Chem. Res. Toxicol. 1995,8, 545-551
545
Linear Free Energy Relationships for Reactions of Electrophilic Halo- and Pseudohalobenzenes, and Their Application in Prediction of Skin Sensitization Potential for S N AElectrophiles ~ David W. Roberts” Unilever Research Port Sunlight Laboratory, Quarry Road East, Bebington, Wirral, L63 3JW, United Kingdom, and Laboratoire de Dermatochimie associb au CNRS, Universitb Louis Pasteur, Clinique Dermatologique, CHU F-67091, Strasbourg, France Received November 30, 1994@
Published kinetic data and linear free energy relationships for nucleophilic aromatic substitution reactions (S& reactions) are analyzed so as to derive a reactivity parameter (RP), defined as Co-(o,m,p) 0.45a*(ipso), quantifying the relative reactivities of S N A ~ electrophiles toward aniline in ethanol. I t is shown that the dataset of Landsteiner and Jacobs, in which 20 S N Aelectrophiles ~ were classified on the basis of experimental evidence as either skin sensitizing and reactive to aniline or nonsensitizers and unreactive to aniline, can be equivalently classified on the basis of R P values and that predictive criteria can be defined as follows: sensitizing/aniline reactive: R P > 3.80; nonsensitizing/unreactive to aniline: RP < 3.65. These predictive criteria based on calculated R P values a r e applied to seven further SNA~ electrophiles to predict which are sensitizers and which are not. There is close agreement between the predictions and the biological data. This validates the applicability of the reactivity parameter RP, derived from linear free energy relationships, to prediction of skin sensitization potential for S N Aelectrophiles. ~ More generally, the applicability of physical organic chemistry principles to structure-activity relationships in contact allergy is further demonstrated.
+
- NG- 6
Introduction Electrophilic halobenzenes and pseudohalobenzenes form a long known class of contact allergens (skin sensitizers), as exemplified by 2,4-dinitrochlorobenzene (DNCB),l which is well-known to dermatologists and has been widely used as the “standard allergen’’ in studies on the biological mechanisms of allergy. Some of the earliest work on structure-activity relationships in skin sensitization, in particular the pioneering work of Landsteiner and Jacobs in the 1930s ( l ) was , with compounds of this type. Several decades later, DNCB and other 2,4dinitrohalo- and 2,4-dinitropseudohalobenzeneswere the subject of a n ambitious study reported by Godfrey and Baer (2), in which attempts were made to establish quantitative relationships between physicochemical parameters and allergenic potential. Both of the above studies provide datasets which can profitably be analyzed in the light of more recently developed insights into structure-biological activity relationships. In the Landsteiner and Jacobs dataset, the compounds are classified as either sensitizers or nonsensitizers. The Godfrey and Baer dataset covers a narrower range of compounds, but differences in the degree of biological activity are quantified. Because of these differences in the form of the biological data, it is best to consider the two datasets separately. The present paper analyzes the findings of Landsteiner and Jacobs from the perspective of subsequent developments in physical organic chemistry, with the aim of
* Address correspondence to Dr. Roberts a t the Unilever Research Port Sunlight Laboratory. @Abstractpublished in Advance ACS Abstracts, April 15, 1995. Abbreviations: DNCB, 2,4-dinitrochlorobenzene;NBI, nonbonding interaction(s); RP, reactivity parameter.
-x-
Yl,Y2..
Y’ ,Y2..
Y l ,Y*..
Figure 1. Nucleophilic aromatic substitution.
developing predictive criteria, based on calculated rather than experimental parameters, for skin sensitization potential of electrophilic halobenzenes and pseudohalobenzenes.
The Findings of Landsteiner and Jacobs Although the biological mechanism of sensitization was not a t that time understood in any detail, Landsteiner and Jacobs in the 1930s had already come to the view that sensitization to chemicals involved covalent bonding to protein ( I ). The compound 2,4-dinitrochlorobenzene was well-known as a strong skin sensitizer, and it was known that some related halogenated and nitrated benzene derivatives were sensitizers while others were not. Aromatic halogen compounds are typically unreactive, but the presence of strong electron attracting groups can active the halogen so as to make it easily substituted via an addition-elimination pathway as shown in Figure 1. Nitro groups can also act as pseudohalogens in this reaction. To test the hypothesis that chemical reactivity is necessary for skin sensitization, Landsteiner and Jacobs compared the results of guinea pig sensitization tests on a series of halogenated and nitrated benzenes with the results of experiments in which reactions with aniline, chosen as a model for protein, were attempted (I). The
0893-228x/95/2708-0545$09.00/00 1995 American Chemical Society
546 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
Roberts
Reactive to aniline, positive in sensitizatlon tests
NO2
X 2, X = NO2; 3, X = CI
l F , lCI, l B r , 11, lNO2
NO2
NO2
4
5
NO2 6
Unreactive to anillne, negative in sensitization tests
6"' CI
X I
Q NO2
NO2
CI
QNo2 CI
A A &
7 , X = NO2; 8, X = CI
CI
NO2
9
O2N
11
6'l CI
I
10
NO2
NO5
12
13
CI
Cl
c,&cl
CI
CI
CI
CI
14
15
16
Figure 2. Landsteiner and Jacobs dataset.
