the Defence Research and Development Establishment, Gwalior-474002,India. Accepted for publication January 23,1991. Abstract 0 A series of amide analogues of N,Ndiethyl-mtoluamide (DEET) and N,Ndiethylphenylacetamide (DEPA) were synthesized to
study their mosquito repellency in relation to chemical structure and physical properties. None of these parameters (vapor pressure, lipophilicity, and molecular length) could be related to protection time. However, when the protection time of all five amides derived from the same carboxylic acid was expressed in terms of all three parameters together, excellent correlation was obtained.
Amides in general and aromatic amides in particular are well-known'-3 repellents for mosquito and other pestiferous insects. It has been demonstrated in our laboratorye that the repellency of N,N-diethylphenylacetamide(DEPA) is almost on par with that of the popular mosquito repellent NJVdiethyl-rn-toluamide (DEET). A series of amides derived from aromatic acids was synthesized and evaluated in our laboratory in order to investigate structur+activity relationships for designing better repellents than DEET and DEPA. Such exercises have been undertaken earlier by several investigators.1.3 In one such study,l the authors tried to relate the repellent potency of Nfl-diethylbenzamides with their boiling points, polarizability, and partition coefficients, but only boiling point (vapor pressure) could be related with the repellency. In none of the other cases could a good relationship between repellency and other physical parameters, except volatility, be established. McIver7 suggested that lipophilicity is also important in influencing repellency, while Sugawara et a1.s and Wright9 demonstrated that molecular size and shape have profound influences on the repellent activity of a compound. In almost all these correlation studies, the physical parameters used were either theoretically calculated values or secondary parameters, and the correlation studies were done with one parameter at a time. The present investigation relates the repellency of a few aromatic acid and alicyclic amides, expressed in terms of protection time against mosquitoes, with experimentally determined values of the three parameters, namely vapor pressure (VP), lipophilicity (log P), and molecular length (ML), by multiple linear regression equation. Table I indicates the structures, molar refractivity (MR), protection time (PT), VP, log P, and ML of the amides. Molar refractivity (MR) values are obtained from the literature.10
Experimental Section The GLC analysis was done on a Perkin-Elmer 3920B equipped with a 2 m x 2 mm i.d. S.S. column packed with 10%stabilized DEGS (Analabs)on chromosorb WHP. Nitrogen was used as the carrier gas at a flow rate of 30 mumin. The oven, iqjection port, and the flame ionization detectors were maintained at 210,240, and 270 "C, respectively. The reversed-phase HPLC (RPHPLC) was performed at room temperature on a Shimadzu LC-6A chromatograph, with a Shimadzu ODS (5 cm x 4 mm) column. The elution profile was monitored at 254
OO22-3549/91/1100-1055$02.50/0 0 1997, American Pharmaceutical Association
nm, with a UV-vis detector. Melting points were determined by the capillary tube method and are uncorrected. Boiling points were determined using a short path distillation apparatus and are uncorrected. The IR spectra obtained on a Perkin-Elmer model 577 were used to confirm the assigned structures. Synthesis of Amides-Forty amides of aromatic and cyclohexyl carboxylic acids were synthesized by reacting the appropriate acid chloride with different amines, including ethyl, dimethyl, diethyl, diisopropyl amines, and piperidine. The products so obtained were purified by vacuum distillation or recrystallization. The purity of each compound was found to range from 95 to 99%, based on analysis by TLC (using silica gel G TLC plates, with the solvent system benzene:acetone, 4:1, v/v) and also GLC. Determination of Saturated Vapor Pressure-Vapor pressures were measured by a static method in an isoteniscope." Prior to VP measurements, each amide was deaerated by a freezing and thawing process. The temperature was maintained constant in a thermostated water bath to an accuracy of 20.025 "C. The pressures were measured from the difference in the heights of the mercury columns of the isoteniscope by a cathetometer (OSAW, India), corrected to 0.001 cmHg. Our aim was to obtain reliable VP data a t room temperature (30 "C). As most of the compounds do not have sufficient volatility at this temperature, the VPs were determined a t three or more higher temperatures. Applying the method of least squares, the VP values at 30 "C were obtained using the following equation:
log VP
=
A - BIT
(1)
where A and B are constants and T is the absolute temperature. Determination of Lipophilicity-The lipophilicity of each compound was determined12 by RPHPLC. The mobile phase consisted of water and methanol in various proportions ranging from 40 to 70% methanol, with a constant flow rate of 1mumin. The capacity factor, k', for each compound at various solvent compositions was plotted against methanol composition to obtain k' at 100%water, which is used as a measure for the relative lipophilicity (log P). Molecular Length Measurements-Molecular lengths (MLs) of the amides were measured &er constructing three-dimensional molecular models at the energy minimum with the organic chemistry set of Beqjamidblaruzen HGS molecular structure models (W. A. Beqjamin Inc., CAI. The MLs were measured based on the design of a rectangle fitted to the amide molecule. Biological Testing-Each repellent was applied at a dose of 1 mg/cm2 on the external surface of the human fist over an area of -150 cm2.The treated surface of the hand was exposed to 200 females (5-7 days old) of the day-biter mosquito Aedes uegypti released in a 75 x 60 x 60 cm test chamber for 5 min at an interval of 0.5 h. The protection time (PT)was determined as in the method described by Sharma and co-workers.13 All the calculations for the correlations were made on a personal computer, using a program for multiple regression analysis.
