Chem. Res. Toxicol. 1991,4,41-49
41
Species Speciflcity in the Chemical Mechanisms of Organophosphorus Anticholinesterase Activityt Kendall B. Wallace* and Jon Robin Kemp Department of Pharmacology, School of Medicine, University of Minnesota, Duluth, Minnesota 55812 Received July 26, 1990
Structure-activity relationships reveal that the two principal determinants of anticholinesterase activity for various organophosphorus insecticides are steric hindrance and the electrophilic strength of the phosphorus atom. The objective of the present investigation was to distinguish between the molecular properties governing species-related differences in organophosphorus sensitivity by comparing the physical-chemical relationships for the inhibition of brain acetylcholinesterase isolated from rats, chickens, or rainbow trout. A homologous series of five dialkyl p-nitrophenyl phosphates consisting of methyl through n-butyl and isopropyl were synthesized and characterized both chemically and biologically. Structure-activity correlations revealed that whereas steric hindrance is the principal factor governing inhibitory potency for rats and hens, the electrophilicity of the phosphorus atom is the principal determinant of anticholinesterase activity in trout. The inductive effect of successive methylene substitutions on the phosphoryl group is reflected by 13C and 31PNMR spectroscopy which correlates with anticholinesterase activity in trout, but not in rats or hens. The results provide the first indication for species-related differences in the molecular regulation of anticholinesterase activity, suggesting that the trout enzyme possesses a relatively weak nucleophilic center within a dimensionally restricted esteratic subsite. Species-specific distinctions in the molecular properties governing anticholinesterase activity provide novel design strategies for improving the selectivity of toxic organophosphorus insecticides.
Introduction The organophosphorus compounds represent a broad class of insecticides used widely for the eradication of assorted household and agricultural pests. Selected organophosphorus agents are also approved for the clinical treatment of head lice in humans. Unfortunately, the effective employment of these compounds is confounded by the frequent intoxication of beneficial nontarget organisms, including numerous documented cases of human fatalities. Contributing to this selectivity is the broad range of sensitivity of different organisms to acute poisoning by organophosphorus compounds, with fish being relatively resistant and rodents and birds very sensitive. Despite large differences in the various pharmacokinetic steps involved in the disposition and metabolism of organophosphorus agents, the idiosyncrasies among species in sensitivity to organophosphorus toxicity is mediated, for the most part, by the sensitivity of acetylcholinesterase (AChE)' to inhibition by the active oxygen analogue of the corresponding phosphorus triester ( 11. Such differences in the sensitivity of AChE from different species to in vitro inhibition by organophosphorus agents have been documented in numerous comparative studies (2-9). Correlations between the in vitro inhibition of AChE and the acute LD50 measured in vivo provide further evidence confirming the central role of the sensitivity of AChE as the principal determinant of acute toxicity of organophosphorus insecticides in various organisms (10-14). Although the research described in this article was funded, in part, by the US.Environmental Protection Agency (CR810963),the manuscript was not subjected to Agency review; therefore, this article does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. * Author to whom correspondence should be addressed.
The chemistry of organophosphorus-mediatedinhibition of AChE is well established and provides a framework for investigating species differences in the molecular properties associated with enzyme inactivation. The inhibitors associate with AChE via Coulombic forces between the electron-deficient phosphorus atom and a nucleophilic center within the esteratic subsite of the enzyme. The formation of this reversible complex, as reflected by the dissociation constant (&), is limited by steric hindrance possibly reflecting the finite dimensions of the esteratic subsite of AChE (5,9-11,15-17). Once formed, the complex undergoes rapid nucleophilic substitution wherein the enzyme becomes irreversibly phosphorylated at the nucleophilic center, releasing the leaving group of the respective inhibitor. The centrality of the electronic properties of the phosphorus atom in distinguishing among inhibitor potency for a diverse array of structurally dissimilar organophosphorus agents is well documented (IO, 12, 14, 16, 18). The heterogeneity of AChE from different sources likely reflects differences in both the physical properties and allosteric regulation of the enzyme. Regardless, differences in sensitivity to inhibition by organophosphorusagents are a manifestation of differing kinetics involved in the interaction of the inhibitor with the active site of AChE, possibly reflecting distinct molecular features of the enzymes from different species. Structure-activity correlations have been employed to establish the principal physical-chemical properties responsible for discriminating between different organophosphorus inhibitors (5,lO-12, 14-19). However, since the association of organophosphorus compounds with AChE is dependent not only Abbreviations: AChE, acetylcholinesterase; LD50,median lethal dose.
1991 American Chemical Society
42 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
on the electrophilicity of the phosphorus atom of the inhibitor but also on the nucleophilic properties of the active site, species-related differences in the electronic properties of the esteratic subsite of AChE may account for the differences in the sensitivity of the enzyme to in vitro inhibition. These differences in the nucleophilic strength may also affect the rate of phosphorylation, and thus irreversible inactivation, of AChE from different species. Similarly, steric hindrance as it affects the association of organophosphorus agents with AChE is dependent on both the molecular volume of the inhibitor and the ability of the enzyme to accommodate the inhibitor as determined by the finite dimensions of the esteratic subsite of the enzyme. Consequently, it has been suggested that the resistance of selected strains of mites and certain species of fish is conferred by the relatively smaller dimensions, and thus greater steric exclusion, of the esteratic subsite of AChE from the resistant species (9, 20). The objective of the present investigation was to gain further insight into the hypothesis that differences in the sensitivity of AChE from different species to in vitro inhibition by organophosphorus agents are a manifestation of distinct differences in the molecular properties of the enzymes. This was accomplished by comparing and contrasting structure-activity correlations between various physical-chemical properties of a homologous series of five dialkyl-substituted p-nitrophenyl phosphates and the kinetic constants describing the association and phosphorylation of AChE isolated from brain tissue of rats, chickens, and rainbow trout.
