Eur. J. Med. Gem. 23 (1988) 63-68 0 Elsevier, Paris

63

Original paper

Polymethylenedioxy bis(2-hydroxyiminomethylpyridinium) as in vitro reactivators of organophosphorous inhibited eel acetylcholinesterase Pierre DEMERSEMAN’, Daniel KIFFER’, Laurent DEBUSSCHE’, Claude LION3, RenC ROYER’ and Henri SENTENAC-ROUMANOU4

‘Service de Chimie de I’lnstitut Curie, ER213 du CNRS, 26, rue d’Ulm, F-75231 Paris Cedex 0.5, ‘Centre d’Etudes du Bouchet, B.P. 3, F-91710 Vert-le-Petit, 31TODYS, Laboratoire de Chimie Organique Physique arsocie’ au CNRS, 1, rue Guy-de-La-Brosse, F-75005 Paris, and 4Service des Recherches, Groupe 9, Direction des Recherches, Etudes et Techniques, 26, bd. Victor, F-75996 Paris Arm&es, France

(Received June 2, 1987, accepted September 15, 1987)

Summary - The synthesis and in vitro AChE reactivating potency of 5 new bridged pyridinium-2 carbaldoximes are described. Tested as reactivators and protectors in vitro against 5 organophosphorous inhibitors, they show a particularly interesting activity against paraoxon and tabun. These oximes themselves are reversible AChE inhibitors.

Resume - PolymCthylhedioxy bis(bydroxyiminomCthyl-2 pyridinium), rkactivateurs in vitro d’acCtylcbolinestCrase (E.E.) inhibke par des poisons organophosphorks. La synthdse et le pouvoir re’activateur d’AChE in vitro de 5 nouvelles pyridinium carbaldoximes-2 pantees sont d&its. Essaye’es en tant que re’activateurs et protecteurs in vitro contre 5 poisons phosphor&, elles montrent une activite’ particulit?rement interessante contre le paraoxon et le tabun. Ces oximes sont elles-me^mes des inhi- biteurs reversibles de I’AChE.

acetylcholinesterase reactivators / organophospborow poisons / bridged pyridinium oximes

Introduction

Organophosphates inhibit acetylcholinesterase (AChE) irre- versibly by phosphorylating a serine hydroxyl at the enzyme active site. Nucleophilic oximes, derived from pyridinium- aldoxime salts, can displace the phosphorous group thus restoring the enzymatic activity. The therapeutic use of such cholinesterase ‘reactivators’ to treat poisoning by organophosphate agents is well known [l].

We recently described [2, 31 a continuous flow method, adapted from Ellman’s technique, with eel AChE immobi- lized on fiberglass paper to follow the reactivating process of organophosphate-inhibited AChE by some salts of new 4-carbaldoxime imidazo-pyridinium reactivators in the presence of substrate. We demonstrated that the rate of reactivation varies greatly with the structures of both the oximes and the organophosphorous group bound to the esteratic site of the enzyme.

Contrathionm (Z[(hydroxyimino) methyll-l-methyl-pyri- dinium methylsulfate), TMB-4 or trimedoxime (1,3-bis[4- [(hydroxyimino)methyl]-pyridinium-1 -yl]-propane dibrom- ide) and pyrimidoxime (4-[(hydroxyimino)methyl]-l-[3-(3- methyl-I&imidazolium-l-yl) propyll-pyridinium dibromide)

afford a good protection against sarin (isopropyl methyl- phosphonofluoridate), Vx (S-2-(diisopropylamino)ethyl o- ethyl methylphosphonothioate) and paraoxon (diethyl 4- nitrophenylphosphate). The same reactivating oximes only produce poor results against tabun (ethyl N,N-dimethyl phosphoramidocyanidate) and they are of no therapeutic value against soman (I ,2,2-trimethylpropyl methylphospho- nofluoridate).

The presence of a benzyl group in 2-[(hydroxyimino) methyl]-1-benzylpyridinium bromide [4], giving a greater hydrophobic character, enhances the ability to reactivate tabun-inhibited AChE compared with TMB-4 and toxo- gonine (1, I’-bis[4-(hydroxyimino) methyll-pyridinium-l- methyl]-ether dichloride). Other 2-hydroxyiminomethylpyri- dinium salts such as PAM-2 Cl, HI-6 or HGG-12 are ineffic- ient against tabun poisoning of bovine and human erythrocy- te AChE in vitro [5] and in vivo on various animal species [6]. Likewise, non-quaternary oximes, such as a-ketoaldoximes, are inactive against tabun (F. Degorre, D. Kiffer and F. Terrier, submitted).

