Synthesis, Absolute Configuration, and Analysis of Malathion

were prepared via two independent pathways using strychnine to resolve the asymmetric ... epidemic malathion poisoning of 28OO Pakistani spraymen...
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Chem. Res. Toxicol. 1993,6, 718-723

718

Synthesis, Absolute Configuration, and Analysis of Malathion, Malaoxon, and Isomalathion Enantiomers Clifford E. Berkmant and Charles M. Thompson; Department of Chemistry, Loyola University of Chicago, 6525 North Sheridan Road, Chicago, Illinois 60626

Scott R. Perrin Regis Chemical Company, 8210 Austin Avenue, Morton Grove, Illinois 60053 Received May 10,1993@

Syntheses of the enantiomers of malathion, malaoxon, and isomalathion are reported herein. Malathion enantiomers were prepared from (R)-or @)-malic acid in three steps. Enantiomers of malathion were converted to the corresponding enantiomers of malaoxon in 52% yield by oxidation with monoperoxyphthalic acid, magnesium salt. The four isomalathion stereoisomers were prepared via two independent pathways using strychnine to resolve the asymmetric phosphorus moiety. The absolute configurations of the four stereoisomers of isomalathion were determined by X-ray crystallographic analysis of an alkaloid salt precursor. A high-performance liquid chromatography technique was developed to resolve the four stereoisomersof isomalathion, and to determine their stereoisomeric ratios. l a : R-malathlon; x = S,Y = o 1b: S-mslsthlon; X = S,Y = 0

Introduction Malathion (1) is one of the most widely used organophosphorus insecticides, and contains an asymmetric carbon center on the succinyl ligand. During insect metabolism, malathion is bioactivated to malaoxon (2) via oxidative desulfuration. Isomalathion (3) results from the thermal isomerization of malathion and has been identified in certain commercial formulations. In the transformation to both malaoxon and isomalathion, the stereogenic carbon center present initially on the succinyl ligand of malathion is maintained (Figure 1). Further, the isomerization to 3 forms an asymmetricphosphorus atom, leading to four possible stereoisomers. Whereas malathion is virtually nontoxic, the racemates of malaoxon and isomalathionare potent inhibitors of acetylcholinesterase (AChE),’ a neurotransmitter mediating enzyme (1-6). The acute toxicity of 2 and 3 to warm-blooded mammals is not fully understood, but most likely results from depression of the central nervous system as a consequence of AChE inhibition. Isomalathion was implicated in the 1976 epidemic malathion poisoning of 28OO Pakistani spraymen (including 5 deaths) during a malaria control program (2, 5).

Owing to malathion’s extensive use, the marked inhibitory potency of racemates 2 and 3, and the putative influence of stereochemistry upon the biological activity, we deemed that asymmetric syntheses of these materials would be warranted. Since 1-3 possess the same center of asymmetry on the succinyl moiety, a common synthetic pathway was envisioned. However, due to the additional phosphorusstereogenic center in isomalathion, a modified synthetic approach to these compounds was envisioned. The 0,O-diethyl malathion and 0,O-diethyl malaoxon stereoisomers were synthesized previously (7 1, but prior t

Currentaddress: IGEN Research Institute, 130 5th Ave. N., Seattle,

WA 98109.

* Abstract published in Advance ACS Abstracts, September 1,1993.

1 Abbreviations: AChE, acetylcholinesterase;MMPP, monoperoxyphthalic acid, magnesium salG m-CPBA, m-chloroperoxybenzoic acid; PEX, potassium ethyl xanthate;DMS, dimethyl sulfate.

CH3Yp’\

/

CH30

il,

Ls

a:Rmalaoxon; X = 0,Y = 0

s

2

C02E1

Zb: Smalaoxon; x

E

0,Y E O

3a: (lS, 3R).lsomalathlon; X = 0, Y L S 3 b ( l R , 3R).lsomalathlon; X = 0, Y = S X: (1R. 3S)-lsomalathlon; X = 0, Y = S 3d: (IS, JS)-laomalnthlon; X = 0, Y = S

Figure 1. Structures of malathion, malaoxon, and isomalathion.

to our preliminary account (8),the relevant 0,O-dimethyl stereoisomers of 1-3 had not been prepared. In this prior report, we outlined a method for the preparation of the malaoxon and isomalathion stereoisomers via the first synthesis of enantio-enriched malathion (8,9).Here, we report a detailed description of those syntheses along with a more expedient route to the isomalathionStereoisomers. Complete characterization of the four stereoisomers of isomalathion was obtained through correlations determined via single-crystal X-ray analysis and verified by an analytical HPLC methods2

Materials and Methods All reagents used in this study were purchased from Aldrich Chemical Co. (Milwaukee,WI), and were purified when necessary by standard literature methods. Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected. Analytical TLC was conducted on E. Merck aluminum-backed, 0.2-mm silica gel 60 Fm,TLC plates. Flash chromatography was performed with Kieselgel 60, 230-400 mesh (Merck, Germany). Elemental analysis was performed by Midwest Microlab Ltd. (Indianapolis, IN). Proton (lH), carbon (W),and phosphorus (alp)NMR spectra were recorded on a Varian VXR 300-MHz instrument in deuterated chloroform (CDC1,; except 10 in DzO). Pertinent proton frequencies are tabulated in the following order: chemical In a prior report (I&, isomalathionconfiations were assigned as subscripts (e.g., RpRc), and the P-X (X = 0, S) *-linkage was treated as a higher priority than P-X-Me 0-linkages.The more conventional numericatom stereocenternomenclaturewith the phosphorus r-bondas the lowest priority is applied here. Configurations of isomalathion stereoisomerspresented in this study correlate with those of the prior report as follows: RpRc = 1S,3R; RpSc = 1S,3S; SpRc = 1R,3R; SpSc = 1R,3S.