Results and Discussion Vapor Pressure ValuesVapor pressure is one of the crucial properties in influencing the efficacy of any repellent. An ideal repellent1 should be neither non-volatile nor too highly volatile. An attempt was made to correlate the repellency with VP alone by a Chi-square test with an arbitrarily chosen value of 0.02 Journal of Pharmaceutical Sciences I 1055 Vol. 80, No. 1 1 , November 1991
Table I-Physical and Biological Properties of Various Repellent Amldes
Number
X
Rl
R,
Empirical Formula
MW
Log
VP, cmHg at 30 "C
ML, A"
MR
PT, h
0
@-N < R1 X
R2 4-OCH3
CH3
H CH3
C2H5
C2H5
C2H5
Iso-C~H~ Iso-C~H~
CHSCH,
H CH3
C2H5
C2H5
C2H5
ISO-C~H,
H CH3
C2H5
'ZH5
Iso-C~H~ Iso-C~H~
CH3
H CH3
C2H5
'ZH5
C2H5
ISO-CH C5H13
2-CI
C10H13N0
C9HllNO C9Hll NO cl lH15N0 c1 3H 1gN0
Iso-C~H~
C10H13N0
C10H13N0
C12H1,NO C14H21N0
C14H17N0 H CH3
CgHloNOCI CgHloNOCI C, H14NOCI C2H5 C2H5 I s o - C ~ H ~ I s o - C ~ H ~ C, jH1,NOCI C13H14NOCI C5H10e C2H5
CH3
2-OC2H5
C10H13N0
C13H19N0
C5H10a
3-CH3
C14H21N02
C14H17N0
CH3
C2H5
C1ZH17N02
C1ZH17N0 I s o - C ~ H ~ Ci4HziNO
C5H10a
H
C10H13N02
C14H17NOZ
C5H10a
4-CH3
C10H13N02
CH3
H CH3
C2H5
C2H5
C2H5
Iso-C3H7
Iso-C3H7
C11H15N02 C13H19N02
C15H23N02 C14H19N02
C5H108
H CH3
7
C11H15N02
'ZH5
Iso-C3H7
C10H13N0
C1OH13NO CizHi7N0 Ci4HziNO clqH 1
179 179 207 235 21 9
1.14 1.60 2.50 2.80 2.40
0.0062 0.0039 0.0037 0.0155 0.1486
13.36 12.16 13.36 13.36 14.08
5.101 5.101 6.029 6.957 6.315
0.08 1.oo 1.oo 1.17 0.75
163 163 191 219 203
1.38 1.72 2.38 2.50 2.80
0.0063 0.0110 0.0244 0.0159 0.0313
11.28 10.48 11.28 11.28 12.40
4.948 4.948 5.876 6.803 6.162
0.08 4.00 2.83 0.50 1.oo
149 149 177 205 189
0.90 1.20 1.80 2.50 2.20
0.0015 0.0015 0.1015 0.0116 0.0559
10.64 10.00 10.64 10.64 1 1.88
4.484 4.484 5.412 6.340 5.698
0.58 1.67 4.00 3.00 3.00
163 163 191 219 203
1.32 1.60 2.34 3.00 2.60
0.0013 0.0055 0.0260 0.0151 0.0001
10.08 10.48 10.80 10.48 12.16
4.948 4.948 5.876 6.803 6.1 62
0.67 3.00 5.00 2.67 1.42
183.5 183.5 211.5 239.5 223.5
0.95 1.54 2.10 3.00 2.80
0.0006 0.0076 0.0602 0.7728 0.0281
10.64 10.00 10.64 10.64 1 1.88
4.976 4.976 5.903 6.831 6.190