From our investigation on new ‘reactivators’ against soman and tabun inhibition, we describe here the synthesis of a new series of symmetrical bis-[2-[(hydroxyimino)

64

methyl]-pyridinium] salts la-e where the two pyridinium moieties are 3-linked by an alkyldioxy group in order to enhance the hydrophobic properties and keep the oxime in the 2-position [7].

d: n.5

e:n=6

1

These new oximes combine the structural elements required for activity WYSUS phosphorylated AChE. Ten years ago, closely related molecules were described [8] as reactivators of sarin-inhibited eel AChE. But these molecules were 5-linked to keep the advantage of the hydroxyimino- methyl group in the 2-position and of the diquaternary structure, thus preventing possible steric hindrance around the nucleophilic group. The new oximes were tested as reactivators and protectors of immobilized electric eel AChE inhibited by paraoxon, tabun, sarin, soman and Vx.

Chemistry

The synthesis of the symmetrical bis-pyridinium salts la-e is outlined in Scheme 1.

The oxidation of the commercially available (3-hydroxy-2 pyridyl) methanol 2 with activated manganese dioxide was carried out in dimethoxyethane and provided 3-hydroxy-2- pyridinecarboxaldehyde 3 in better yields than those pre- viouslv reported when the reaction was performed in ethanol [9]. - -

Q: 0 II:,, 2 2

5

Scheme 1.

a: n-2

b: n-3

c:n=4

d : n= 5

e:n.6

This hydroxyaldehyde 3, upon treatment with the appro- priate w,u’-dibromoalkane in dimethylformamide solution afforded the bridged dialdehydes 4a-e in the presence of potassium carbonate. The transformation of the dialdehydes into the corresponding oximes 5a-e was achieved with hydroxylamine in a hydroalcoholic solution. Their quatern- arization was accomplished with iodomethane in refluxing ietrahydrofuran (THF) to yield the required double salts la-e.

The 2 structure of the oximes 5a-e and salts la-e (i.e., situation of the hydroxyl group with respect to the proton of the hydroxyiminomethyl group) was established by measurement of the difference between the lH NMR frequencies of the hydroxyl group and that of the proton of the hydroxyiminomethyl group [lo]. This 2 configuration was also unequivocally demonstrated by measurement of the 15N-H coupling constant of the aldehydic proton with the 15N enriched oxime group [II]. In the series considered herein, the determination of the Z----E configuration by the IR spectrum of the deuteriated oxime [12] is problema- tical, particularly when the alkanoic chain is short.

Biochemistry

The interactions of oximes with inhibited AChE and with the active enzyme are described by eqn. l-3.

K EI + R ti EIR

kz (1)

EIR + E (2)

K E+R tiER (3)

where R is the tested oxime, EI is the phosphonylated or phosphorylated AChE, E is the active immobilized enzyme, EIR is the reversibly formed complex between reactivator and inhibited enzyme, ER is the reversibly formed complex between enzyme and reactivator.

Reversible AChE inhibition

Type 1 oxime AChE inhibition is a reversible, time indepen- dent, but concentration dependent process. The Ki of oximes was determined at a constant substrate concentration with various oxime concentrations, and at a constant oxime concentration with 5 x 10m4-5 x low3 M substrate concentrations. The kinetic analysis of experimental results

Table I. Reversible inhibition constant Kc of oximes la-e for immobilized AChE.

Compd.

la lb lc Id le

a/LM.

n Ki”

2 0.5 3 0.43 4 0.37

0.10 2 0.10

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Table II. Kinetic parameters for reactivation of organophosphorous-inhibited AChE.