0893-228~/93/2106-0718$04.00/0 0 1993 American Chemical Society

Malathion, Malaoxon, and Isomalathion Enantiomers

Chem. Res. Toxicol., Vol. 6, No. 5, 1993 719

(t,J =7.2,3H), 1.25 (t,J =7.2,3H),2.84(dd, J =5.4, 17.1, lH), 3.00 (dd, J =9.0,17.1, lH), 3.77 (dd, J =3.0,15.3,6H), 4.03-4.22 and the number of hydrogens. Proton and carbon frequencies (m, 5H); NMR 6 13.9, 14.0, 37.7, 37.8, 44.98, 45.03, 54.21, 54.27, 61.08, 62.04, 169.90, 169.99; NMR 6 96.15. of spectra obtained are relative to chloroform ('H, 7.24 ppm; lac, 77.0 ppm) as an internal standard (except lOab, relative to H-O@')-Malathion (lb) was prepared from 4b as described for D, 'H 4.65 ppm, and dioxane, '3C 66.5 ppm). Phosphorus SIP la: [ a I n D = -80.0' (c = 1.25, CHC&). The NMR spectral data chemical shifts are relative to phosphoric acid (HsPOd) in CDC13 of this material were identical to those of la. as an external standard. (K)-Malaoxon (2a). (R)-Malathion(la)(0.200g, 0.606mmol) HPLC was performed using an AB1 (Analytical Kratos was dissolvedin 3 mL of CH2Cl2and added to a stirring suspension Division, Ramsey, NJ) series 400 Spectroflow pump, and a of technical grade (80%)monoperoxyphthalic acid, magnesium Spectroflow783 programmable absorbance variable wavelength salt (MMPP; 0.187 g, 0.606 mmol), in 2 mL of CH2C12 at room detector equipped with a flow cell of 8.0-mm path length and temperature. The mixture was heated to reflux for 24 h, cooled 12-pL in dead volume. All results from the UV-vis detector (215 to room temperature, and partitioned between 20 mL of ethyl nm, sensitivity range 0.1 AUFS) were recorded by a Hewlettether and 20 mL of saturated aqueous sodium bicarbonate. The Packard 3396A integrator (Avondale,PA). Injections were made aqueous layer was extracted with 20 mL of ether, and the organic by a Rheodyne 7125 injector (Bodman Chemical, Aston, PA) layers were combined, extracted with brine, dried over sodium fitted with a 20-pL sample loop. Separation of the isomers and sulfate, and concentrated to an oil. Purification via flash the determination of stereoisomeric ratios were performed on a chromatography using silica (petroleum ether:ether = 1:2) gave 25-cm X 4.6-mm4.d. Chiralpak AD (Regis Chemical Co., Morton a colorless oil (0.098 g, 52% yield): Rj = 0.13 (petroleum ether: Grove,IL) analytical column. Isopropyl alcohol and hexane used ether = 1:2); [a]=D = +46.7O (c = 0.555, CHC&);'H NMR 6 1.23 (mobile phase) were analytical grade and were purchased from (t,J = 7.0,3H), 1.27 (t,J = 7.0,3H), 2.90 (dd, J = 5.4, 17.1, lH), Bodman Chemical (Aston, PA, EM Science). The mobile phase 3.06 (dd, J = 8.7,17.1, lH), 3.81 (dd, J = 4.8,12.9,6H), 4.08-4.24 was degassed prior to use by sonication with a Bransonic Model (m, 5H); I3CNMR 6 13.91,14.03,38.11,38.18,42.38,42.43,54.07, 32 (A. Daigger Scientific, Wheeling, IL). Elution was isocratic. 54.14,54.22,61.06,62.09,169.84,170.00, 170.08;31PNMR628.3. Caution: The organophosphorus compounds used in this (5)-Malaoxon (2b) was prepared from l b as described for 2a: study are hazardous and should be handled in a well-ventilated [(Y]"D = -43.5O (c = 0.75, CHCh). The NMR spectral data of hood by trained personnel. this material were identical to those of 2a. (@-Diethylmalate and (5)-diethylmalate were prepared (1S,3@- and (lR,3R)-isomalathion diastereomers (3a, 3b) as described from (R)- and (&diethyl malate, respectively (IO). were prepared from la via dealkylation with potassium ethyl xanthate and realkylation with dimethyl sulfate as previously Diethyl (5)-O[(Trifluoromethyl)sulfonyl]malate (4a). reported (4): [ a I n D = +41.3' (c = 1.25, CHCl3). Trifluoromethanesulfonicanhydride (2.00 mL, 11.9 mmol) was dissolved in 10 mL of CH2Cl2 under an Ar(g) blanket and chilled (1R,35)- and (lS,3S)-ieomalathion diastereomers (3c, 3d) were prepared from l b via dealkylation with potassium ethyl to -78 'C. In a separate flask, (5')-diethylmalate (2.00 g, 10.6 mmol) and 2,6-lutidine (1.24 mL, 10.6 mmol) were dissolved in xanthate and realkylation with dimethyl sulfate as previously 10 mL of CHzClz at room temperature, and added dropwise to reported (4): [ a I n ~ -40.0' (C = 0.75, CHC&). the anhydride solution over 15 min. After the addition was S M e t h y l [2-(Ethoxycarbonyl)pyrrolidino]phosphorocomplete, the reaction mixture was brought to 0 OC for 1h and choridothioate (6a,b). A 5-mL benzene solution of ethyl allowed to warm slowly to room temperature until TLC indicated L-prolinate (0.85 g, 5.94 mmol) and triethylamine (0.82 mL, 5.94 the consumption of (&-diethylmalate (ca. 2 h). The reaction mmol) was added dropwise over 15 min to a 7.5-mL benzene mixture was concentrated to an oil followed by the addition of solution of 5'-methylphosphorodichloridothioate (5) (0.98g,5.94 ether to precipitate the lutidinium salt. The salt was filtered in mmol) at room temperature. The reaction was halted at 1h by vacuo and washed three times with 50 mL of ether. The fitrate the addition of 15 mL of ether. Filtration of the amine salt and was concentrated to an oil (3.47 g, 100% crude yield) and used concentration in vacuo left an oil, which was purified by flash immediately for conversion to enantio-enriched malathion (la): chromatography using silica (ether) to give a 1:l ratio of [ a I z 6=~+32.6' (C 1.39, CHCl3); 'H NMR 6 1.29 (t,J = 7.2, diastereomers 6a and 6b in 77 % yield Rf = 0.42 and 0.3 (ether). 3H), 1.34 (t,J =7.2,3H), 3.06 (d, J = 5.7,2H),4.22 (dq, J - 2.1, Data for 6a (nonpolar band): [a]% = -52.6O (c = 1.04, CHCh); 7.2,2H),4.33 (dq, J=2.1,7.2,2H),5.49(t,J=6.0,lH);'SCNMR 'H NMR 6 1.28 (t, J = 7.1, 3H), 1.95-2.3 (m, 4H), 2.50 (d, J = 6 13.79, 13.92, 36.81, 61.82, 63.17, 78.80, 112.45, 116.23, 120.47, 18.2,3H), 3.38-3.46 (m, 2H), 4.19 (9,J = 7.1, 2H), 4.48 (dt, J = 124.70, 166.03, 167.52. 3.6,8.1,1H); NMR 6 37.84,37.94. Data for 6b (polar band): Diethyl (R)-0-[(trifluoromethyl)sulfonyl]malate (4b) [ C Y ] ~ D -48.2' (C = 1.15, CHCh); 'H NMR 6 1.30 (t,J = 7.2,3H), was prepared as indicated above for 4a starting with 1.98-2.29 (m, 4H), 2.40 (d, J = 18,3H), 3.51 (9,J = 6.6,2H), 4.21 (dq, J =1.8,7.2,2H),4.35 (dt,J = 3.3,8.5,1H);31PNMR638.17, (R)-diethylmalate: [(Y]%D = -30.1' (c = 1.71, CHCls). The NMR spectral data were identical to those of 4a. 38.21. Sodium 0,Odimethyl phosphorodithioate was prepared (&S)-Diethyl Mercaptosuccinate (7). A 1:2 water/acetone by reaction of equimolar concentrations of (CHsO)zP(S)SH(II) solutionof sodium hydrogen sulfide (0.142 g, 2.5 mmol) was added and sodium carbonate in methanol. Following concentration of to a 2.0-mL acetone solution of diethyl bromosuccinate (0.5 g, 1.98 mmol) (7)at 0 OC. After 2 h, the acetone was removed by this solution in uacuo,crystallizationwas achieved by the addition of ethyl ether. rotary evaporation, and the product extracted from the aqueous solution with three 10-mL portions of ether. Kugelrohr distil(R)-Malathion (la). Triflate 4a (3.47 g, 10.6 mmol) was lation (55 OC, 1mmHg) gave a colorless oil in 44% yield 1H dissolved in 20 mL of THF and chilled to 0 OC under Ar(g) NMR 6 1.22 (t, J = 7.1, 3H), 1.26 (t, J = 7.1, 3H), 2.16 (d, J = atmosphere. Sodium 0,O-dimethyl phosphorodithioate (2.86 g, 9.4, lH), 2.71 (dd, J = 6.0,16.9, lH), 2.96 (dd, J = 9.0,16.9, lH), 15.9 mmol) was dissolved in 10 mL of THF, chilled to 0 OC, and 3.70 (dt, J = 6.0,9.3, lH), 4.12 (q, J = 7.1,2H), 4.18 (4, J = 7.1, added dropwise to the triflate solution over 15min. The reaction 2H). mixture was allowed to warm slowly to room temperature, stirred for 2 h, and partitioned between 50 mL of ethyl ether and 50 mL S-[ 1,2-Bis(ethoxycarbonyl)ethyl] S M e t hyl [2-(Et hoxyof water. The ether layer was separated and the aqueous layer carbonyl)pyrrolidino]phosphorodithioate (8). Phosphorore-extracted with 50 mL of ether. The organic layers were chloridothioate 6a (0.10 g, 3.68 mmol) was dissolved in 0.5 mL combined, extracted with 50 mL of brine, dried over sodium of THF followed by the addition of diethyl mercaptosuccinate (0.083 g, 4.05 mmol) and triethylamine (0.056 g, 4.05 mmol). The sulfate, and concentrated to an oil. Purification via flash chromatography using silica (petroleum ether:ether = 1:l)gave solution was stirred at room temperature under argon for 20 h a colorless oil (2.79 g, 80% yield): Rf = 0.20 (petroleum ether: whereupon the mixture was filtered, concentrated to an oil, and ether = 1:l); [a]% = +79.7' (c = 1.25, CHCls); 1H NMR 6 1.22 purified by flash chromatography (ether) to afford a 34.5 % yield. shift (6 in parts per million), multiplicity