Materials and Methods Caution. Extreme precautions must be observed during the synthesis and subsequent handling of these highly toxic anticholinesterase agents. The use of proper laboratory attire, including vinyl gloves, are a necessity a t all times to prevent accidental exposure. Periodic blood samples should be withdrawn from all personnel working with large quantities of these agents-plasma ChE levels should be measured to monitor for potential exposure. Occasional blood samples should be submitted t o an independent medical laboratory for further confirmation of potential exposure. Ethanol, n-propanol, 2-propanol, n-butanol, phosphorus trichloride, sodium p-nitrophenoxide, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Sulfuryl chloride was obtained from Eastman Kodak Co. (Rochester, NY). Methanol and all solvents were HPLC or reagent grade (Fisher Scientific Co., Fair Lawn, NJ). Sodium carbonate and anhydrous magnesium sulfate were products of Mallinckrodt, Inc. (Paris, KT) and Sargent-Welch Scientific Co. (Skokie, IL), respectively. Triton X-100 and acetylthiocholine iodide were purchased from Sigma Chemical Co. (St. Louis, MO). Commercial 0,O-diethyl 0-p-nitrophenyl phosphate (Paraoxon) was 95% pure as supplied by Chem Service (West Chester, PA). Dimethyl, diethyl, di-n-propyl, diisopropyl, and di-n-butyl chlorophosphates were synthesized by a modification of the method of Fiszer and Michalski (22)and McIvor et al. (23). All reagents were dissolved in equal volumes of hexane. Three equivalents of the appropriate alcohol were added dropwise to one equivalent of cold (5-10 "C) phosphorus trichloride; as the reaction is exothermic, an ice bath was used t o maintain the reaction temperature below 10 "C. Immediately after the addition of alcohol, 1 equiv of sulfuryl chloride was introduced dropwise to the newly formed trialkyl phosphite. The hexane was removed under reduced pressure (water pump aspiration) and the dialkyl chlorophosphate purified by vacuum distillation. The various dialkyl p-nitrophenyl phosphates were then prepared from the respective dialkyl chlorophosphates and sodium p-nitrophenoxide by modifying the methods of Fletcher et al. (24), Schrader (25),and DeRoos and Toet (26). Specifically, sodium p-nitrophenoxide was suspended in ethanol (1.5 M), and neat
Wallace and Kemp dialkyl chlorophosphate was added dropwise, producing a faint exothermic reaction. The mixture was then heated for 90 min a t 87 "C under a reflux apparatus. After cooling t o room temperature, the mixture was filtered to remove sodium chloride and the ethanol removed by rotoevaporation under reduced pressure (water pump aspirator). Forgoing vacuum distillation, the resulting crude product was dissolved in methylene chloride and washed with a 5% aqueous solution of sodium carbonate to remove unreacted p-nitrophenoxide. The methylene chloride was dried with anhydrous magnesium sulfate, filtered, and then removed by rotoevaporation. The resulting dialkyl p-nitrophenyl phosphate was then heated under reduced pressure (0.2 mmHg) to remove traces of unreacted dialkyl chlorophosphate (modified from ref 24). Purities of the unbranched dialkyl p-nitrophenyl phosphates were greater than 99% as determined by GC-FID and confirmed by GC-MS and HPLC-UV. Diisopropyl p-nitrophenyl phosphate was greater than 95% pure. T h e only impurity detected in any of the preparations was the corresponding dialkyl chlorophosphate which was found to be inactive in inhibiting AChE. Prior to chemical and/or biological characterization, each inhibitor was again washed in methylene chloride with a 5% aqueous solution of sodium carbonate to remove hydrolysis products. Infrared spectra were obtained between silver chloride plates by using a Beckman IR-33 spectrophotometer. Ultraviolet spectra were monitored with a Beckman DU-7 spectrophotometer. Nuclear magnetic resonance spectra (IH, 13C,and 31P)were obtained on a n IBM AF 200-MHz FT-NMR. The gas chromatograph (Hewlett-Packard Model 5890A) was equipped with a flame ionization detector (GC-FID). The gas chromatograph-mass spectrometer (GC-MS) was a Hewlett-Packard 5995C quadrapole, E1 70-eV system. The column on both systems was a 25-m (0.32 m m i d . ) SE-54 siloxane supplied by Hewlett-Packard. Highresolution GC-MS (EI, 70 eV, resolution 2 6000) data were obtained from the University of Minnesota Mass Spectrometry Laboratory on a VG Model 7070 mass spectrometer. Splitless injections were employed with 5-8 psi head pressure and 2 mL/min He carrier flow. The temperature program consisted of a 5-min initial hold a t 100 "C followed by a gradient up t o 250 "C a t 10 "C/min, with a final hold for 10 min. The high-pressure liquid chromatography (HPLC) procedure was adapted from Sultatm et al. (21);the system consisted of either a Beckman pump (Model llOA) or a Spectra Physics Model SP8700 solvent delivery system equipped with a Rheodyne 7105 injector. A Regis silica column (5 pm, 250 X 4.6 i.d.) was employed for separation and the eluent monitored a t 254 nm (Kratos Spectroflow 783 UV detector). The mobile phase consisted of 99% methylene chloride-1 % acetonitrile delivered a t a flow rate of 1 mL/min. For reverse-phase HPLC, an octadecyl sulfate (ODS) column (Regis, 5 pm, 250 X 4.6 mm i.d.) was employed; the mobile phase included 50% deionized water-50% acetonitrile, delivered a t 1 mL/min. For purposes of comparison, commercial diethyl p-nitrophenyl phosphate (Paraoxon, Sigma Chemical Co.) was included in the physical-chemical analyses. These data provided a standard against which the synthetic products could be compared to c o n f i i the spectral characterization (UV, IR, and NMR) and a reference standard for the chromatographic analyses (GC and HPLC). The first-order rate constants describing the alkaline hydrolysis of each of the five organophosphorus agents were taken from Ooms and Breebaart-Hansen (27). T h e octanol-water partition coefficients (log Po,,.,) were calculated according to the method of Leo and Weininger (28). The kinetic constants describing the in vitro inactivation of AChE from the respective species have been published previously (9). The means of three repetitions of the measurement of the bimolecular inhibition constant (kJ,the dissociation constant (&), and the first-order phosphorylation rate constant (k,) were employed for all correlations. For purposes of clarification, k,is equal to k /Kd. AT1 regressions were performed by least squares and the slopes evaluated by analysis of variance. A probability of p < 0.05 was used as the criterion for statistical significance.