0.58 5.00 3.00 1.oo 1.oo
193 193 221 249 233
2.40 3.80 2.50 3.00 2.60
0.0003 0.0264 0.0012 0.0144 0.0030
10.64 10.00 10.64 10.64 1 1.88
5.565 5.565 6.493 7.420 6.779
0.08 2.83 3.50 1.08 1.33
163 163 191 21 9 203
1.oo 1.48 2.40 3.10 2.44
0.0058 0.0020 0.1043 0.0014 0.1814
11.76 10.40 11.76 11.76 12.40
4.948 4.948 5.876 6.803 6.162
1.00 2.17 6.00 1.00 2.58
169 169 197 225 209
2.28 2.19 3.12 1.78 3.20
0.0168 0.0136 0.1638 0.2843 0.0315
11.68 10.52 11.68 11.68 12.24
5.042 5.042 5.970 6.898 6.256
0.50 3.00 4.00 2.00 2.00
0
8
CH3
H CH3
ClOH19NO ClOH19NO
C2H5
C2H5
C11H15N0
C2H5
ISO-CH C5Hlia
Iso-C~H~
C14H23N0 C14H23N0
* Represents both R, and R, groups.
cmHg and PT of 2 h. Twenty-five out of 40 compounds were within this generalization (,$ = 4.87 at p < 0.05, df = 1). Solids in general were found to be poor repellents against mosquitoes. Lipophilicity V a l u ee r n e lipophilicities (log P) of the homologous compounds increased progressivelywith the number of carbon atoms in the chain, as expected. Compounds having 1056 I Journal of Pharmaceutical Sciences Vol. 80, No. 1 1, November 1991
moderate log P values, ranging between 1.5 and 2.5, appear to have relatively better repellency (2=: 6.83 at p < 0.05, df = 21, which is in agreement with the literature.' Twenty-fiveout of the 40 compounds examined conform to the above conclusion. Molecular Lengths-Compounds having a ML >10.6 A" exhibit relatively longer protection periods (i.e.,PT > 2 h; ,$
Table ICProtectIon Time Estimation for Varlous Repellent Amldes against Mosqultoes by Use of Equation 5
ne
Repellents pAnisamides pToluamides Benzamides mToluamides eChlorobenzamides eEthoxybenzamides Phenylacetamides Cyclohexylacetamides a
rb
0.996 0.970 0.995 0.948 0.697 0.423 0.870 0.991
FC
36.82 5.233 30.791 2.987 0.315 0.073 1.042 18.30
a
b
C
d
0.647 -4.088 0.625 -0.613 4.125 -0.387 0.824 1.985
00.121 12.222 01.475 3.399 -2.533 0.705 2.308 1.782
-0.505 -2.058 -0.81 1 3.329 -3.883 -0.604 -2.107 -2.182
6.383 56.018 13.005 -24.018 31.155 11.113 29.699 24.846
Number of data points. Multiple correlation coefficient. F test value.