aniline experiments were carried out by heating the test compound with aniline in absolute alcohol as solvent, in sealed tubes in a steam bath for 2 h. If less than 5% of the test compound reacted under these conditions, the compound was classed as unreactive, and if more than 90% reacted it was classed as reactive. Their findings are summarized in Figure 2. For the reactive compounds the position in the ring a t which the substitution reaction takes place is indicated by a n arrow. As can clearly be seen from Figure 2, Landsteiner and Jacobs found a complete correspondence between ability to react with aniline and ability to sensitize. At the time when Landsteiner and Jacobs published these results, the use of linear free energy relationships for reactivity correlations had not yet been developed. The aim of the present work is to determine whether the reactivelsensitizing compounds are distinguishable from the nonreactivelnonsensitizing compounds on the basis of the Hammett and Taft constants of the substituents, and if so whether predictive application is possible.
Linear Free Energy Relationships for Electrophilic Halo- and Psuedohalobenzenes Referring to the reaction mechanism shown in Figure 1, it can be seen that the displacement of halogen or
pseudohalogen, if it occurs, proceeds in two steps: the
addition of the nucleophile to produce an intermediate species in which a negative charge is delocalized in the ring, and the elimination of the leaving group from this intermediate. In reactions of this type (SN&reactions), it is usually the first step which is rate-determining, particularly when the substituent corresponding to the incoming nucleophile is, or gives rise to by deprotonation, a poorer leaving group than the original substituent ( 3 ) . In the case of aniline, which is a weak base, the first step is likely to be rate-determining. It seems reasonable to assume, given the complete correspondence between ability to sensitize and ability to react with aniline, that the rate-determining step is the same for the biological nucleophiles involved in sensitization as it is for aniline. Referring to Figure 1 the effect of a Y substituent on reactivity will depend on its ability to stabilize the developing negative charge in the intermediate (or, more precisely, in the transition state leading to it). This can be modeled by the Hammett u- constant, and therefore the combined effect of all the Y substituents may be modeled by Xu-(o,m,p). In studies where the meta and para Y substituents are varied, Hammett relationships based on u- values have been demonstrated, with Q values ranging from 3.5 to 5 (4-6). To apply the same approach to the Landsteiner and Jacobs set of compounds, it is necessary to model the effects of ortho substituents on reactivity. Although u- values for ortho substituents are available from the compilation by Perrin et al. (7), they are not applicable in all situations, particularly when, as in the case of a nitro group, the greater activating electronic effect in the ortho position can be opposed by adverse nonbonding interactions (NBI) between occupied orbitals of the ortho substituent and of the attacking nucleophile. For present purposes it seems reasonable to use the tabulated u- value for the o-chloro substituent, but it is necessary to consider further how to deal with the o-nitro substituent. The u- value given by Perrin et al. (7) for the o-nitro group is 1.72, as compared with 1.26 for thep-nitro group. Thus in the absence of NBI effects, the o-nitro group should be substantially more activating than the p-nitro group. The relative activating effects of 0- and p-nitro groups toward various nucleophiles have been discussed in some detail by Bunnett and Morath (8). For reactions with anionic nucleophiles, the ortho/para ratio [i.e., Monitro)/k(p-nitro)] is usually < l, indicating that the o-nitro group is less activating than thep-nitro group. However for amine nucleophiles the ratio is usually found to be '1, and this can be attributed to a combination of reduced NBI effects due to the nucleophile being uncharged and a stabilizing effect of intramolecular hydrogen bonding involving a hydrogen atom attached to nitrogen in the attacking nucleophile and an oxygen atom of the o-nitro group. This effect, it was argued, outweighs the NBI effect. Consistent with this rationalization, the magnitude of the ortho/para ratio, as determined from rate constants for reactions of ortho and para chlorobenzenes with piperidine, was found to be negatively correlated with solvent hydrogen bonding ability, having a value of 80 for xylene as solvent, 46 for benzene as solvent, 2.9 for 99.8% ethanol as solvent, and 1.4 for water (with 1% dioxane) as solvent (8). With aniline as nucleophile in S N A reactions ~ the ortholpara ratio for nitro group activation is somewhat larger than it is with piperidine as nucleophile-by a factor of 713 (ref 9, p 304), based on
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 547
Skin Sensitization-SNAr Electrophiles