Compd. Inhibitor of immobilized AChE

tabun paraoxon vx sarin

KP kzb knC r* Kra kzb kd r* Kr” kzb kd r* KP kzb knC r*

la 4.83 0.076 1.57x lo4 0.9917 0.4 0.131 3.2x 105 0.9999 4.30 0.034 7.9 x 103 0.981 e lb 2.16 0.350 1.65x 105 0.9994 0.57 0.380 6.6x lo5 0.9987 8.32 0.272 3.3 x lo4 0.985 24.8 0.093 3.75x 103 0.9916 XC 2.62 0.130 4.9 x104 0.9985 0.64 0.285 4.5x105 0.9970 3.38 0.066 1.9x 10” 0.9990 e Id 3.02 0.082 2.7 x lo4 0.9989 0.12 0.084 7.1 x105 0.9999 e e le 3.09 0.040 1.3 x104 0.9957 0.57 0.290 5.0~10~ 0.9995 e e

“PM. bmin-l. CM-1 x min-I. *Correlation coefficient. eUnexploitable results.

shows that the inhibition is strictly non-competitive and increases with the length of the alkyl chain between the two pyridinium rings (see Ki Table I).

Reactivation of phosphonylated eel AChE

the oxime and the soman-inhibited enzyme. For these reasons, our experiments gave no determinations of the kinetic parameters for the reactivation of the soman- inhibited enzyme. Thus, the percent reactivation was deter- mined at 30 min with oximes introduced immediately after soman inhibition (Table III).

The immobilized AChE is fully inhibited using organo- phosphorous agents, then reactivated by oximes la-e for 5 min. The experimental methods for inhibition, reacti- vation and the kinetic studies were carried out as previously described [2, 31. The kinetics of phosphonylated-AChE reactivation proved to be a function of the concentration of the added test compounds. The reactivation potency is expressed as k,, (k,/K,) which is the bimolecular rate constant for reactivation in the limit of low reactivator concentration.

Table III. Reactivation of soman-inhibited AChE.

Reactivation data are divided into two groups according to the structure of the phosphorylated eel AChE. Table II shows that oximes la-e exhibit the highest k,i reactivation values for the phosphorylated enzyme (inhibitor: tabun or paraoxon). At the present time, they are the most potent reactivators known against these inhibitors.

Compd. o/o reactivation

la 3.22 lb 11.8 1C 28.7 Id 22.1 le 19.3

Immobilized enzyme was inhibited by 2 x 10-a M soman without substrate. Oximes were injected for 30 min at 5 x 1O-4 M concen- tration just after inhibitor. Then the substrate was introduced again and the reactivated activity was read after the steady state.

Kinetic data obtained with the methylphosphonylated enzyme (inhibitor: Vx or sarin) reported in Table II are not so favorable. Some oximes (n < 5) gave experimental results which could lead to valid kinetic results with Vx- inhibited AChE. With sarin-inhibited AChE, only oxime lb, n = 3, had significant values; these results were valid within a precise concentration range (3.3 x 10-6-10-5 M). Above these concentrations, the kobs values for reactivation were minimized. Generally, with methylphosphonylated AChE, the plot of kobs veTsus oxime concentration gives a Gaussian curve which can be used for kinetic studies only in its initial phase.

Antagonism of AChE inhibition

The strong competitive reversible inhibition of AChE by oximes la-e suggested that it might be possible to protect the enzyme’s active site from irreversible phosphorylation by preinhibition of the immobilized AChE with these oximes. AChE was about 50% reversibly inhibited by oximes injected continuously before the phosphorous inhibition.

Table IV indicates the protective index (PI) which is the ratio of the kobs inhibition with and without oximes. The concentration of the inhibitors and the inhibition time were selected in order to induce about 95% enzyme inhibition without oxime.

Reactivation of soman-inhibited AChE

Soman-inhibited AChE differs from other inhibited enzymes If the inhibition mechanism is the same for all the organo- in several respects: 1) spontaneous dealkylation of the pina- phosphorous inhibitors tested here, these results show colyl group of the inhibited enzyme (‘ageing’ phenomena) that with the same percent protection by the same oxime, takes place; 2) the pinacolyl moiety covers the anionic the protective index was different for every inhibitor. binding site, and retards Coulombic interactions between Controls with TMB4 and HI-6, using identical concentra-

66

Table IV. Antagonism of irreversible AChE inhibition.

Compd. [RI” % Ei Protective index

standard. The microanalyses for the elements indicated were in good agreement with the calculated values (* 0.4%). Manganese dioxide was a commercially available product (Merck no. 805958).