(8,

singlet; d, doublet;

t, triplet; q, quartet; m, multiplet), coupling constant (Jin hertz),

720 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 The stereoisomerswere not separated: Rf= 0.27 (ether); lH NMR 6 1.22-1.32 (m, 9H), 1.94-2.23 (m, 4H), 2.37 (dd, J = 1.2, 15.3, 3H), 3.09-3.15 (m, 2H), 3.43-3.51 (m, 2H), 4.10-4.46 (m, 8H). 19-Methylstry chninium S-[(R) - 1,2-Bis(ethoxy carbon y 1)ethyl] @Methyl Phosphorodithioate (9a,b). Malathion la (2.0 g, 6.06 mmol) and (-)-strychnine (2.23 g, 6.66 mmol) were dissolved in 30 mL of methanol, and heated to reflux for 16 h. The solvent was removed, and the remaining waxy solid was taken up in 25 mL of chloroform and diluted with 480 mL of ethyl acetate, followed by 70 mL of ether. The solution was warmed to effect solution and stored at 0 "C. After 4 days, the first crop of crystals (1.64 g) was recovered by filtration and triturated with 75 mL of ethyl ether. The filtrate was diluted further with ether (125mL), and stored at room temperature for 2 days, which produced a flocculent second crop of crystals. 31P NMR analysis showed a 9a to 9b diastereomeric ratio of 7:3 for the first crop, whereas a 1001ratio of the 9b to 9a diastereomers was found for the second crop. The first crop was recrystallized from a 1:6 methanol/ether solution, and showed a 93:7 ratio of 9a to 9b. A third recrystallization gave a 1OO:l ratio of 9a to 9b by dissolving 1.37 g in 80 mL of methanol followed by the addition of 320 mL of ethyl ether. Data for 9a: mp 181-182 'c (8);[(YIz2D = +16.1' (C = 0.31, CHC13);'H NMR 6 1.18 (t,J = 7.2,3H), 1.20