data for reactions of 3- and 5-nitro-2-chloropyridines (10, 11). This can be attributed to a further reduction in the NBI effect as a consequence of a n earlier transition state when aniline, which is more polarizable than piperidine, is the nucleophile. Thus, for modeling the chemical experiments carried out by Landsteiner and Jacobs using aniline in absolute alcohol, it seems appropriate to multiply the ortholpara activation ratio of 2.9 for piperidine with nitrochlorobenzenes in 99.8% ethanol by the anilinelpiperidine 713 ratio, giving a calculated ortholpara ratio for aniline of 6.8. A a- value for the o-nitro group in reactions with aniline in absolute alcohol can now be calculated as follows: K(o-NO2) = a-(p-N02)
+ (log 6.8)/~
Table 1. Kinetic Data (12,131for Reactions of l-X-Substituted-2,4-dinitrobenzenes krel (to X = I) for reaction with: u* MeOpiperidine PhSX NOn OSOzC&Me-p SOzCsHs SOCsH5
F c1 OCsH4NOz-p Br
I OCsH51 SCsH4NO2-p
4.25 3.62a 3.25 3.24 3.21 2.96 2.91 2.84 2.46 2.43 2.33
2590
3100 5.2 13.3 3.4 1 0.88 0.45
1345
890 100 3.2 4.7 3300 4.3 3.0 4.3 1
27 0.7 0.3 1.5 1 0.01 0.28
" T h e u* value for SO&& is used here, no value for OSOzCsH4Me-p being given in ref 7.
As mentioned above, e values for S N Areactions ~ range from 3.5 to 5. Values toward the lower end of the range apply for alkoxide ion as nucleophile (4, 5). For piperiLogk dine (the most similar nucleophile to aniline for which F NO2 data appear to be available) a e value of 4.95 is reported (6). Using this value in the above equation leads to a up=1.7 value of 1.43 for the o-nitro group. It is possible that an even higher value may be more suitable for modeling reactivity in vivo. The chemical reaction leading ulti- mately to sensitization probably takes place in a cell membrane, whose solvent effects may be better modeled by xylene than by ethanol. On the other hand, membranebound protein may make the i n vivo medium more protic. Carrying out the above calculation, but this time starting 2 3 4 o* from the piperidine ortholpara ratio figure of 80, correFigure 3. Linear free energy relationship for reactions of sponding to reaction in xylene, leads to a a-value of 1.72 substituted 2,4-dinitrobenzenes with piperidine. (identical with the figure given by Perrin et al. (7)) for the o-nitro group. In view of the absence of information as to the solvation Logk properties of the in vivo reaction medium, the u-(o-NOz) value of 1.43, relevant to reactivity toward aniline in ethanol, is used from this point onward. The conclusions are not significantly affected when the analysis is repeated using the a-(o-NOz) value of 1.72 relevant to reaction in xylene. The dependence of reactivity on the nature of the ipso group, i.e., X in Figure 1, is somewhat complex. In general, since the departure of X occurs subsequently to the rate-determining step, reactivity in SNATreactions is less dependent on the leaving group than in S N ~ , , reactions (3). However, significant variations of reactiv5 2 3 u* ity are observed as X is varied and are usually attributed Figure 4. Linear free energy relationship for substituted 2,4to a combination of electronic effects (stabilization of the dinitrobenzenes with methoxide ion. developing negative charge in the 2-like transition state) and steric effects (non-bonding interactions between X with methoxide ion (121, piperidine (131, and thiopheand the incoming nucleophile Nu). The relative impornoxide ion (12). These data are shown in Table 1. With tance of these two effects depends on the nature of the the relatively hard nucleophiles methoxide ion and nucleophile. piperidine, but not with the soft nucleophile thiophenoxWith hard nucleophiles, of low polarizability, such as ide ion, 2,4-dinitrofluorobenzene is the most reactive of methoxide ion, both effects appear to be important, and the electrophiles studied. Taft a* constants may be among the commonly studied X groups the fluoro group appropriate to model the electronic effects of the X groups stands out as being associated with particularly high in the transition state, and these are also shown in Table reactivity, since because of its small size non-bonding 1. interactions are minimized. With softer nucleophiles Figures 3, 4, and 5 show plots of log Krel against 8 for (more highly polarizable), a larger degree of new bond each of the three nucleophiles. When the 2,kdinitroformation can occur a t a greater distance, so that in the fluorobenzene data are omitted, reasonably linear relatransition state leading to 2 non-bonding interactions tionships are found for methoxide and piperidine as between Nu and X are less significant and electronic nucleophiles. For thiophenoxide as nucleophile the linear effects predominate in determining reactivity as X is relationship obtained includes 2,4-dinitrofluorobenzene. varied. These points are illustrated by kinetic data The e values of the three plots are very similar: 1.7 for published for reactions of substituted 2,4-dinitrobenzenes
I1
/
/
/
548 Chem. Res. Toxicol., Vol. 8, No. 4, 1995
Roberts a*(ipso)
Logk
Negative
1
" I
2
3
4
a*
5
Figure 5. Linear free energy relationship for substituted 2,4dinitrobenzenes with thiophenoxide ion.