Results The physical-chemical properties of the synthetic organophosphorus compounds are presented in Tables I-V.
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 43
Species-Specific Anticholinesterase QSAR
Table I. IR and UV Spectra of Dialkyl p-Nitrophenyl Phosphates 0
R
IR ROP=O N=O
CH3 aromatic aromatic
c=c
aromatic aromatic
c=c
aromatic aromatic
c=c
C2H6(synthetic)
~
CH CH3 ROP=O N=O
C2H5 (commercial)
n-C3-7
CH CH3 ROP=O N=O CH CH3 ROP=O
N=O aromatic aromatic
c=c
aromatic aromatic
c=c
aromatic aromatic
c=c
iso-C3H7
n-C4H9
" Concentration:
frequency, cm-' 1225 and 1275; also 930 and 1025 1345 and 1520 1490 and 1590 3085 1450 and 3000 1220 and 1280; also 930 and 1030 1345 and 1520 1490 and 1590 3090 1440 and 2990 1230 and 1280; also 910 and 1020 1345 and 1520 1490 and 1590 3085 1440 and 2990 1230 and 1285; also 920 and 1020 1345 and 1520 1490 and 1590 3090 2980 1230 and 1275; also 920 and 1000 1350 and 1525 1495 and 1595 3090 2990 1230 and 1285; also 920 and 1025 1345 and 1525 1490 and 1595 3085 2970
_____~
5X
CH CH3 ROP=O N=O CH CH3 ROP=O N=O CH CH3
UV" A,,
= 271.9 nm
E M = 8900 M-lVcm-' A,,
= 273.4 nm
E M = 8974 M-l-cm-' A,,
= 273.5 nm
EM = 10342 M-lscm-' A,
= 273.0 nm
E M = 9224 M-'.cm-' A,
= 274.3 nm
EM = 8384 M-l-cm-' A,,
= 273.6 nm
EM = 8410 M-'.cm-'
M in acetonitrile.
Table 11. IH,lac,and
NMR (ppm) of Dialkyl p-Nitrophenyl Phosphates
a
31P
'H NMR' 3.87 (d, 6), 7.36 (d: 2), 8.21 (d: 2) CH3 C2H5(synthetic) 1.33 (t of d, 6), 4.16 (overlap q, 4), 7.34 (d,c 2), 8.20 (d: 2) C2H5(commercial) 1.34 (t of d, 6), 4.22 (overlap q, 4), 7.35 (d,c 2), 8.22 (d: 2) 0.93 (t,'6j, 1:68 (sext, 4), 4.10 (9, 4), 7.35 (d,c 2), n-C3H7 8.21 (d: 2) iso-C3H7 1.30 (overlap d, 6), 4.74 (overlap q, l),7.34 (d: 2), 8.20 (d: 2) n-C4H9 0.89 (t, 6), 1.38 (sext, 4), 1.65 (p, 4), 4.14 (q, 4), 7.34 (d: 2), 8.20 (d,c 2)
R
13C NMR" 55.2 (d), 120.4 (d), 125.6 (s), 144.7 (s), 155.3 (d) 15.9 (d), 65.0 (d), 120.4 (d), 125.5 (s), 144.5 (s), 155.5 (d) 16.0 (d), 65.1 (d), 120.4 (d), 125.6 (s), 144.6 (s), 155.5 (d) 9.85'(s), 23.5 (d), 70.5 (d), 120.4 (d), 125.6 b), 144.6 (s), 155.6 (d) 23.5 (overlap d), 74.2 (d), 120.4 (d), 125.5 (s), 144.4 (s), 155.8 (d) 13.4 (s), 18.5 (s), 32.0 (d), 68.8 (d), 120.5 (d), 125.6 (s), 144.6 (s), 155.6 (d)
NMRb -5.45 (p) -7.69 (p) -7.68 (d) -7.48 (P) -9.45 (t) -7.46 (p)
"Solvent = CDC13. Reference = trace 'H or 13C in CDC1, (7.24 ppm). *Solvent = CDCl,. Reference = H3P04 (85% aqueous) external. Complex doublet.