Berkman et al. (m, 4H), 3.34 (8, lH), 3.60 (d, 14.9, 3H), 3.81 (8, 4H), 4.01-4.13 (m, 8H),4.20-4.42 (m, 5H), 6.55 (s, lH), 7.14 ( t , J = 7.5, lH), 7.30 (t,J = 7.4, lH), 7.51 (d, J = 7.3, lH), 8.06 (d, J = 8.0, 1H); l3C NMRG 14.17,14.23,25.29,29.74,38.60,38.63,39.67,42.08,44.39, 44.43,47.07,53.21,53.39,53.48,55.86,58.80,60.66,61.26,61.86, 64.22,64.38,75.08,116.52,122.59,124.85,127.72,130.17,132.75, 136.69,141.66,168.63,171.06,171.63; 31PNMR 6 68.12. Data for compound 9d: mp 158-159 "C (8); [ ( ~ 3 2 % = -61.7' (c = 0.31, CHCls); 'H NMR 6 1.18 (t, J = 7.1, 6H), 1.37 (d, J = 10.3, lH), 1.66 (d, J = 15.1, lH), 2.16 (dd, J = 5.4,13.1, lH), 2.27-2.38 (m, lH), 2.68 (dd, J = 2.7,17.8, lH), 2.93-3.17 (m, 4H), 3.34 (8, lH), 3.62 (d, J = 15.1,3H), 3.83 (e, 4H), 4.01-4.18 (m, 8H), 4.21-4.33 (m, 2H), 4.43 (s, 3H), 6.58 (8, lH), 7.14 (t,J = 7.4, lH), 7.31 (t,

J=7.8,1H),7.52(d,J=7.4,1H),8.07(d,J=7.9,1H);l3CNMR 6 14.17,14.23,25.34,29.72,38.50,39.67,42.06,44.45,44.49,47.10, 53.20,53.39,53.48,55.78,55.80,58.81,60.65,61.28,61.81,64.23, 74.99,116.48,122.64,124.81,127.81,130.12,132.80,136.64,141.65,