= 5.31 at p < 0.02, df = 1). Only 20 out of the 40 compounds scanned obeyed this generalization. The poor generalizations observed above suggest that no single physical property is solely responsible for repellent activity. This is further substantiated by the observed variations in activity among compounds of similar physical properties, like volatility, log P, or ML. In view of the rather poor generalizations observed above, including that of piperidine anisamide which otherwise satisfies all the optimal criteria such as VP, log P, and ML set above, an attempt to express the PT of all 40 amides in terms of two parameters, namely log P and log VP values using eq 2, resulted in a poor correlation (r = 0.351):
PT = a l o g P + b log VP + d
(2)
where a, b, and d are constants. Similarly, eqs 3 and 4 also failed to describethe protection time of a repellent, as evidenced by low
correlation coefficients (r = 0.404 and 0.547, respectively):
PT = a log P + c ML + d
PT = b log VP + c ML + d
(3) (4)
Further, an effort was made to incorporateall three parameters simultaneously to express the protection time using eq 5:
PT=alogP+blogVP+cML+d
(5)
where a, b, c, and d are constants. The correlation coefficient for all 40 amides improved (r = 0.551)compared with that obtained using eqs 2-4. However, when all the amides derived from the same carboxylic acid were considered, the correlation improved significantly, even in the above equations (i.e., eqs 2-4; r = 0.5-0.98; complete data not presented). An effort was made to correlate the F T s of all the amides derived from same carboxylic acid with all three parameters as per eq 5. This resulted in considerably enhanced correlations (Table 11) for all the amides, except the ortho-substituted derivatives. Failure in the case of ortho-chlorobenzamides and orthoethoxybenzamides to get good correlation by eq 5 may be due to the well-known ortho substituent (steric) effect,14 where Hammett type of equations do not hold. Hansch and coworkers15 opined that molar refraction (MR) and molecular weight (MW) are the approximate measures of the “steric b u l k . The MR values have been used previously in some biological QSAR studies.16 Substitution either of MR or MW for ML in eq 5 resulted in significantly improved correlation in the case of orthochlorobenzamides (eqs 6 and 7):
PT = 1.924 log P + 3.035 log VP - 6.895 MR + 42.932 n = 5, r = 0.989, F = 15.49
(6)
PT = 2.942 log P + 2.476 log VP - 0.234 MW + 48.847 n = 5,r = 0.985, F
=
10.78
(7)
However, in the case of orthoethoxybenzamides, there was no better correlation even by replacing ML with either MR or MW (r < 0.4). This can’t be explained a t present. Studies1.3.7-9 carried out so far on repellents evaluate only individual parameters for correlation with repellency. The present work examines the role of either two or three important factors a t a time (e.g., log P, VP, ML, MR, MW) in the structure-activity correlation of insect repellents. In the case of compounds having non-ortho substituents, the first three parameters showed good correlation for the repellent activity, whereas in ortho-substituted chlorobenzamides, either MR or MW (which are the approximate measures of “steric bulk” according to Hansch et al.15), along with the other two parameters (viz.,log P and VP) could account for the repellent activity.
References and Notes 1. Skinner, W. A.; Johnson, H. L. In Drug Design, Volume 10; Ariens, E. J., Ed.; Academic: New York, 1980;pp 277304. 2. Rao, S.S.;Rao, K. M.; Ramachandran, P. K. J . Sci. Znd. Res. 1988,47,722-735. 3. McGovern, T. P.;Schreck,C. E.; Jackson, J. Mosq.News 1984,44, 11-16. 4. Kumar, S.;Prakash, S.; Sharma, R. K., Jain, S. K.; Kalyanasundaram,M.; Swamy, R. V.; Rao, K. M. Znd. J . Med. Res. 1984, 80,541445. 5. Mathu, K. K.; Gu ta, G. P.; Kaushik, M. P.; Sikdar, A. K.; Rao, K. M. Znd. J. Med)Res. 1986,83,466470. 6. Prakash, S.;Kumar, S.; Su anarayana,M. V. S.;Sharma, R. K.; Rao, K. M. Znt. J . Cosmet.%. 1988,10,23-28. 7. McIver, S.B. J . Med. Entomol. 1981,18,357361. 8. Sugawara, R.; Tominaga, Y.; Suzuki, T. Insect Biochem. 1977,7, 483-485. 9. Wright, R. H. Sci. Am. 1975,233(1), 104-111. 10. Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; John Wiley & Sons: New York, 1979. 11. American Society for Testin and Materials Annual Book of Standards, Part 24; ASTM: I%hiladelphia,PA, 1978;p 740. 12. Braumann, T.J. Chromutogr. 1986,373,191-225. 13. Sharma, R.K.; Jain, S. K.; Kumar, S.; Rao, K. M. Znd. J . Hosp. Pharm. 1984,21,26-29. 14. Hansch, C. In Drug Design, Volume 2;Ariens, E. J.; ed.; Academic: New York, 1971;p 309. 15. Hansch, C.; Leo, A.; Unger, H. S.; Kim, H. K.; Nikaitani, D.; Lien, E. J. J . Med. Chem. 1973,16(11), 1207-1216. 16. Hansch, C.; Coats, E. J . Phurm. Sci.1970,59,731-743.
Acknowledgments We thank Brig K. M. Rao, Director, DRDE, Gwalior, for his active encouragement of this work. We are ateful to Dr. P. K. RamachanRao for their fruitful su ges dran, Emeritus Scientist, and Dr. tions throughout this investi ation. Acknowledgmentsare also dSue to Dr. R. Vi’ayaraghavan,Mr. %.Pandey, and Mr. A. C. Pandey for their help in tie statistical analysis of our results.