Table I summarizesthe IR and UV spectra, demonstrating strong bands for the substituted phosphates as well as for the nitro and aromatic ?r systems. UV spectra include the maximum absorbance (A,-obtained from scans between 220 and 340 nm) for the homologous series, with the corresponding molar extinction coefficient; these data were obtained by using the same concentration for all compounds (5 X M dissolved in acetonitrile). The NMR data for a number of nuclei (lH, 13C,and 31P) for the series of dialkyl p-nitrophenyl phosphates illustrate the inductive effects of successive methylene substitutions on the a-carbon and phosphorus atoms (Table 11); shifts in the 31Pspectra with successive methylene substitutions are indicative of the progressive decrease in electroposi-
tivity of the phosphorus atom. The 'H NMR data for the dialkyl-substituted p-nitrophenyl phosphates correlate with the proton data obtained for the corresponding dialkyl chlorophosphates (data not shown). The '3C NMR data were obtained by decoupling the protons from the phosphorus atom. However, the carbon atoms are still coupled to phosphorus. To enhance detection of the substituted 1- and 4-carbons of the aromatic system, the pulse delay was increased to 10 s. This provided sufficient time for the 1-and 4-carbons to relax between pulses. Coupling occurs with carbon atoms that are three or less atoms removed from phosphorus: dimethyl and diethyl as well as aromatic carbons 1, 2, and 6 couple with phosphorus to yield a doublet. The diisopropyl p-nitrophenyl phos-
Wallace and Kemp
44 Chem. Res. Toxicol., Vol. 4 , No. I , 1991 Table 111. GC-MS Data for Dialkyl p-Nitrophenyl Phosphates 0
relative retention time molecular R (GC)' ion, m / z 247 0.926 CH3 275 CZH5 0.998 n-C,H, 1.088 303 303 iso-CSH7 1.011 n-C,Hg a
1.194
m / z (relative abundance) 247 (12), 230 (17), 200 (9), 135 (9), 109 (loo), 96 (54), 79 (30) 275 (go), 247 (51), 232 (391, 220 (461, 219 (40), 149 (75), 139 (5% 127 (291, 109 (10% 99 (38), 81 (45) 303 (14), 261 (8), 232 (18), 220 (loo), 139 (7), 123 (9), 109 (13) 303 (21), 261 (16), 246 (60), 220 (99), 219 (94), 203 (39), 189 (1% 163 ( 1 4 , 139 (26), 123 (20), 109 (501, 43 (100) 331 (7), 276 (18), 220 (loo), 109 (4)
331
Reference = commercial diethyl p-nitrophenyl phosphate ( t =~18.62 m i d .
Table IV. Relative' Retention Times (by HPLC-UV) and Calculated Octanol/Water Partition Coefficients (log P o / w ) for the Series of Dialkyl p-Nitrophenyl Phosphates 0 Rob RO'
R CHS C2H5
n-C3H7 iso-C3H7 n-CdHg p-nitrophenol
1-
0e
silica 1.012 1.000 0.772 1.050 0.683 0.489
N
0
ODS 0.688 1.003 1.877 1.613 3.935 0.646
2
log Poiw* 1.38 2.06 2.74 2.52 3.60
Reference = commercial diethyl p-nitrophenyl phosphate ( t =~ 14.21 min on silica and 7.72 min on ODS). *Calculated by the method of Leo and Weininger (28).
Table V. Regression of PhysicalChemical Constants for the Series of Dialkyl p-Nitrophenyl Phosphates regression equation" rz significance* 0.951 p < 0.005 tR(GC) = 13.891 + 2.250(10g Po w) tR(silica) = 18.450 - 2.281(log 0.645 ns tR(0DS) = -13.730 + 11.302(10g Polw) 0.897 p < 0.05 I3C NMR = 58.042 - l l . O l ( k h y d ) 0.849 p < 0.05 31PNMR = -5.699 + 2.287(khYd) 0.957 p < 0.005
60,w)
"Least-squares linear regression of the data for the five synthetic dialkyl-substituted p-nitrophenyl phosphates. Abbreviations: tR, retention time (min) on the respective column; POiw, calculated octanol/water partition coeficient; khyd,first-order alkaline hydrolysis rate constant from Ooms and Breebaart-Hansen (27). * ns, not statistically significant ( p < 0.05).
phate splits into two overlapping doublets (Table 11). In contrast, the y-and &carbons on the larger dialkyl chain organophosphates do not couple, and singlets are observed. In support of this decoupling phenomenon observed for dialkyl-chain y- and &carbons, the aromatic carbons 3-5 are not coupled to phosphorus and are observed as singlets. Chemical shifts for 31PNMR were measured with respect to an external reference of phosphoric acid (H3P04= 0 PPm). Table I11 presents mass spectra (MS) data for each organophosphate. The molecular ion as well as the major ionic fragments (109 for all products) are presented with the relative abundance expressed for each ion with respect to the base peak. The MS data combined with the chromatographic data reveal that the purity of the synthetic inhibitors exceeds 99% with the exception of diisopropyl p-nitrophenyl phosphate (>95% pure). The only detectable impurity in any of the products was the corresponding dialkyl chlorophosphate, which was inactive in inhibiting AChE in our assay system. The relative re-
tention times of the dialkyl p-nitrophenyl phosphates as observed by GC and HPLC are provided in Tables I11 and IV, respectively. The relative retention time for each organophosphate is reported with respect to commercial diethyl p-nitrophenyl phosphate (Paraoxon) as a positive control. As expected, on the nonpolar GC and ODS-HPLC columns the dialkyl p-nitrophenyl phosphates exhibit increasing retention times with successive methylene substitutions. Conversely, a polar (silica) HPLC column (Table IV) allows the larger dialkyl p-nitrophenyl phosphates to elute from the column earlier than the smaller chain (i.e., more polar) compounds. However, the elution of diisopropyl p-nitrophenyl phosphate deviates from this trend, as the longest retained organophosphate on a silica column. Unlike the GC and HPLC-ODS columns, resolution of the synthetic organophosphates on silica is not solely on the basis of polarity as further evidenced by the poor correlation of the retention time (tR) on silica with log Poi? (Table V). The inductive effects of various substituents affect the electropositivity of the phosphorus atom and the strength of the P-0-Ar bond. These alterations are reflected in the IR and NMR spectra as well as in the susceptibility of the phosphotriester compounds to alkaline hydrolysis. The proportionality between changes in the l3CUand 31P NMR spectra and the hydrolysis rate constants is illustrated by the significant correlations between these parameters (Table V). The data suggest that successive methylene substitutions decrease the electropositivity of the phosphorus atom as reflected by the larger 31PNMR shifts and that associated with this is an increase in the strength of the phosphoester bond as reflected by the slower rate of alkaline hydrolysis. Correlations between the retention of the unbranched, straight-chain synthetic organophosphates on different chromatographic columns and the inhibition kinetics for brain AChE are illustrated in Table VI. Interestingly, despite the lack of a correlation between the retention of the inhibitors on nonpolar GC or HPLC-ODS columns and the respective ki for any of the three species of AChE, there was a significant correlation between tR on nonpolar columns and the Kd for rat and hen AChE, but not for trout AChE. The retention of the unbranched inhibitors on the polar silica HPLC column correlated with both kiand Kd for rat and hen, but not trout AChE. In contrast, there was no significant correlation between t R and k, for any of the three columns or three species examined. As is evident from Figure lB, the observed correlations were only valid for the unbranched dialkyl-substituted compounds; none of the above-mentioned regressions were statistically significant when diisopropyl p-nitrophenyl phosphate was included in the analyses.