168.66, 171.02, 171.43; 3lP NMR 6 67.18. 19-Methylstrychninium 0,s-Dimethyl Phosphorodithioate (lOa,b). Isolation of loa. A modificationof a prior procedure (8)is reported here. (-)-Strychnine (24.40g, 0.073 mol) was added to a stirring solution of O,O,S-trimethylphosphorodithioate(12.04 g, 0.07 mol) in methanol (180 mL) and heated to reflux for 24 h. (t,J=7.1,3H),1.38(dt,J=2.8,10.5,1H),1.69(d,J=16.1,1H), Upon cooling, the solution deposited the first crop of crystals of 2.17 (dd, J = 4.6,14.2, lH), 2.33 (dt, J = 7.3,13.6, lH), 2.68 (dd, loa. The mother liquor was retained (uide infra). The crystals J = 2.8, 17.7, lH), 2.95-3.18 (m, 4H), 3.34 (8, lH), 3.62 (d, J = were triturated with methanol and recrystallized twice from 100% 14.9,3H), 3.83 (s,4H),4.00-4.20 (m,8H),4.21-4.49 (m,5H),6.57 methanol: mp 202-203 'C (8);[cy]% = +15.8' (c = 0.545, MeOH); (8, lH), 7.15 (t, J = 7.7, lH), 7.32 (t, J = 7.4, lH), 7.50 (d, J = ~HNMR61.38(dt,J=3.1,10.7,1H),1.56(d,J=14.8,1H),1.97 7.1, lH), 8.08 (d, J = 8.0, 1H); 13C NMR 6 14.19, 14.24, 25.30, (d, J = 2.8, lH), 2.02 (d, J = 14.1, 3H), 2.14 (dt, J = 7.6, 13.7, 29.76,38.62,38.65,39.68,42.11,44.40,44.44,47.08,53.22,53.41,lH), 2.44-2.59 (m,2H), 2.97 ( d d , J = 8.2, 17.8,1H),3.27 (s,3H), 53.50,55.87,55.91,58.81,60.66,61.27,61.84,61.88,64.23,64.38,3.30 (e, lH), 3.44 (d, J = 14.6,3H), 3.56-3.76 (m, 3H), 3.95-4.25 64.42,75.13,116.56,122.54,124.87,127.68,130.22,132.73,136.74, (m, 5H), 4.35 (dt, J = 3.2, 8.3, lH), 6.28 (e, lH), 7.12 (t,J = 7.6, 141.68, 168.63, 171.08, 171.66; 31P NMR 6 68.10. Data for 9b: lH), 7.25 (t, J = 7.7, lH), 7.37 (d, 7.6, lH), 7.78 (d, 8.1, 1H);13C mp 159-160 'C (8);[cy]% = +32.2' (c = 0.25,CHC13); 'H NMR NMR6 13.53,13.58,24.18,28.87,38.82,40.68,46.03,48.83,52.82, 6 1.18 (t,J=7.1,3H), 1.19 (t,J = 7.1,3H), 1.38 ( d t , J = 2.9,10.5, 53.09,53.18,54.40,58.39,61.63,63.78,64.10,74.71,76.36,115.82, lH), 1.67 (d, J = 15.4, lH), 2.17 (dd, J = 5.1, 13.3, lH), 2.33 (dt, 122.94, 125.43, 129.08, 129.93,132.68, 135.60, 140.65,171.32; 31P J=6.8,13.9,1H),2.68(dd, J = 2.8,17.7,1H),2.95-3.18(m,4H), NMR S 78.87. 3.35 (8, lH), 3.62 (d, J = 15.0,3H), 3.84 (s,4H),4.00-4.17 (m, 8H), Isolation of lob. The mother liquor from 10a was stored at 4.21-4.35 (m, 2H), 4.43 (d, J = 13.1, 3H), 6.59 (8, lH), 7.15 (t, J 0 "C overnight and gave amorphous crystals that were recrys= 7.5, lH), 7.31 (t, J = 7.8, lH), 7.50 (d, J = 7.1, lH), 8.07 (d, tallized from warm 95% ethanol. Following filtration of this J = 8.0, 1H); 13C NMR 6 14.20, 14.24, 25.29, 29.75, 38.53, 39.70, first crop, the mother liquor was concentrated in uacuo to a solid 42.10,44.43,44.47,47.11, 53.20, 53.39,53.48, 55.82, 58.82,60.65, and again recrystallized from warm 95 % ethanol to give a second 61.25, 61.16, 64.24, 64.32, 75.09, 116.52, 122.59, 124.82, 127.76, crop of crystals. The two crops were combined and recrystallized 130.16,132.77,136.67, 141.67,166.65,171.03, 171.42, 171.50; 31P once more from warm 95% ethanol to give lob: mp 253 'C (8); NMR 6 67.20. Anal. Calcd for C31HdlN20gPS2: C, 56.01; H, [a]22~ = -13.5' (c = 0.63, MeOH); 1H NMR 6 1.36 (d, J = 10.7, 6.22; N, 4.21. Found: C, 56.26; H, 6.23; N, 4.32. lH), 1.55 (d, J = 15.7, lH), 1.91 (d, J = 12.2, lH), 2.01 (d, J = 19-MethylstrychniniumS-[(S)-1f-Bis( et hoxycarbony1)14.1,3H),2.12 ( d t , J = 7.8,13.7, lH), 2.44-2.57 (m, 2H), 2.96 (dd, ethyl] 0-Methyl Phosphorodithioate (9c,d). Malathion l b J = 8.2, 18.1, lH), 3.27 (8, 4H), 3.44 ( d , J = 14.5,3H), 3.53-3.76 (2.0 g, 6.06 mmol) and (-)-strychnine (2.23 g, 6.66 mmol) were (m,3H),3.93-4.25(m,5H),4.34(dt,J=3.1,8.3,1H),6.28(s,lH), dissolved in 30 mL of methanol, and the mixture was brought 7.12 (t, J = 7.6, lH), 7.24 (t,J = 7.6, lH), 7.36 (d, J = 7.6, lH), to reflux for 16 h. The solvent was evaporated, and the waxy 7.76 (d, J = 8.1,lH); '3C NMR 6 13.56,13.60,24.21,28.89,38.84, solid was taken up in a minimum of methanol followed by the 40.72,46.06,52.83,53.11,53.20,54.44,58.40,61.63,63.80,64.10, addition of 300 mL of ethyl acetate and 100 mL of ether. The 74.72,76.36,115.83,122.98,125.45,129.09,129.95,132.67,135.63, solution was briefly heated to reflux, and following return to 140.66, 171.29; 3lP NMR 6 78.79. room temperature, the solution deposited the first crop of crystals Method A. General Procedure for the Synthesis of (ca.l.Og), whichwere isolated by vacuum filtration and triturated Isomalathion Stereoisomers (3) from Strychnine-Desmethwith 120 mL of ether. The filtrate deposited a second crop of yl Malathion Salts (9). To a 0.12 M stirring solution of 9 in crystals at room temperature that were filtered and washed with methanol/acetone (1:3) was added dimethyl sulfate (1 equiv), ethyl ether. 3lP NMR analysis of the first crop showed a 7:3 ratio and the solution was heated to reflux for 3 h. After cooling, 3 of 9c to 9d, whereas the second crop gave a 1001 diastereomeric mL of ethyl ether was added to precipitate the sulfate salt. The ratio of 9d to 9c. The first crop was recrystallized from 450 mL reaction mixture was filtered, concentrated to an oil, and purified of a 1:6 methanol/ether solution to give 0.82 g of an 85:15 via flash chromatography using silica (petroleum ether/ether, diastereomeric ratio of 9c to 9d. Further recrystallization from 1:2) to give a colorless oil. 300 mL of a 1:3methanol/ether solution gave a 946 diastereomeric Method B. General Procedure for the Synthesis of ratio of 9c to 9d. A final recrystallization to afford a 1OO:l Isomalathion Stereoisomers (3) from Strychnine-0,sdiastereomeric ratio of 9c to 9d was achieved by dissolving 0.550 Dimethyl Phosphorodithioate Salts (10). To a 0.2 M stirring g in 16 mL of ethylene glycol and 27 mL of absolute ethanol suspension of 10 in CHaCN, 4 (1equiv; 0.78 M CH&N solution) followed by the addition of 250 mL of ethyl ether. Data for was added dropwise over 5 min. After 1 h, the solution was compound 9c: mp 189 'c (8);[(YIuD = -45.1' (c = 0.26, CHCl,); 1HNMR61.17(t,J=7.1,3H),1.19(t,J=6.9,3H),1.37(d,Jdiluted 2-fold with ethyl ether to precipitate the strychninium triflate salt. The mixture was filtered in uacuo, and the filtrate = 10.5, lH), 1.66 (d, J = 15.6, lH), 2.18 (dd, J = 5.5, 14.3, lH), was concentrated in vacuo to an oil and purified by gravity 2.33 (dt, J = 7.0,13.9, lH), 2.67 (dd, J = 2.7,17.8, lH), 2.94-3.17