Figure 6. Discriminant analysis plot.
piperidine and 1.9 for the other two nucleophiles, giving a n average value of 1.8.
halogen and NO2 groups, there is the possibility of either a halogen or a n NO2 group being displaced. The Ca-(o,m,p) parameter alone is not sufficient to distinguish between these possibilities. Thus some of the compounds containing both NO2 and C1 groups react a t a n NO2 group position, even though the Ca-(o,m,p) parameter is larger for reaction a t a C1 group position, indicating that the larger a*(i)value of the NO2 group outweighs the smaller Za-(o,m,p) value for reaction a t this position. Estimates of the relative importance of a*(i) and Ca-(o,m,p) can be obtained as follows: A reactivity parameter (RP) is defined as:
Discriminant Analysis On the basis of the foregoing, it seems reasonable to look for a combination of a*(ipso) and Ca-(o,m,p) to discriminate between the sensitizinglaniline-reactive compounds and the nonsensitizinglaniline-unreactive compounds. Table 2 lists 8 ( i )and Za-(o,m,p) values for all the compound studied. The Za-(o,m,p) values are calculated from the individual substituent constants given by Perrin et al. (7), except that the value of 1.43, a s derived above, is used for the o-nitro substituent. In some cases different values are given by Perrin et al. for a substituent, depending whether it is applicable to phenol ionization or to aniline protonation. For the substituents considered here these differences are insignificant, and by arbitrary choice the values corresponding to the aniline system are used. Figure 6 shows a plot of a*(i)against Ca-(o,m,p) for the compounds studied by Landsteiner and Jacobs. It can be seen that the compounds which sensitize and react with aniline are grouped in a separate region of the plot from the compounds which do not sensitize and which do not react with aniline. It seems clear that the discrimination between reactivelsensitizing and unreactivelnonsensitizing is based more on the Zo-(o,m,p) parameter than on the u*(i)parameter, and indeed for this set of compounds the two classes could be distinguished on the basis of Za-(o,m,p) alone. However, the need for o*(i) becomes apparent when the mode of reaction is considered. For compounds containing both
A theoretical value for a can be derived simply by dividing the e value for reactions in which the ipso group is varied by the e value for reactions in which the meta and para substituents are varied. As discussed above, although experimental e values are not available for aniline as nucleophile, it seems reasonable to assign 5 as the former e value and 1.8 as the latter. Thus a value of 0.45 is obtained for a. A check on the applicability of the RP based on a value of 0.45 for a can be made by deriving an empirical a value range from the experimental findings of Landsteiner and Jacobs, as follows: Compound 5, l-chloro-2,4,5-trinitrobenzene, reacts with aniline in absolute alcohol by displacement of the 5-nitro group rather than by displacement of the 1-chlorogroup. Therefore, the RP value for 5-nitro group displacement must be greater than that
Table 2. Parameters for Discriminant Analysis compd
L-(o,m,p)
lNOz
2.69 2.69 2.69 2.69 2.69
7 8 9 10 11
1.26 1.26 1.93 1.67 1.11
5 (displacement of C1) 6 (displacement of C1)
3.43 2.54
1F 1c1 1Br 11
a*(ipso) compd Reactive/Sensitizing 3.21 2 2.96 3 2.84 4 2.46 5 4.25 6 UnreactiveNonsensitizing 4.25 12 2.96 13 2.96 14 2.96 15 2.96 16 For Nonpreferred Reactions 2.96 2.96
I.o-(o,m,p)
a*(ipso)
4.12 3.10 3.06 3.06 2.60
2.96 2.96 2.96 4.25 4.25
1.48 0.74 1.04 1.28 2.32
4.25 4.25 2.96 2.96 2.96
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 549
Skin Sensitization-SNAr Electrophiles Table 3. Ranking Based on RP Values compd
2 5 4 1NOz
6 16 12 9 7 10
RP
compd
RP
Reactive and Sensitizing 5.45 3 4.97 1F 1c1 4.97 1Br 4.60 4.51 1L Unreactive and Nonsensitizing 3.65 13 3.39 15 3.26 8 3.17 11 3.00 14
4.43 4.13 4.02 3.97 3.80 2.65 2.61 2.59 2.44 2.37