S.g
Journal of Pharmaceutical Sciences I 1057 Vol. 80, No. 11, November 1991
study their mosquito repellency in relation to chemical structure and physical properties. None of these parameters (vapor pressure, lipophilicity, and molecular length) could be related to protection time. However, when the protection time of all five amides derived from the same carboxylic acid was expressed in terms of all three parameters together, excellent correlation was obtained.
Amides in general and aromatic amides in particular are well-known'-3 repellents for mosquito and other pestiferous insects. It has been demonstrated in our laboratorye that the repellency of N,N-diethylphenylacetamide(DEPA) is almost on par with that of the popular mosquito repellent NJVdiethyl-rn-toluamide (DEET). A series of amides derived from aromatic acids was synthesized and evaluated in our laboratory in order to investigate structur+activity relationships for designing better repellents than DEET and DEPA. Such exercises have been undertaken earlier by several investigators.1.3 In one such study,l the authors tried to relate the repellent potency of Nfl-diethylbenzamides with their boiling points, polarizability, and partition coefficients, but only boiling point (vapor pressure) could be related with the repellency. In none of the other cases could a good relationship between repellency and other physical parameters, except volatility, be established. McIver7 suggested that lipophilicity is also important in influencing repellency, while Sugawara et a1.s and Wright9 demonstrated that molecular size and shape have profound influences on the repellent activity of a compound. In almost all these correlation studies, the physical parameters used were either theoretically calculated values or secondary parameters, and the correlation studies were done with one parameter at a time. The present investigation relates the repellency of a few aromatic acid and alicyclic amides, expressed in terms of protection time against mosquitoes, with experimentally determined values of the three parameters, namely vapor pressure (VP), lipophilicity (log P), and molecular length (ML), by multiple linear regression equation. Table I indicates the structures, molar refractivity (MR), protection time (PT), VP, log P, and ML of the amides. Molar refractivity (MR) values are obtained from the literature.10
Experimental Section The GLC analysis was done on a Perkin-Elmer 3920B equipped with a 2 m x 2 mm i.d. S.S. column packed with 10%stabilized DEGS (Analabs)on chromosorb WHP. Nitrogen was used as the carrier gas at a flow rate of 30 mumin. The oven, iqjection port, and the flame ionization detectors were maintained at 210,240, and 270 "C, respectively. The reversed-phase HPLC (RPHPLC) was performed at room temperature on a Shimadzu LC-6A chromatograph, with a Shimadzu ODS (5 cm x 4 mm) column. The elution profile was monitored at 254
OO22-3549/91/1100-1055$02.50/0 0 1997, American Pharmaceutical Association
nm, with a UV-vis detector. Melting points were determined by the capillary tube method and are uncorrected. Boiling points were determined using a short path distillation apparatus and are uncorrected. The IR spectra obtained on a Perkin-Elmer model 577 were used to confirm the assigned structures. Synthesis of Amides-Forty amides of aromatic and cyclohexyl carboxylic acids were synthesized by reacting the appropriate acid chloride with different amines, including ethyl, dimethyl, diethyl, diisopropyl amines, and piperidine. The products so obtained were purified by vacuum distillation or recrystallization. The purity of each compound was found to range from 95 to 99%, based on analysis by TLC (using silica gel G TLC plates, with the solvent system benzene:acetone, 4:1, v/v) and also GLC. Determination of Saturated Vapor Pressure-Vapor pressures were measured by a static method in an isoteniscope." Prior to VP measurements, each amide was deaerated by a freezing and thawing process. The temperature was maintained constant in a thermostated water bath to an accuracy of 20.025 "C. The pressures were measured from the difference in the heights of the mercury columns of the isoteniscope by a cathetometer (OSAW, India), corrected to 0.001 cmHg. Our aim was to obtain reliable VP data a t room temperature (30 "C). As most of the compounds do not have sufficient volatility at this temperature, the VPs were determined a t three or more higher temperatures. Applying the method of least squares, the VP values at 30 "C were obtained using the following equation:
log VP
=
A - BIT
(1)
where A and B are constants and T is the absolute temperature. Determination of Lipophilicity-The lipophilicity of each compound was determined12 by RPHPLC. The mobile phase consisted of water and methanol in various proportions ranging from 40 to 70% methanol, with a constant flow rate of 1mumin. The capacity factor, k', for each compound at various solvent compositions was plotted against methanol composition to obtain k' at 100%water, which is used as a measure for the relative lipophilicity (log P). Molecular Length Measurements-Molecular lengths (MLs) of the amides were measured &er constructing three-dimensional molecular models at the energy minimum with the organic chemistry set of Beqjamidblaruzen HGS molecular structure models (W. A. Beqjamin Inc., CAI. The MLs were measured based on the design of a rectangle fitted to the amide molecule. Biological Testing-Each repellent was applied at a dose of 1 mg/cm2 on the external surface of the human fist over an area of -150 cm2.The treated surface of the hand was exposed to 200 females (5-7 days old) of the day-biter mosquito Aedes uegypti released in a 75 x 60 x 60 cm test chamber for 5 min at an interval of 0.5 h. The protection time (PT)was determined as in the method described by Sharma and co-workers.13 All the calculations for the correlations were made on a personal computer, using a program for multiple regression analysis.