Malathion, Malaoxon, and Isomalathion Enantiomers chromatography using silica (petroleum ether/ethyl ether, 1:l) to give a colorless oil. (lS,3R)-Isomalathion (3a). Method A from 9a (0.313,0.471 mmol) gives 0.122 g (79%). Method B from 10a (1.10 g, 2.17 mmol) and 4a (0.700 g, 2.17 mmol) gives 0.178 g (25%): [a12% = +58.6' (C = 0.64, CHCls);'H NMR 6 1.23 (t, J = 7.1,3H), 1.27 (t, J = 7.1, 3H), 2.37 (d, J = 16.8, 3H), 2.94 (dd, J = 5.2, 17.1, lH), 3.08 (dd,J = 8.8,17.1, l H ) , 3.85 (d,J = 13.7,3H), 4.09-4.26 (m,5H); '3C NMR d 13.13,13.17,13.96,14.07,37.97,38.02,43.35, 43.40, 54.00, 54.11, 61.11, 62.18, 169.89; 3lP NMR 6 56.92. (lR,3R)-Isomalathion(3b). MethodAfrom9b(0.313g,0.471 mmol) gives 0.098 g (63%). Method B from 10b (1.10 g, 2.17 mmol) and 4a (0.700 g, 0.217 mmol) gives 0.220 g (31%): [a]2% =+42.3'(~=0.64,CHCls);'HNMR61.23(t, J=7.1,3H),1.27 (t, J = 7.1, 3H), 2.36 (d, J = 16.9, 3H), 2.97 (dd, J = 5.2, 17.2, lH), 3.08 (dd,J = 8.9,17.1, l H ) , 3.84 (d,J = 13.6,3H),4.0&4.25 (m,5H); 13C NMR 6 13.38,13.43,13.98,14.08,38.16,38.21,43.24, 43.27,53.83,53.93,61.12,62.18,169.93, 170.01;31PNMR658.38. (lR,3S)-Isomalathion (3c). MethodAfrom9c(0.200g,0.301 mmol) gives 0.066 g (66%). Method B from 10b (1.57 g, 3.11 mmol) and 4b (1.00 g, 3.11 mmol) gives 0.612 g (60%): [a]2%= -57.6O (c = 0.50, CHCl3). The NMRspectraldataof thismaterial were identical to those of 3a. (lS,3S)-Isomalathion (3d). MethodA from9d (0.300g, 0.452 mmol) gives 0.107 g (72%). Method B from 10a (1.57 g, 3.11 mmol) and 4b (1.0 g, 3.11 mmol) gives 0.523 g (51%): = -44.8" (c = 0.58, CHCls). The NMRspectraldataof this material were identical to those of 3b.

Results and Discussion Compounds 1-3 have a common stereogenic center at the succinyl carbon. Therefore, a divergent synthetic strategy was sought that uses a central intermediate containing this center. Within this strategy, enantiomers of malathion were first desired, since the stereoisomers of both 2 and 3 could be subsequently prepared through simple functional group transformations as shown for (R)malathion (eqs 1 and 2).

CH,0-~,s~C02E1

{

CHIO

l a : Rmalathlon

0

CO Et

CH,0-!\sLC02Et

(1)

20: R-malaoxon 0

COEt

CH,0*!IsLCo2Et CHIS

(2)

3a: (lS,3R)-lsomalathlon and 3 b (1 R,3R)-isomalalhlon

a. MMPP, b. PEX then OMS

An earlier method described the preparation of enantioenriched 0,O-diethyl malathion by reaction of diethyl sodium phosphorodithioate with (R)- or @)-diethyl bromosuccinate (7).Our application of this reaction with dimethyl sodium phosphorodithioate and (SI-diethyl bromosuccinate seemed straightforward, but the identical reaction conditions failed to provide malathion. Variations in solvent, cation, temperature, order of reagent addition, and time also failed to give malathion. Apparently, an elimination reaction predominated in the case of the dimethyl analog as evidenced by the formation of diethyl maleate and diethyl fumarate. An additional concern of this modified procedure is that two inversions of configuration are conducted (formation of the bromide with NaBr and S N displacement ~ by the thioate), which may reduce the enantio-enrichment. We desired to install the carbon-sulfur bond in a single inversion reaction, and envisioned conversion of the (R)-

Chem. Res. Toxicol., Vol. 6, No. 5, 1993 721

or (SI-diethyl malate hydroxyl to a suitable leaving group followed by direct displacement. The tosylate, mesylate, acetate, trifluoroacetate, and triflate of @)-diethyl malate (4a)were synthesized (10)and reacted directly with freshly prepared dimethyl phosphorodithioate, sodium salt (11). Despite complete failure of the tosylate, mesylate, and acetate to give product, the triflate reacted cleanly at -78 "C to afford @)-(+)-malathion (la) in 67% overall yield from @)-malic acid (eq 3). Likewise, @)-(-)-malathion

Smallc mold

la: R.malathlon

4.

(lb) was prepared from (R)-malic acid. All physical and spectral properties for la and lb were identical, except optical rotations that were approximately equal and opposite. Attempts to confirm the enantiomeric purity of la and lb by spectral means or derivatization failed. The synthesis of enantio-enriched malaoxon was achieved in a manner similar to that reported for the racemic material (12). m-Chloroperoxybenzoic acid (mCPBA) oxidation of the malathion enantiomers (la, lb) led to a 25% yield of the corresponding malaoxon enantiomers. Since the recovery was poor, alternative oxidanb were examined including HzOz, (CF&02)20, t-BuOOH, KI04, and MMPP. These reagents generally resulted in extensive decomposition with the exception of MMPP (9),which furnished the corresponding enantiomers of 2 in 50% yield (eq 1). Diastereomeric mixtures of isomalathion (3a, 3b or 3c, 3d) were prepared from malathion enantiomers (la, lb) via dealkylation with potassium ethyl xanthate (PEX) followed by realkylation with dimethyl sulfate (DMS) ( 4 ) as shown for 3a and 3b (eq 2). However, all attempts at a preparative separation of these mixtures failed, and consequently this approach was abandoned. Our recent successful synthesis of isoparathion methyl via the Lewis acid promoted methanolysis of precursory diastereomeric proline phosphorodithioates (13) encouraged us to modify this procedure for the preparation of isomalathion enantiomers, despite the fact that this approach would require several reactions at the stereogenic centers. Diastereomeric proline phosphorodithioates 8 that bear methylthio and diethyl thiomaleate ligands were prepared according to Scheme I. Methyl phosphorodichloridothioate (51, prepared from thiophosphoryl chloride, was reacted with ethyl L-prolinate to give the diastereomeric phosphorochloridothioates 6a and 6b (14) that were separable by flash chromatography using silica. Scheme I. Failed Synthesis of Isomalathion from Proline Diastereomer Precursors 1.pro11n. CH,S-P(0)CII

5

0

-=%

II

P

F0ZEt

$O,Et

CH,S

W6b

a. dkthyr mercaptosucclnste, Na+ salt, b. MeOH, acld

8

k lhloleeter cleavage and recovered 8

Racemic diethyl mercaptosuccinate (7) was prepared by reaction of diethyl bromosuccinate with sodium hydrogen sulfide. In preliminary experiments, the nonpolar phosphorochloridothioate diastereomer 6a was reacted with racemic diethyl mercaptosuccinate to afford the