Table 4. Predictive Criteria Based on RP
RP = x o - ( o , m , p ) + 0.45o*(ipso) mediction
criterion
positive range of uncertainty negative
RP > 3.80 3.65 < RP < 3.80 RP < 3.65
for 1-chloro group displacement, i.e.:
3.06
+ 4 . 2 5 ~> 3.43 + 2 . 9 6 ~ a
>
0.29
Compound 11 is reactivelsensitizing whereas 12 is unreactivelnonsensitizing. Therefore, the RP value for 11 must be larger than that for 12,i.e.:
2.69
+ 2 . 4 6 ~> 1.48 + 4 . 2 5 ~ a < 0.68
Thus the value of a is between 0.29 and 0.68; i.e., any a value in this range would enable aniline-reactive1 sensitizing and aniline-unreactivehonsensitizingcompounds to be distinguished from one another on the basis of their RP values. This supports the validity of the theoretical a value of 0.45, calculated as a ratio of e values, which falls within this range. On that basis a reactivity parameter RP defined as:
seems appropriate for predicting whether or not a given S N Aelectrophile ~ is expected to be reactive to aniline and to sensitize. In Table 3 the compounds are ranked in decreasing order of their RP values calculated as above. Compound 11 is the sensitizingheactive compound with the lowest RP value and 16 is the nonsensitizing/ unreactive compound with the highest RP value. On the basis of the RP values of 11 and 16,the predictive criteria shown in Table 4 can be defined. These predictive criteria are applicable to aniline reactivity as defined by Landsteiner and Jacobs and to the outcome of sensitization testing according to the method used by them. However, bearing in mind that this test method was designed to be more generally applicable, if the present analysis is to have other than purely theoretical value, the predictive criteria should be applicable to data from other test methods. Before proceeding to test the applicability of the discriminant analysis against data on further compounds, it is necessary to consider how RP values should be
Table 5. Estimation of ~ 7 p - Nfor ) Nitrogen Heteroaromatics log krel (nucleophile)
electrophiles
ref
4-chloropyrimidine 7.17 9, p 318 2-chloropyridine (piperidine) 4-chloropyridine -1.0 15, p 175 4-chloronitrobenzene (methoxide) 4-chloro-3-nitropyridine0.46 15, pp 276,267 2,4-dinitrochlorobenzene(pyridine)
a-(p-N) 1.45 1.01
1.35
calculated for S N Aelectrophilic ~ nitrogen heteroaromatic compounds.
Hammett Constants for Nitrogen Heteroaromatics For nitrogen heteroaromatic compounds, it is necessary to consider the question of finding appropriate a- values to model the effects of the ring nitrogens on S N A ~ reactivity. The assignment of Hammett constants to heteroatoms in heteroaromatic rings has been discussed by Jaffe and Jones (241, who conclude that it is not possible to specify generally applicable Hammett constants. For present purposes, some idea of appropriate u- values to use may be obtained by consideration of published relative rate data, in particular, as summarized by Shepherd and Fedrick (25)and by Illuminati ( 9 ) . Relative rate data on reactions of pairs of S N A ~ electrophiles with piperidine, pyridine, or aniline in ethanol (e = 4.95) and with methoxide ion in methanol (e = 3.97) can be used to calculate values for a-(N) as follows:
a-(N) = (log kr& o-(N) = o-(NO,)
(when N replaces CH)
+ (log krel)/e (when N replaces CNO,)
The e values used for these calculations are 4.95 (6) for piperidine, pyridine, and aniline and 3.97 ( 5 )for methoxide ion as nucleophile. Table 5 summarizes the calculations for u-(p-N). The values derived from the piperidine and pyridine data seem the more relevant for present purposes and suggest, in agreement with earlier qualitative generalizations (see ref 15, p 2831, that nitrogen in the para position is somewhat more activating than p-NOz. That the converse appears to be true for methoxide as nucleophile may possibly be attributable to solvation effects. With a hard negatively charged nucleophile, the transition state will