Results and Discussion Vapor Pressure ValuesVapor pressure is one of the crucial properties in influencing the efficacy of any repellent. An ideal repellent1 should be neither non-volatile nor too highly volatile. An attempt was made to correlate the repellency with VP alone by a Chi-square test with an arbitrarily chosen value of 0.02 Journal of Pharmaceutical Sciences I 1055 Vol. 80, No. 1 1 , November 1991
Table I-Physical and Biological Properties of Various Repellent Amldes
Number
X
Rl
R,
Empirical Formula
MW
Log
VP, cmHg at 30 "C
ML, A"
MR
PT, h
0
@-N < R1 X
R2 4-OCH3
CH3
H CH3
C2H5
C2H5
C2H5
Iso-C~H~ Iso-C~H~
CHSCH,
H CH3
C2H5
C2H5
C2H5
ISO-C~H,
H CH3
C2H5
'ZH5
Iso-C~H~ Iso-C~H~
CH3
H CH3
C2H5
'ZH5
C2H5
ISO-CH C5H13
2-CI
C10H13N0
C9HllNO C9Hll NO cl lH15N0 c1 3H 1gN0
Iso-C~H~
C10H13N0
C10H13N0
C12H1,NO C14H21N0
C14H17N0 H CH3
CgHloNOCI CgHloNOCI C, H14NOCI C2H5 C2H5 I s o - C ~ H ~ I s o - C ~ H ~ C, jH1,NOCI C13H14NOCI C5H10e C2H5
CH3
2-OC2H5
C10H13N0
C13H19N0
C5H10a
3-CH3
C14H21N02
C14H17N0
CH3
C2H5
C1ZH17N02
C1ZH17N0 I s o - C ~ H ~ Ci4HziNO
C5H10a
H
C10H13N02
C14H17NOZ
C5H10a
4-CH3
C10H13N02
CH3
H CH3
C2H5
C2H5
C2H5
Iso-C3H7
Iso-C3H7
C11H15N02 C13H19N02
C15H23N02 C14H19N02
C5H108
H CH3
7
C11H15N02
'ZH5
Iso-C3H7
C10H13N0
C1OH13NO CizHi7N0 Ci4HziNO clqH 1
179 179 207 235 21 9
1.14 1.60 2.50 2.80 2.40
0.0062 0.0039 0.0037 0.0155 0.1486
13.36 12.16 13.36 13.36 14.08
5.101 5.101 6.029 6.957 6.315
0.08 1.oo 1.oo 1.17 0.75
163 163 191 219 203
1.38 1.72 2.38 2.50 2.80
0.0063 0.0110 0.0244 0.0159 0.0313
11.28 10.48 11.28 11.28 12.40
4.948 4.948 5.876 6.803 6.162
0.08 4.00 2.83 0.50 1.oo
149 149 177 205 189
0.90 1.20 1.80 2.50 2.20
0.0015 0.0015 0.1015 0.0116 0.0559
10.64 10.00 10.64 10.64 1 1.88
4.484 4.484 5.412 6.340 5.698
0.58 1.67 4.00 3.00 3.00
163 163 191 219 203
1.32 1.60 2.34 3.00 2.60
0.0013 0.0055 0.0260 0.0151 0.0001
10.08 10.48 10.80 10.48 12.16
4.948 4.948 5.876 6.803 6.1 62
0.67 3.00 5.00 2.67 1.42
183.5 183.5 211.5 239.5 223.5
0.95 1.54 2.10 3.00 2.80
0.0006 0.0076 0.0602 0.7728 0.0281
10.64 10.00 10.64 10.64 1 1.88
4.976 4.976 5.903 6.831 6.190
0.58 5.00 3.00 1.oo 1.oo
193 193 221 249 233
2.40 3.80 2.50 3.00 2.60
0.0003 0.0264 0.0012 0.0144 0.0030
10.64 10.00 10.64 10.64 1 1.88
5.565 5.565 6.493 7.420 6.779
0.08 2.83 3.50 1.08 1.33
163 163 191 21 9 203
1.oo 1.48 2.40 3.10 2.44
0.0058 0.0020 0.1043 0.0014 0.1814
11.76 10.40 11.76 11.76 12.40
4.948 4.948 5.876 6.803 6.162
1.00 2.17 6.00 1.00 2.58
169 169 197 225 209
2.28 2.19 3.12 1.78 3.20
0.0168 0.0136 0.1638 0.2843 0.0315
11.68 10.52 11.68 11.68 12.24
5.042 5.042 5.970 6.898 6.256
0.50 3.00 4.00 2.00 2.00
0
8
CH3
H CH3
ClOH19NO ClOH19NO
C2H5
C2H5
C11H15N0
C2H5
ISO-CH C5Hlia
Iso-C~H~
C14H23N0 C14H23N0
* Represents both R, and R, groups.