722 Chem. Res. Toxicol., Vol. 6, No. 5, 1993

Berkman et al.

desired diastereomeric pair of proline phosphorodithioates 8 as confirmed by NMR. Despite an extensive investigation of conditions, all attempts to replace the proline moiety with a methoxy group failed, including Lewis acid methanolysis (13). Thin-layer chromatography and smell suggestedthat expulsion of one of the thiolgroups probably occurred. Although several phosphorothioates had been resolved through fractional crystallization of the alkaloid salts of cinchonidine, a-phenylethylamine, quinine, brucine, and strychnine (15),only scant examples report the resolution of phosphorodithioates with alkaloids. Since an efficient synthesis of malathion enantiomers was established, alkaloid resolution of the stereogenic phosphorus should be possible. Dealkylation of l b with strychnine formed the methylstrychninium desmethyl @)-malathionsalt (9c, 9d). Repeated fractional crystallization of this diastereomeric mixture afforded the desired resolved, crystalline salts 9c and 9d (Scheme 11). Similarly, R-malathion (la) was converted to the strychnine salts 9a and 9b (not shown). Throughout this procedure, 31PNMR (Table I) was used to evaluate the diastereomer ratios of the recrystallized samples. Doping experiments were conducted and showed that 2 % of a contaminating isomer could be observed by 3IP NMR. We were unable to find a suitable peak or peaks in the l3C or lH NMR that could distinguish between the stereoisomers. Scheme 11. Strychnine Resolution of Desmethyl (@-Malathion and Its Conversion into the Corresponding Isomalathion Enantiomers S

(In,

03

COzEt

It

CH,O/[

stereogeniccenter, yielded the four individual isomalathion stereoisomers corresponding to the configurations known from the precursor salts (Scheme 11). Approximately 1020% of 0-alkylation with DMS occurred to give malathion, which was separated by column chromatography using silica and recycled. We also were fortunate to obtain crystals of 9a that were suitable for X-ray crystallographic analysis (16), which both identified the absolute configuration of this isomer as "S"a t phosphorus (Figure 21, and allowed the relative assignment of the remaining configurations. A more direct route was sought for multigram preparation of 3 for biological evaluation. Since the diastereomeric methylstrychninium salts of 0,s-dimethyl phosphorodithioate were resolved previously by fractional we presumed that reaction of these crystallization salts with either triflate enantiomer (4a or 4b) would provide the individual stereoisomers of 3 (Scheme 111).In this approach, control over the stereogenic carbon and phosphorus centers could be individually achieved and still preserve chiral integrity. In part, this method proved to be superior to the previous method (Scheme 11) since the time required for fractional crystallization was minimized and the yields for the resolved salts of 10 (45%) were an improvement over the preparation of 9 (20%). Again, 3lP NMR was used to analyze the isomer composition throughout the recrystallization process. The coupling reaction proceeded in 2540% yield, lower than the alkylation reaction of 9 with DMS.

\

CH,O

I b 5-mnlathlon 1. stlychnlm

C27

C26

Figure 2. ORTEP diagram of (S)-S-[(R)-1,2-bie(ethoxycarbony1)ethyllO-methylphosphorothioate. The methylstrychninium counterion is not shown. Scheme 111. Strychnine Resolution of 0,SDimethyl Phosphorothioate and Its Conversion into the Corresponding Isomalathion Enantiomers 30: (IS, 3R)-lsomalathlon

3d: (lS, 3S)iromalathlon

3c: ( I R , 3S)-isomalalhlon

Table I. Physical Data. compound [al2% la [(It)-malathion] +79.7 (1.25) l b [(a-malathion] - 80.0(1.25) 2a [(It)-malaoxon] +46.7 (0.55) 2b [(S)-malaoxon] - 43.5 (0.75) 3a [(lS,3It)-isomalathion] +58.6 (0.64) 3b [(lR,3It)-isomalathion] +42.3 (0.64) 3c [(lS,3S)-isomalathionl - 57.6 (0.50) 3d [(lS,3S)-isomalathion] - 44.8 (0.58) 9a [(1S,3R)-saltl +16.1 (0.31) 9b [(lIt,3It)-salt] +32.2 (0.25) 9c [(lIt,3S)-saltI 45.1 (0.26) 9d [(lS,3S)-~altI - 61.7 (0.58) loa [(lR)-salt] +15.8 (0.54) 10b [(lS)-~alt] - 13.5 (0.63)

-

SIP

34:(IS,3S)-laomalathion

NMR ( 8 ) mpb ("C) 96.15 96.15 27.96 27.96 56.92 58.38 56.92 58.38 68.10 67.20 68.12 67.18 78.87 78.79

CHIO

3 b (IR, 3R).Isomslathlon

0 , S-dlmathyl phoaphorodithloate CH,O

3 ~ (IR, : 3S).1aomaiathlon

lob

a. 8ftyChnln~,b. fracflonal c ~ ~ f n l l l z 8 t l o n

181-182 159-160 189 158-159 202-203 253

a Rotationsperformed in CHCk except 10 in MeOH, concentrations are given in parentheses, g/100 mL. Reference 8.

Alkylation of the strychnine salts 9a-9d with DMS, presumed to occur without disturbing the phosphorus

Routine purity analyses of the isomalathion stereoisomers of 3 were conducted by 31PNMR, specific rotations, and HPLC (Tables I and 11). In conjunction with the 31P NMR studies and single-crystal X-ray analysis, HPLC analyses substantiated not only the stereoisomeric ratios but also the assignment of absolute configuration. All four isomalathion stereoisomers were chromatographically resolved using the Chiralpak AD chiral stationary phase (Figure 3). Identification of the peaks arising

Chem. Res. Toxicol., Vol. 6, No. 5, 1993 723

Malathion, Malaoxon, and Zsomalathion Enantiomers

purchase of the Varian VXR 300-MHz NMR, and Regis Chemical Co., Morton Grove, IL, for the use of the HPLC and chromatographic supplies. The authors also wish to express their thanks to Dr. Christopher J. Welch (Regis Chemical) for insightful discussions and helpful contributions to the paper.