be later and the partial charge a t the para position will be greater. This charge can be stabilized by solvation to a greater extent when the para substituent is nitro. Overall, for present purposes it seems reasonable to assume that the relevant o-(p-N) value is in the range 1.35-1.45. Table 6 summarizes the calculations for a-(o-N). The trends in the ortholpara activating effect of o-nitrogen appear to follow a similar pattern to those for o-nitro. Thus the calculated u-(o-N) value for methoxide as nucleophile is lower than that for piperidine as nucleophile, which in turn is lower than that for aniline as the nucleophile. This can be attributed to adverse NBI effects between the lone pair on the o-nitrogen and the attacking nucleophile being greatest when the nucleophile is charged and least when the nucleophile is most
Roberts
550 Chem. Res. Toxicol., Vol. 8, No. 4, 1995 Table 6. Estimation of a-(o-N) for Nitrogen Heteroaromatics ref
2-chloro-5-nitropyridine 4.11 (piperidine) 4-chloronitrobenzene 2.26 4-chloronitrobenzene 2-chloropyridine (methoxide) 2-chloro-3-nitropyridine3.52 (piperidine) 2-chloronitrobenzene 2-chloro-5-nitropyridine -1.48 2,4-dinitrochlorobenzene(aniline) 2-chloropyrimidine -0.80 2-chloro-3-nitropyridine (piperidine) 2-chloro-5-nitropyridine -0.75 2,4-dinitrochlorobenzene(pyridine)
u-(o-N)
9, p 318
0.83
15, p 175
0.61
15, p 175
0.71
CI clQ(x
C H,OMe
CI 16
17
CI Cl
X = CI. 18 X
COzH, 19
15, pp 276, 267 1.13* 15, p 281
1.19”
15, p 282
1.04d
a krel for each pair = Mfirst compound named)/k(second compound named). b Calculated as o-(o-NOz,aniline) - (log K,i)/Q = 1.43-0.30. Calculated as o-(o-NOz,piperidine) - (log krel)/~= 1.35-0.16; value of 1.35 for u-(o-NOz,piperidine) obtained from ortho/para activation ratio of 713 (ref 9, p 304) and @(piperidine) value of 4.95 (61, using u-(p-NOz) value of 1.26. Calculated as o-(o-NOZ,pyridine)- (logk,,1)/@ = 1.19-0.15; value of 1.19 for d o NO2,pyridine) calculated from log krel value of -0.28 for 2-chloro3-nitropyridine vs 2-chloro-5-nitropyridine(ref 15, p 282).
polarizable, together with activating intramolecular hydrogen-bonding effects when the nucleophile is uncharged. The overall degree of variation in the a-(o-N) values is such that it does not seem appropriate to take a n average value, but rather to accept a wider range of uncertainty by working on the basis that the appropriate o-(o-N) value is no larger than 1.19 and no lower than 0.71. It would appear from the d N ) values calculated above that nitrogen para to the leaving group is more activating than nitrogen ortho to the leaving group irrespective of the nucleophile. This accords with the generally held view that 4-substituents are more reactive than the corresponding 2-substituents in pyrimidines (13). However, it has been shown (16)that the reverse applies for reactions of chlorine-substituted pyrimidines in isooctane (which, as a hydrophobic nonprotic solvent may possibly be a good model for the in vivo reaction medium) with piperidine as the nucleophile. This situation is analogous to the effect of the solvent on ortholpara activation ratios for nitro groups (8) and is likewise attributable to the intramolecular hydrogen-bonding effect being enhanced in magnitude when the opportunity for hydrogen bonding with the solvent is removed.
Predictive Applicability The predictive applicability of the discriminant analysis can now be tested against data, published subsequently to the paper of Landsteiner and Jacobs, on the related compounds 16-22, whose structures are shown in Figure 7. Compound 16, tetrachloroisophthalonitrile, has a &-(o,n,p) value of 3.62 and a u*(ipso) value of 2.96, giving an RP value of 4.95. This places it in the predicted reactivelsensitizing category. More than 40 years after Landsteiner and Jacobs’s paper appeared, 16 was in fact reported as a n occupational allergen, causing contact dermatitis in workers using it as a wood preservative (17).