cmHg and PT of 2 h. Twenty-five out of 40 compounds were within this generalization (,$ = 4.87 at p < 0.05, df = 1). Solids in general were found to be poor repellents against mosquitoes. Lipophilicity V a l u ee r n e lipophilicities (log P) of the homologous compounds increased progressivelywith the number of carbon atoms in the chain, as expected. Compounds having 1056 I Journal of Pharmaceutical Sciences Vol. 80, No. 1 1, November 1991
moderate log P values, ranging between 1.5 and 2.5, appear to have relatively better repellency (2=: 6.83 at p < 0.05, df = 21, which is in agreement with the literature.' Twenty-fiveout of the 40 compounds examined conform to the above conclusion. Molecular Lengths-Compounds having a ML >10.6 A" exhibit relatively longer protection periods (i.e.,PT > 2 h; ,$
Table ICProtectIon Time Estimation for Varlous Repellent Amldes against Mosqultoes by Use of Equation 5
ne
Repellents pAnisamides pToluamides Benzamides mToluamides eChlorobenzamides eEthoxybenzamides Phenylacetamides Cyclohexylacetamides a
rb
0.996 0.970 0.995 0.948 0.697 0.423 0.870 0.991
FC
36.82 5.233 30.791 2.987 0.315 0.073 1.042 18.30
a
b
C
d
0.647 -4.088 0.625 -0.613 4.125 -0.387 0.824 1.985
00.121 12.222 01.475 3.399 -2.533 0.705 2.308 1.782
-0.505 -2.058 -0.81 1 3.329 -3.883 -0.604 -2.107 -2.182
6.383 56.018 13.005 -24.018 31.155 11.113 29.699 24.846
Number of data points. Multiple correlation coefficient. F test value.
= 5.31 at p < 0.02, df = 1). Only 20 out of the 40 compounds scanned obeyed this generalization. The poor generalizations observed above suggest that no single physical property is solely responsible for repellent activity. This is further substantiated by the observed variations in activity among compounds of similar physical properties, like volatility, log P, or ML. In view of the rather poor generalizations observed above, including that of piperidine anisamide which otherwise satisfies all the optimal criteria such as VP, log P, and ML set above, an attempt to express the PT of all 40 amides in terms of two parameters, namely log P and log VP values using eq 2, resulted in a poor correlation (r = 0.351):
PT = a l o g P + b log VP + d
(2)
where a, b, and d are constants. Similarly, eqs 3 and 4 also failed to describethe protection time of a repellent, as evidenced by low
correlation coefficients (r = 0.404 and 0.547, respectively):
PT = a log P + c ML + d
PT = b log VP + c ML + d
(3) (4)
Further, an effort was made to incorporateall three parameters simultaneously to express the protection time using eq 5:
PT=alogP+blogVP+cML+d
(5)
where a, b, c, and d are constants. The correlation coefficient for all 40 amides improved (r = 0.551)compared with that obtained using eqs 2-4. However, when all the amides derived from the same carboxylic acid were considered, the correlation improved significantly, even in the above equations (i.e., eqs 2-4; r = 0.5-0.98; complete data not presented). An effort was made to correlate the F T s of all the amides derived from same carboxylic acid with all three parameters as per eq 5. This resulted in considerably enhanced correlations (Table 11) for all the amides, except the ortho-substituted derivatives. Failure in the case of ortho-chlorobenzamides and orthoethoxybenzamides to get good correlation by eq 5 may be due to the well-known ortho substituent (steric) effect,14 where Hammett type of equations do not hold. Hansch and coworkers15 opined that molar refraction (MR) and molecular weight (MW) are the approximate measures of the “steric b u l k . The MR values have been used previously in some biological QSAR studies.16 Substitution either of MR or MW for ML in eq 5 resulted in significantly improved correlation in the case of orthochlorobenzamides (eqs 6 and 7):