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 45
Species-specific Anticholinesterase QSAR
Table VI. Regression of Kinetic Inhibition Constants as Functions of Retention of Straight-Chain Dialkyl p-Nitrophenyl Phosphates on GC or HPLC Columns" column regression equation r2 significanceb GC-siloxane log (rat ki) = 7.137 - 0.227(tR) 0.894 ns log (hen ki) = 7.149 - 0.236(t~) 0.863 ns log (trout ki) = 8.291 - 0.457(t~) 0.877 ns 0.980 p < 0.05 log (rat &) = 3.045 + 0.179(t~) log (hen &) = -4.202 0.258(t~) 0.914 p 50.05 log (trout Kd) = 0.086 + 0.168(t~) 0.626 ns 0.524 ns log (rat k ) = 1.055 - 0.046(t~) log (hen = -0.005 + 0.019(tR) 0.598 ns 0.474 ns log (trout k,) = 5.476 - 0.296(t~) HPLC-silica log (rat ki) = -0.004 + 0.220(tR) 0.988 p < 0.01 0.980 p < 0.05 log (hen ki) = -0.315 + 0.231(tR) 0.807 ns log (trout ki) = -5.637 + 0.404(t~) 0.968 p < 0.05 log (rat Kd) = 2.491 - 0.164(tR) 0.983 p < 0.01 log (hen Kd) = 3.877 - 0.246(t~) log (trout Kd) = 5.336 - 0.159(t~) 0.664 ns log (rat k ) = -0.492 + 0.052(tR) 0.782 ns log (hen I!,) = 0.531 - 0.013(tR) 0.320 ns log (trout k,) = -3.445 + 0.254(tR) 0.410 ns HPLC-ODS log (rat ki) = 3.321 - 0.043(tR) 0.876 ns 0.857 ns log (hen ki) = 3.183 - 0.045(tR) log (trout ki) = 0.430 - 0.075(t~) 0.651 ns 0.945 p < 0.05 log (rat Kd) = -0.020 + 0.034(t~) 0.910 p < 0.05 log (hen &) = 0.0133 + 0.049(t~) 0.840 ns log (trout Kd) = 2.835 + 0.037(t~) log (rat k ) = 0.279 - 0.009(tr) 0.533 ns log (hen = 0.319 - 0.004(tR) 0.586 ns log (trout k,) = 0.237 - 0.038(tR) 0.220 ns
5)
5)
a Only the data for the four straight-chain analogues were included in the least-squares linear regressions. None of the correlations were statistically significant (p < 0.05) when the data for diisopropyl p-nitrophenyl phosphate was included in the regressions. Abbreviations: t ~ , retention time in min; k,,bimolecular inhibition constant in (mM-min)-'; &, dissociation constant in wM;k,, first-order phosphorylation rate constant in m i d . bns, not statistically significant (p < 0.05).
Table VII. Regression of Ultraviolet and Infrared SpectrophotoscopicData against Inhibition Constants for the Series of Dialkyl-Substituted p -Nitrophenyl Phosphates" indeaendent variable regression eauation r2 significanceb 0.588 ns log (rat ki)= 178.965 - 0.646(A,,) UV absorbance maximum, A, (nm) log (hen ki) = 177.422 - 0.641(A,,) 0.555 ns p < 0.05 log (trout ki) = 377.512 - 1.386(Am,) 0.825 0.792 p < 0.05 log (trout k,) = 325.591 - 1.194(Am,) log (rat ki) = 105.849 - O.O71(u) 0.610 ns IR (CH,) frequency, u (cm-') log (hen ki) = 106.531 - 0.641(v) 0.595 ns 0.757 p < 0.01 log (trout ki) = 207.467 - 0.143(u) log (trout kp) = 198.183 - 0.136(u) 0.890 p < 0.05 log (rat ki) = 165.226 - 0.133(u) 0.633 ns IR (R-0-P) frequency, u (cm-') log (hen ki) = 173.203 - 0.139(u) 0.670 ns log (trout hi) = 243.896 - 0.200(u) 0.438 ns a The equations describe the least-squares linear regression of the data for all five dialkyl-substituted p-nitrophenyl phosphates. Abbreviations: ki,bimolecular inhibition constant in (mM.min)-' as taken from Kemp and Wallace (9);k,, first-order phosphorylation rate constant in m i d taken from the same source; A, ultraviolet absorption maximum in nm. bns, not statistically significant ( p < 0.05).
Table VIII. Correlation between Kinetic Inhibition Constants and Alkaline Hydrolysis Rate Constants for the Series of Dialkvl-Substituted ~ - N i t r o ~ h e nPhosDhates" vl dependent variableb regression equation r2 significance' 0.779 p < 0.05 bimolecular inhibition constant, ki (mM-min)-' log (rat ki) = 3.291 + 1.085(khyd) 0.749 ns log (hen ki) = 3.125 + 1.086 (khyd) 0.938 log (trout ki) = 0.597 + 2.155(khyd) p < 0.01 0.861 p < 0.05 dissociation constant, Kd (rM) log (rat Kd) = -0.081 - 1.019(khyd) log (hen Kd) = 0.253 - 1.061(khyd) 0.740 ns 0.464 ns log (trout &) = 3.059 - 0.599(khyd) log (rat k,) = 0.191 + 0.061(khyd) 0.095 ns phosphorylation rate constant, k, (min-*) log (hen k,) = 0.384 + 0.048(khyd) 0.158 ns p < 0.01 0.945 log (trout k,) = 0.792 + 1.902(khyd) "The hydrolysis rate constants [khyd= log ( k i / k O )are ] taken from Ooms and Breebaart-Hansen (27). "he from Kemp and Wallace (9). ens, not statistically significant (p < 0.05).