References Toia, R.F., March, R.B., Umetsu, N., Mallipudi, N. M., Allahyari, R., and Fukuto, T. R. (1980) Identification and toxicological evaluation ofimpuritiesintechnicalmalathionandfenthion. J.Agric. Food Chem. 28,599-604. Iyer, V., and Parmar, B. S. (1984) The isomalathion problem a review. Int. J. Trop. Agric. 11, 199-204. Wester, R.C., and Cashman, J. R.(1989) Antiinfective properties of malathion. In Sulfur-Containing Drugs and Related Organic Chemicals: Chemistry, Biochemistry and Toxicology (Damani, L. A., Ed.) Vol. 3, pp 181-196, Ellis Horwood Ltd.,Chicheeter, U.K. Thompson, C. M., Frick, J. A., Natke, B. C., and Hansen, L. K. (1989) Preparation, analysis, and anticholinesterase properties of 0,O-dimethyl phosphorothioate isomerides. Chem. Res. Toxicol. 2, 386-391. Baker, E. L., Zack, M., Miles, J. W., Alderman, L., Warren, McW., Dobbin, R. D., Miller, S., and Teeters, W. R. (1978) Epidemic malathion poisoning in Pakistan malaria workers. Lancet I, 31-34. Metcalf, R. L., and March, R. B. (1953)The isomerization of organic thionophosphate insecticides. J. Econ. Entomol. 46, 288-294. Hassan, A., and Dauterman, W. C. (1968) Studies on the optically active isomers of 0,O-diethyl malathion and 0,O-diethyl malaoxon. Biochem. Pharmacol. 17, 1431-1439. Berkman, C. E., and Thompson, C. M. (1992) Synthesis of chiral malathion and isomalathion. Tetrahedron Lett. 33, 1415-1418. Jackson, J. A., Berkman, C. E., and Thompson, C. M. (1992) Stereoselectiveand chemoselectiveoxidation of phosphorothionatee using MMPP. Tetrahedron Lett. 33, 60616064. Cohen, S. G., Neuwirth, Z., and Weinstein, S.Y. (1966)Association of substrates with a-chymotrypsin, diethyl a-acetoxpuccinate, and diethyl malate. J. Am. Chem. SOC. 88,5306-5315. Kabachnik, M. F., and Mastryukova, T. A. (1953)Organophosphorus comDounds.Dialkvl dithioDhosDhates. Izu.Akad. Nauk SSSR.Otd. . . Khim. Nauk 121-i25. (12) Bellet, E. M., and Casida, J. E. (1974)Products of peracid oxidation of organothiophosphorus compounds. J.Agric. Food Chem. 22,207-

-

40 5 0 6 0 7 0 8 0 9 0

time (min)

HPLC

Figure 3. Representative

showing the separation of

isomalathion stereoisomers. The order Table 11. HPLC Analyses of the Stereoisomeric Ratios of Isomalathion**

1R,3R 1S,3R 1S,3S 1R,3S 3b [(lR,3R)-isomalathion] 94.1 3.1 0.7 1.8 16.9 3a [(lS,3R)-isomalathion] 0.8 93.0 2.4 3.8 18.6 3d [(lS,3S)-isomalathionl 13.2 2.5 84.0 0.3 21.8 3c [(lR,3S)-isomalathion] 1.0 0.8 98.2 24.5 compound

a Column, Chiralpak AD; mobile phase, 10:90 IPA/hexane; flow rate, 0.4mLlmin; injection volume, 20& wavelength, 215 nm; sample concentration, 3.0 mg/mL. In chromatographic elution order. c Capacity factor for each isomalathion stereoisomer.

*

from individual isomalathion stereoisomers was based upon comparison with purified samples obtained by fractional crystallization and available from a previous study (8) (Table 11). Confirmation of absolute configuration was obtained by the measurement of the capacity factor for each stereoisomerically enriched isomalathion sample.

Summary Stereoisomers of both 2 and 3 were derived from malathion. Improved yields for the transformation of 1 to 2 were achieved with MMPP in place of m-CPBA. Two independent, successful methods are reported for the preparation of3 from enantiomers of malathion. The first method aided the determination of absolute configuration through X-ray crystallography. The second method has the advantage of simplicity with greater sufficiency. The preparation of the stereoisomers of 1-3 now permits a more thorough investigation of their toxic action (18,19).

Acknowledgment. Kind financial support was provided by the National Institute of Environmental Health Sciences (Grant ES04434) and is gratefully acknowledged. The authors thank Loyola University of Chicago for the

211.

(13) Ryu, S., Jackson, J. A., and Thompson, C. M. (1991) Methanolysis of phosphoramidates with boron trifluoride-methanol complex. J. Org. Chem. 56,4999-5002. (14) Koizumi, T., Kobayashi, Y., Amitani, H., and Yoshii, E. (1977) A practical method of preparing optically active dialkyl phenyl phosphates. J. Org. Chem. 42, 3459-3460. (15) Valentine, D. (1984) Preparation of the enantiomers of compounds containing chiral phosphorus centers. In Asymmetric Synthesis (Morrison.J. D.. and Scott. J. W. Eds.) Vol. 4.-. . ~ 0263-312. Academic Press Inc.; Orhhdo, FL. ' (16) Berkman, C. E., Femandez, E. J., Thompson, C. M., and Pavkovic, S. F. (1993) Structure of. N-methylstjchninium' (S)-[S-1, (R); 2-dicarbethoxyethyll-0-methylphosphorodithioate.Acta Crystallogr. C49, 554-556. (17) Hilgetag,G. Optishaktivedithiophosphorsaureeater.(1969)Z.Chem. 9, 310-311. (18) Berkman, C. E., Ryu,S., Quinn, D. A., and Thompson, C. M. (1993) Kinetics of the post-inhibitory reactions of acetylcholinesterase poisoned by chiral isomalathion. A surprising non-reactivation induced by the Rp stereoisomers. Chem. Res. Toxicol. 6, 28-32. (19) Berkman, C.E.,Quinn,D. A.,andThompson, C.M. (1993)Interaction of acetylcholinesterase with the enantiomers of malaoxon and isomalathion. Chem. Res. Toxicol. (following paper in this issue).