PT = 1.924 log P + 3.035 log VP - 6.895 MR + 42.932 n = 5, r = 0.989, F = 15.49
(6)
PT = 2.942 log P + 2.476 log VP - 0.234 MW + 48.847 n = 5,r = 0.985, F
=
10.78
(7)
However, in the case of orthoethoxybenzamides, there was no better correlation even by replacing ML with either MR or MW (r < 0.4). This can’t be explained a t present. Studies1.3.7-9 carried out so far on repellents evaluate only individual parameters for correlation with repellency. The present work examines the role of either two or three important factors a t a time (e.g., log P, VP, ML, MR, MW) in the structure-activity correlation of insect repellents. In the case of compounds having non-ortho substituents, the first three parameters showed good correlation for the repellent activity, whereas in ortho-substituted chlorobenzamides, either MR or MW (which are the approximate measures of “steric bulk” according to Hansch et al.15), along with the other two parameters (viz.,log P and VP) could account for the repellent activity.
References and Notes 1. Skinner, W. A.; Johnson, H. L. In Drug Design, Volume 10; Ariens, E. J., Ed.; Academic: New York, 1980;pp 277304. 2. Rao, S.S.;Rao, K. M.; Ramachandran, P. K. J . Sci. Znd. Res. 1988,47,722-735. 3. McGovern, T. P.;Schreck,C. E.; Jackson, J. Mosq.News 1984,44, 11-16. 4. Kumar, S.;Prakash, S.; Sharma, R. K., Jain, S. K.; Kalyanasundaram,M.; Swamy, R. V.; Rao, K. M. Znd. J . Med. Res. 1984, 80,541445. 5. Mathu, K. K.; Gu ta, G. P.; Kaushik, M. P.; Sikdar, A. K.; Rao, K. M. Znd. J. Med)Res. 1986,83,466470. 6. Prakash, S.;Kumar, S.; Su anarayana,M. V. S.;Sharma, R. K.; Rao, K. M. Znt. J . Cosmet.%. 1988,10,23-28. 7. McIver, S.B. J . Med. Entomol. 1981,18,357361. 8. Sugawara, R.; Tominaga, Y.; Suzuki, T. Insect Biochem. 1977,7, 483-485. 9. Wright, R. H. Sci. Am. 1975,233(1), 104-111. 10. Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; John Wiley & Sons: New York, 1979. 11. American Society for Testin and Materials Annual Book of Standards, Part 24; ASTM: I%hiladelphia,PA, 1978;p 740. 12. Braumann, T.J. Chromutogr. 1986,373,191-225. 13. Sharma, R.K.; Jain, S. K.; Kumar, S.; Rao, K. M. Znd. J . Hosp. Pharm. 1984,21,26-29. 14. Hansch, C. In Drug Design, Volume 2;Ariens, E. J.; ed.; Academic: New York, 1971;p 309. 15. Hansch, C.; Leo, A.; Unger, H. S.; Kim, H. K.; Nikaitani, D.; Lien, E. J. J . Med. Chem. 1973,16(11), 1207-1216. 16. Hansch, C.; Coats, E. J . Phurm. Sci.1970,59,731-743.
Acknowledgments We thank Brig K. M. Rao, Director, DRDE, Gwalior, for his active encouragement of this work. We are ateful to Dr. P. K. RamachanRao for their fruitful su ges dran, Emeritus Scientist, and Dr. tions throughout this investi ation. Acknowledgmentsare also dSue to Dr. R. Vi’ayaraghavan,Mr. %.Pandey, and Mr. A. C. Pandey for their help in tie statistical analysis of our results.
S.g
Journal of Pharmaceutical Sciences I 1057 Vol. 80, No. 11, November 1991