Although there was no relationship between the UV or IR(CH,) absorption maxima and ki for either rat or hen AChE, a significant regression was observed for trout which was ascribed to reflect changes in k, (Table VII). No
kinetic inhibition Constants are
correlations were observed for the IR (R-0-P) spectra. Regression of the alkaline hydrolysis rate constants for the homologous series of inhibitors with the various kinetic inhibition constants reveals contrasting relationships for
46 Chem. Res. Toxicol., Val. 4, No. 1, 1991
Wallace and Kemp
Table IX. Correlation between Kinetic Inhibition Constants and NMR Spectroscopy Data for the Series of Dialkyl-Substituted p -Nitrophenyl Phosphatesn independent variable regression equationb r2 significanceC 13C NMR, 6 (ppm) log (rat ki) = 7.983 - 0.083(6) 0.654 ns log (hen ki)= 7.860 - 0.084(6) 0.637 ns log (trout ki) = 10.838 - 0.179(6) 0.924 p < 0.01 log (trout k,) = 9.825 - 0.158(6) 0.929 p < 0.01 31PNMR, b (ppm) log (rat hi) = 5.494 + 0.408(6) 0.601 ns log (hen ki) = 5.299 + 0.404(b) 0.566 ns log (trout ki) = 5.503 + 0.880(8) 0.855 p < 0.05 a The data for all five dialkyl-substituted p-nitrophenyl phosphates were regressed against the corresponding kinetic inhibition constants, which were taken from Kemp and Wallace (9). Abbreviations: ki, bimolecular inhibition constant in (mM.min)-'; k,, first-order phosphorylation rate constant in min-'. not statistically significant (p < 0.05).
log Polw 3
2
1
4
z
2.0
A A
o
1.5
5
-1.0
v
s
1.o
0 Rat (r2=0.025)
A
-
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: 1B
9
0
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0
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M
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nn
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z
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-
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"
.
.
-10
,
I
.
,
-9
,
, -8
.
, -7
.
Hen (r2=0.084)
, -6
.
, -5
31P-NMR (ppm)
Figure 2. Least-squares linear regression of the first-order
phosphorylation rate constant (k,) for rat, hen, and trout brain AChE as a function of the shifts in 31PNMR for all five synthetic dialkyl-substitutedp-nitrophenyl phosphates.
v
u"
1
/
J
r
9
/ v
"
0.0-
/
b
A
I
I
18
20
22
GC Rt (min)
Figure 1. Least-squares linear regression of the dissociation constant (&) as a function of the octanol/water partition coef-
ficient (log Polw;panel A) and retention time of the synthetic dialkyl-substitutedp-nitrophenyl phosphates on a siloxane GC column (panel B). The open symbols depict the respective values for the unbranched analogues,and the closed symbols reflect the corresponding values for the diisopropyl analogue. The regression coefficient squared (?) for the plots of the data for the unbranched substituents (open symbols) are 0.967 and 0.890 for rat and hen AChE as a function of log Polwand 0.980 and 0.914, respectively, for the plot of the retention on GC. Including the diisopropyl analogue (closed symbols) negated the statistical significance of all regressions.
rat and trout brain AChE (Table VIII). Whereas kbyd correlated with kiand K d but not k, for the rat, the significance of hhyd for trout AChE was manifested in k,. The regression of khyd against trout K d was not statistically significant; nor were the regressions for any of the kinetic constants for hen AChE. The relationship between the electronic properties of the organophosphates and the inhibition of trout, but not rat or hen, brain AChE is further illustrated by the regressions with the NMR spectral data (Table IX). Although neither 13C nor 31PNMR data correlated with ki for rat or hen AChE, both NMR spectra yielded statistically significant
relationships when regressed against the kifor trout AChE. This relationship apparently reflects the inductive effects on altering h, for trout brain AChE as evidenced from the positive correlations between either 13C or 31P NMR spectral shifts and trout k, (Table IX and Figure 2). No statistically significant regressions existed between the NMR spectral data and hi or k, for either rat or hen brain AChE.