For compound 17,it is necessary to adjust the u- value for the nitro group to take into account steric inhibition of resonance. From a comparison of the rate constants
c’xxx CI
CI
X = OPO(OMe)2, 20
22
X = OCH2C02CH2CH20C4Hg, 21
Figure 7. Further S N A electrophiles. ~
tabulated by Illuminati (ref 9, p 360) for reactions of aniline (e taken as 4.95) with the related compounds 2-chloro-3-cyano-4,6-dimethyl-5-nitropyridine (log k = -4.38 a t 30 “C) and 2-chloro-3-cyano-5-nitropyridine (log k = -2.41 a t 10 “C; E, = 9.8 kcabmol-I) the u- value for the sterically inhibited nitro group is calculated to be 0.76. Using this value, the RP for compound 17 is found to be in the range 3.96-4.44, depending on the value assigned to d 0 - N ) . Thus, compound 17 is assigned to the predicted reactivelsensitizing category. Consistent with this analysis, compound 17 is listed in a textbook of occupational allergy (18)as causing skin sensitization in workers in the pharmaceutical industry using it as a synthesis intermediate. The RP calculated for compound 18 is in the range 4.76-4.86, on which basis the prediction is clearly positive. For compound 19 (presumed to be ionized a t physiological pH) the calculated RP is 4.48-4.58, again corresponding to a clearly positive prediction. Both compounds 18 and 19 were reported in 1981 as sensitizers in guinea pig tests (19). Compound 20 has an OPO(0Me)Z group meta to the leaving group. The d m e t a ) value for this group position is not available, but is not expected to be large on the basis of the listed d p a r a ) value of 0.04 for the same group. A value of 0.10 is arbitrarily chosen as the uvalue for this substituent. The true value is unlikely to be significantly larger. Similarly, no d m e t a ) value is available for the OCH&O&HzCHzOC4Hg group in compound 21,and a value of 0.20 is arbitrarily assigned (cf. 0.11 for m-OCH3 and 0.39 for m-COCH3). The true value is unlikely to be significantly larger. Thus, the RP values are estimated as 2.99-3.47 for compound 20 and 3.093.57 for compound 21. These values correspond to clearly negative predictions. Guinea pig sensitization tests on these compounds were reported in 1981 (19). The test method was a modified version of that used by Landsteiner and Jacobs, designed to be better a t detecting sensitization potential in weak sensitizers. Compound 20 was negative, and compound 21 was marginal (one animal out of 9 giving a positive response). Compound 22, 2,4-dichloropyrimidine, is listed by Foussereau and Benezra, in their textbook on occupational allergy (18).They quote Graul as proposing that 22,used as a n intermediate in the synthesis of cytosine,
Chem. Res. Toxicol., Vol. 8, No. 4, 1995 551
Skin Sensitization-SNAr Electrophiles
References
Table 7. Predictions Based on RP Values compd
RP
predicted
lit. (37-19)
16 17 18
4.95 3.96-4.44 4.76-4.86 4.48-4.58 2.99-3.47 3.09-3.57 3.76-4.34
sensitizer sensitizer sensitizer sensitizer nonsensitizer nonsensitizer probable sensitizer
sensitizer sensitizer sensitizer sensitizer nonsensitizer marginal (in more sensitive test) proposed sensitizer
19
20 21 22
is the allergen responsible for sensitizing workers in the pharmaceutical industry manufacturing adenosine. Unfortunately, due to a misprint in the list of references, it has not so far been possible to trace the original paper so as to ascertain what, if any, experimental evidence exists regarding the sensitization potential of this compound. Its RP value is calculated to be in the range 3.76-4.34, which spans the borderline between the definite positive criterion and the range of uncertainty for reactivity and sensitization. However, the lower limit of the RP range (3.76) is only marginally below the value required (3.80) for a positive prediction, and on balance therefore 22 would be predicted as more likely than not to be a sensitizer. Table 7 summarizes the predictions and the findings for compounds 16-22. Agreement is good in all cases where experimental data are available. This validates the applicability of the reactivity parameter RP, derived from linear free energy relationships, to prediction of skin sensitization potential for S N A ~ electrophiles. More generally, the applicability of physical organic chemistry principles to structure- activity relationships in contact allergy is further demonstrated. I t may be noted that although hydrophobicity is well recognized as a n important parameter in quantitative structure-allergenicity correlations (ZO),it has not been found necessary to use a hydrophobicity parameter in the present study. This should not be taken to indicate that hydrophobicity is unimportant in sensitization by S N A ~ electrophiles. Over the set of compounds considered in the present study the variation in electrophilic reactivity is much larger than the variation in hydrophobicity. As a result, it is the reactivity parameter which dominates in discriminating between sensitizing and nonsensitizing compounds. In a separate paper an analysis will be presented of the Godfrey and Baer data ( 2 )for a different set of S N Aelectrophiles, ~ in which differences in biological activity are quantified and a structure-allergenicity relationship based on hydrophobicity is derived. In the present study electrophilic reactivity has been modeled by a combination of Hammett and Taft substituent constants. Currently there is considerable research activity aimed a t developing quantum mechanical indices suitable for modeling electrophilicity (211. The compounds considered here could provide a useful dataset for evaluation of such indices.
(1) Landsteiner, K.,and Jacobs, J. (1936) Studies on the sensitisation of animals with simple chemical compounds. 11. J . Ezp. Med. 64, 625-639. (2) Godfrey, H.P., and Baer, H. (1971) The effect of physical and chemical properties of the sensitising substance on the induction and elicitation of delayed contact sensitivity. J . Immunol. 106, 431-441. (3) Bunnett, J . F. (1958) Mechanism and reactivity in aromatic necleophilic substitution reactions. Quart. Rev. 12, 1-16. (4) Miller, J . (1956) Electrophilic and nucleophilic substitution in the benzene ring and the Hammett equation. Aust. J . Chem. 9,6173. (5) Bunnett, J. F., Moe, H., and Knutson, D. (1954)Activation of the nucleophilic displacement of chlorine from 4-substituted-2-nitrochlorobenzenes and 4-substituted-2,6-dinitrochlorobenzenes by methoxide ion. J . Am. Chem. SOC. 76, 3936-3939. (6) Berliner, E., and Monack, L. C. (1952) Nucleophilic displacement in the benzene series. J . Am. Chem. SOC. 74, 1574-1579. (7) Perrin, D. D.,Dempsey, B., and Serjeant, E. P. (1981) pK, Prediction for Organic Acids and Bases, Chapman and Hall, London. (8) Bunnett, J. F., and Morath, R. J . (1955) The ortho:para ratio in activation of aromatic nucleophilic substitution by the nitro group. J . Am. Chem. SOC.77, 5051-5055. (9) Illuminati, G. (1964) Nucleophilic heteroaromatic substitution. Adv. Heterocycl. Chem. 3,285-371. (10)Bishop, R. R., Cavell, E. A. S., and Chapman, N. B. (1952) Nucleophilic displacement reactions in aromatic systems. Part I. Kinetics of the reactions of chloronitropyridines with aromatic amines and with pyridine. J . Chem. SOC., 437-446. (11) Chapman, N. B., and Rees, C. W. (1954) Nucleophilic displacement reactions in aromatic systems. Part 111. Kinetics of the reactions of chloronitropyridines and chloropyrimidines with piperidine, morpholine, piperidine and aniline. J. Chem. SOC., 1190-1196. (12) Bartoli, G., and Todesco, P. E. (1977) Nucleophilic substitution. Linear free energy relationships between reactivity and physical properties of leaving groups and substrates. ACC.Chem. Res. 10, 125-132. (13) Bunnett, J . F., Garbisch, E. W., Jr., and Pruitt, K. M. (1957) The “element effect” as a criterion of mechanism in activated aromatic nucleophilic substitution reactions. J . Am. Chem. SOC.79,385391. (14) Jaffe, H. H., and Jones, H. L. (1964) Applications of the Hammett equation to heterocyclic compounds. Adu. Heterocycl. Chem. 3, 209-261. (15) Shepherd, R. G., and Fedrick, J. L. (1965) Reactivity of azines with nucleophiles. Adu. Heterocyclic Chem. 4, 145-423. (16) Mamaev, V. P., Zagulyaeva, 0. A., and Krivopalov, V. P. (1970) Relative reactivities of 2- and 4-chloropyrimidines. Dokl. Akad. Nauk S.S.S.R. 193, 600-601. (17) Johnsson, M.,Buhagen, M., Leira, H. L., and Solvang, S. (1983) Fungicide-induced contact dermatitis. Contact Dermatitis 9,285288. (18) Foussereau, J., and Benezra, C. (1970) Les kczemas allergiques professionels, Masson, Paris. (19) Rao, S. A.,Betso, J . E., and Olsen, K. 3. (1981) A collection of guinea pig sensitisation test results-grouped by chemical class. Drug Chem. Toxicol. 4,331-351. (20) Roberts, D. W., and Williams, D. L. (1982) The derivation of quantitative correlations between skin sensitisation and physicochemical parameters for alkylating agents and their application to experimental data for sultones. J . Theor. Biol. 99, 807-825. (21) Mekenyan, 0.G., and Veith, G. D. (1994) The electronic factor in QSAR: MO-parameters, competing interactions, reactivity and toxicity. SAR QSAR Enuiron. Res. 2, 129-143.
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