Discussion In our first attempts to synthesize the dialkyl p-nitrophenyl phosphates by the method of Fagerlind et al. (29), it was discovered that the products were contaminated by as much as 60% by the corresponding alkoxy-p-nitrophenyl ethers which could not be differentiated from the intended products by IR or UV spectroscopy. This problem was resolved by adapting the synthesis to avoid the distillation step, yielding products which were at least 95% pure (dimethyl, diethyl, di-n-propyl, and di-n-butyl p-nitrophenyl phosphates were greater than 99 % pure). The IR spectra for the series of dialkyl p-nitrophenyl phosphates agree with those observed by DeRoos and Toet (26), and the UV spectrum for diethyl p-nitrophenyl phosphate is consistent with that reported by Fagerlind and associates (29). The electron impact mass spectra of the synthetic organophosphates agree with that reported by Stan et al. (30). It has been suggested that the m/z 109 ionic species observed for both dimethyl and diethyl phosphates is not a homologous fragment for the two dialkyl p-nitrophenyl phosphates, but rather a different ion with the same mlz (30). The data we present suggest two different 109 fragments for each of the individual dimethyl and diethyl
Species-specific Anticholinesterase QSAR
Chem. Res. Toricol., Vol. 4, No. 1, 1991 47
analogues: the separate phosphorus-containingspecies (30) as well as a homologous ion derived from the aromatic portion of the dialkyl p-nitrophenyl phosphates. Since each of the five inhibitors analyzed in this study possesses an m / z of 109, it is considered a homologous fragment; however, an m / z of 109 corresponds to the base peak for both the dimethyl and diethyl analogues, while m/z = 109 is equal to 50% or less of the base peak for the three larger dialkyl p-nitrophenyl phosphates. These data support the existence of two separate ionic species, and we propose the following as homologous fragments obtained from the entire series of compounds studied: NO*
W Z 109
This common nitro rearrangement has been described by Beynon (31) and is supported by our data by the presence of a high-resolution mass peak at 109.0264 amu (Table 111). Proton NMR spectra for 40 organophosphorus pesticides, including commercial Paraoxon (diethyl p-nitrophenyl phosphate), reveal coupling between protons on the methyl carbon and the phosphorus atom, with the a-carbon protons of Paraoxon displaying overlapping quartets (32). Our spectra for both commercial and synthetic Paraoxon exhibit the same overlapping quartets (Table 11). Carbon-13 NMR spectra of the synthetic dialkyl p-nitrophenyl phosphates also indicate coupling between phosphorus and carbon atoms that are displaced three or less atoms from the phosphorus nucleus (Table 11). Protons also couple to the phosphorus atom and are observed on 31PNMR spectra as pentets (quintets) for all of the organophosphates, except for commercial diethyl p-nitrophenyl phosphate and diisopropyl p-nitrophenyl phosphate (Table 11). Meyerson et a]. (33)reported 31PNMR chemical shifts for a series of organophosphates using H3P04 as the standard reference. Dimethyl p-nitrophenyl phosphate exhibited a chemical shift at -4.8 ppm which is in agreement with the value of -5.45 ppm for the dimethyl analogue reported in Table 11. Likewise, Ross and Biros (34) observed similar phosphorus NMR shifts for a wide variety of organophosphates with diethyl p-nitrophenyl phosphate a t 119.7 ppm [reported with respect to phosphorus oxide (ppm = O ) ] . These shifts in the 31PNMR spectra reflect the inductive effects of the alkyl substituents on the phosphorus atom, the electropositivity of the phosphorus atom decreasing with successive methylene substitutions. This is also reflected by the IR data demonstrating less R-O-P bond stretching with successive methylene substitutions and correlates with the progressive decrease in the first-order alkaline hydrolysis rate constants. The importance of the electropositivity of the phosphorus atom in the Coulombic association of the inhibitor with AChE and in determining the rate of enzyme phosphorylation has been substantiated by correlations between the kinetic inhibition constants for fly head AChE and various physical-chemical indicators of the electronic properties of the phosphorus atom. These include IR spectroscopy as a measure of bond stretching, the firstorder hydrolysis rate constants, Taft’s electronic constant (a),and Hammett’s Q constant for assorted structurally diverse organophosphates (10, 11, 16,17). We observed similar correlations between the inhibition of trout brain AChE and the hydrolysis rate constants or the UV or IR spectra and extended these to include correlations between shifts in the 31PNMR spectral with both k, and ki for trout
AChE (Tables VII-IX). The fact that there was a significant relationship between kh, and the Kd for rat AChE (Table VIII) suggests that the electropositivity of the phosphorus atom may be a factor contributing to the association of the inhibitor with the nucleophilic subsite of the enzyme. However, this interpretation is tempered by the failure to observe correlations of Kd with other measures of the electronic properties of the inhibitor (UV or IR spectroscopy, or ‘H, 13C,or 31PNMR). Conversely, the rate of phosphorylation of trout brain AChE is strongly influenced by the electronic properties of the inhibitor as indicated by the significant correlations between the k for trout AChE and UV and IR spectra, khyd, and both and 31PNMR spectral shifts. Conversely, the phosphorylation, and hence inhibition, of rat and hen AChE are not functions of the electronic properties of the phosphorus atom as demonstrated by the absence of correlations between k, or ki and any of the spectral or hydrolysis data. This disparity between species implies basic differences in the nucleophilic character of the respective enzymes. The lack of influence of the phosphorus atom on k, for rat and hen AChE suggests that these enzyme possess a strong nucleophilic center within the esteratic subsite. Thus, once the reversible complex between enzyme and inhibitor is formed, the nucleophilic substitution reaction proceeds rapidly and independently of the electronic properties of the inhibitor. These data for rats and hens contrast with the structure-activity investigations reported for fly head AChE, which demonstrate a strong dependence of enzyme inhibition on the electropositivity of the phosphorus atom (11,12, 16). Conversely, the chemical mechanisms governing the inhibition of trout AChE resemble those reported for other species. It may be proposed that trout brain AChE, like that from houseflies, possesses a relatively weak nucleophilic center, k, being directly proportional to the electrophilic strength of the inhibitor. This weak electronegativity of the esteratic subsite of trout AChE may also be partially responsible for the low affinity (high Kd) of the enzyme for the inhibitors (9),being that the association of the inhibitor with the enzyme occurs via Coulombic forces. The retention of the various dialkyl p-nitrophenyl phosphates on both polar (silica) and nonpolar (ODSHPLC and GC) columns is consistent with the octanol/ water partition coefficients for the respective inhibitors. As expected, successive methylene substitutions are associated with a progressive increase in the hydrophobicity and prolonged retention times on nonpolar columns. Conversely, the highly substituted p-nitrophenyl phosphates elute rapidly from the polar silica HPLC column. Interestingly, diisopropyl p-nitrophenyl phosphate elutes from the silica column later than any other compound in the series, demonstrating that carbon branching decreases the polarity of the organophosphate. This phenomenon is not observed on the nonpolar GC column, nor on the reverse-phase ODS-HPLC column. Indeed, as expected, diisopropyl p-nitrophenyl phosphate possesses a retention time between those of the diethyl and di-n-propyl analogues. However, if one assumes effective carbon chain length (i.e., the number of carbons in the longest unbranched chain) is the principal criterion determining the retention on the silica column, diisopropyl p-nitrophenyl phosphate would be expected to elute from the column at the same time as the diethyl analogue, which is what was observed (Table IV). Steric exclusion of bulky inhibitors from the active site has been implicated as a major factor governing the inhibition of AChE (5, 10, 11,15-17, 19). consequently, it
136
Wallace and Kemp
48 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
has been proposed that resistance to organophosphates may be conferred, in part, by the limiting dimensions of the esteratic subsite of AChE, the resistant species or strain exhibiting greater steric hinderance (9,20). By use of the retention times and log Po as measures of hydrophobicity, it is concluded that, unkke the inhibition of AChE by carbamate compounds, hydrophobic interactions are of little significance to the mechanism of inhibition of AChE. In fact, the data support the contention that hydrophobic substituents hinder, rather than enhance, the association of organophosphates with AChE (26). The correlations between enzyme inhibition and log Po or the various retention times is thus probably not indicative of hydrophobicity but rather is a manifestation of differences in molecular volume. This is especially apparent for the branched diisopropyl p-nitrophenyl phosphate, which possesses a log Polw comparable to that of the di-n-propyl analogue (Table IV) but is only l/lo as potent of an inhibitor against rat and hen AChE (9). For the unbranched dialkyl substituents, there was a significant correlation or the between Kd for either rat or hen AChE and log Polw retention time on GC columns (Figure 1). A similar correlation was observed between Kd and the retention on silica or ODS-HPLC columns (Table VI), suggesting that, for the given series of unbranched dialkyl-substituted organophosphates, column chromatography may provide a convenient means for estimating inhibitor potency against rat or hen brain AChE. However, since no significant correlations were observed when diisopropyl p nitrophenyl phosphate was included in the regressions, it is concluded that the discriminating factor is molecular volume rather than linear dimensions as has been implicated previously (16). Consequently, steric hindrance as it affects the association of the inhibitor with AChE most likely reflects the finite volume of the esteratic subsite on the enzyme. The fact that the Kd for trout AChE was not a function of these parameters may reflect an extremely confining esteratic subsite wherein the molecular volume of the smallest analogue studied, dimethyl p-nitrophenyl phosphate, exceeds the dimensions of the esteratic subsite of trout brain AChE. This may also account for the very low affinity (large Kd) of trout brain AChE for the various inhibitors when compared to the enzyme from either rats or hens (9). In conclusion, the structure-activity analyses reveal several important factors governing the inhibition of AChE by organophosphorus compounds. The data reinforce the reported importance of both steric hindrance and electropositivity of the phosphorus atom as important determinants of inhibitor potency. However, the significance of each of these factors differs between species. Trout brain AChE behaves similarly to that reported for fly AChE wherein inhibitor potency is a function of the electronic properties of the phosphorus atom. Our data demonstrate that this variation in inhibitor potency is, as would be expected, directly attributable to differences in the phosphorylation rate constant (k,). The existence of this relationship suggests that trout brain AChE possesses a fairly weak nucleophilic center wherein the rate of phosphorylation varies in proportion to the electrophilic strength of the phosphorus atom. Conversely, k, does not vary between inhibitors for either rat or hen AChE, suggesting that these enzymes possess a strong nucleophilic center; phosphorylation proceeds rapidly and independently of the electronic properties of the inhibitor. For rat and hen brain AChE, the discriminating factor is steric hindrance as determined by the molecular volume of the corresponding inhibitor. Therefore, in comparison to rat
and hen brain AChE, it appears that the resistance of trout AChE to inhibition by organophosphorus agents is conferred by the relatively weak nucleophilic center within the dimensionally restricted esteratic subsite. Differentiation of the chemical mechanisms of inhibition of AChE provides valuable insights into possible strategies for designing new organophosphorus agents with greatly improved species specificity. By selectively modifying either the steric or electronic properties of the compound, it may be possible to selectively target the intended pest organism and at the same time diminish the hazards to economic species. A prerequisite to accomplishing this objective, however, is that similar structure-activity investigations be performed to elucidate the molecular regulation of anticholinesterase activity in the species of principal interest, particularly in humans. Acknowledgment. We appreciate the expert assistance of Dr. Robert M. Carlson and Robert J. Liukkonen in the synthesis and chemical characterization of the organophosphorus compounds employed in this investigation. The NMR spectroscopy was accomplished with the generous expertise of S.B. Bosard and Drs. R. Caple and D. E. Samkoff, Chemistry Department, University of Minnesota, Duluth. Estimates of log Polwwere generously provided by the US. EPA, Environmental Research Laboratory-Duluth. Susan J. Kurki provided excellent clerical assistance in the preparation of the manuscript. This research was supported, in part, by a Grant-in-Aid from the University of Minnesota Graduate School and by U.S. Environmental Protection Agency Grant CR810963. Registry No. AChE, 9000-81-1; ethanol, 64-17-5; n-propanol, 71-23-8; 2-propanol, 67-63-0; n-butanol, 71-36-3;methanol, 67-56-1; phosphorus trichloride, 7719-12-2; diethyl chlorophosphate, 814-49-3; di-n-propyl chlorophosphate, 2510-89-6; di-2-propyl chlorophosphate, 2574-25-6; di-n-butyl chlorophosphate, 819-43-2; dimethyl chlorophosphate, 813-77-4; sodium p-nitrophenoxide, 824-78-2; dimethyl p-nitrophenyl phosphate, 950-35-6; diethyl p-nitrophenyl phosphate, 311-45-5; di-n-propyl p-nitrophenyl phosphate, 1153-30-6; di-2-propyl p-nitrophenyl phosphate, 3254-66-8; di-n-butyl p-nitrophenyl phosphate, 2255-19-8.
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