3-Hydroxy-2-pyridinecarboxaldehyde 3 soman paraoxon Vx tabun sarin

la 1.6 53 * 2 1.77 6.28 4.14 5.25 N 1

lb 3.2 53 i 3 1.63 8.41 3.45 6.64 lc 1.6 52 5 2 2.13 6.47 3.38 4.95 z: Id 0.35 49 f 2 1.72 3.6 4.06 4.70 N 1 le 0.35 57 * 2 1.60 3.12 3.80 5.57 - 1

&,uM. Oximes were injected previously in order to inhibit reversibly about 50% of AChE activity in the presence of acetylthiocholine. At the steady state, the organophosphorous inhibitors were injected at such a concentration that 95% of the initial enzyme activity was inhibited. The protective index (PI) is the ratio : ki tabs) without oxime/ ki cobs).

The oxidation of hydroxypyridylmethanol 2 was perfcrmed according to a modification of the procedure described by Weiss [14] for the preparation of 3-hydroxy-6-methyl-2-pyridinecarboxaldehyde. Starting from 62.5 g (0.5 mol) of (3-hydroxy-%pyridyl)-methanol-2 in 750 mi of 1,2-dimethoxyethane and 400 g of manganese dioxide at 2OoC for 24 h, the aldehyde 3 (52 g, 85%) is obtamed after~recrystallization from cyclohexane as pale yellow prisms, mp : 80% (mp lit. [9] : 79- SOT).

tions, gave no protection. With the highest oxime concen- tration (2 x low4 M), which induced SO-90% reversible inhibition, the protection was almost complete lc.

Conclusion

This study reveals that bis-[2-[(hydroxyimino)methyl]-pyri- dinium] salts bridged in the 3-position on the pyridinium ring by a polymethylenedioxy group, -0-(CH,),-0--, are relatively potent reactivators of electric eel AChE inhibited in vitro by the chemical warfare agents Vx and sarin. With paraoxon- and tabun-inhibited AChE, these compounds are the best known in vitro reactivators. Against soman-inhibited AChE, the results obtained are about the same as those found with other known oximes (TMB4, HI-6).

The noteworthy differences in reactivity observed with these new oximes on the phosphoryl or phosphonyl group of the inhibited enzyme cannot be explained only by a change in the nucleophilicity of the oxime anion or in the reactivity of the phosphorous atom. Most likely, the recovery of the enzymatic activity is related to the stereochemical requirements of the nucleophilic displacement reaction at the phosphorylated or phosphonylated active site of AChE and with its hydrophobic binding areas around this active site [13].

Oximes la-e are reversible AChE inhibitors, protective against irreversible inhibition by tested organophosphorous inhibitors, except sarin. This antagonism of irreversible phosphonylation and the good reactivation potencies make these new oximes, especially lb and lc, rather attractive for the prevention of poisoning by tabun and soman.

Experimental protocols

Chemistry

Melting points (uncorrected) were determined on a Kofler WME apparatus. Spectral data were obtained with a Perkin-Elmer 1710 Fourier Transform infrared spectrometer and Varian EM 390 NMR spectrometer (90 MHz) using tetramethylsilane (TMS) as the internal

3,3’- (Polymethylenedioxy) bis (2-pyridinecarboxaldehyde) 4u-e General procedare. In a 250 ml conical flask fitted with a reflux condenser were placed 3-hydroxy-2-pyridinecarboxaldehyde 3 (0.08 mol) the appropriate o,w’-dibromoalkane (0.04 mol), anhydrous potassium carbonate (0.08 mol) and dimethvlformamide (100 ml). The mixture was heated‘at 60°C with stirring under nitrogen atmosphere for 2.5 h, allowed to cool to room temperature, then poured into water (500 ml). The bridged dialdehydes 4a-e were extracted with dichloromethane. The combined organic layers were thoroughly washed with water, dried with sodium sulfate and concentrated (50-60 ml).

3,3’- (Ethylenedioxy) bis(2-pyridirtecarboxaldehyde) 4a Buff-colored crystals (yield: 36 %) mp : 159OC; lH NMR (CDCls), il ppm = 4.58 (s, 2 x CHa); 7.40-7.76 (m, 4H); 8.45 (dd, 2H, J = 4.5 Hz and 1.5 Hz); 10.25 (s, 2 x CHO); IR (CDCls): 1713 cm-l (C=O). Anal. (CI~HUNZO~) C, H, N.

3,3’- (Trimethylertedioxy) bis (2-pyridinecarboxaldehyde) 4b Brown crystals (yield: 85%) mp : 10S°C; lH NMR (CDCla), S ppm: 2.46 (t, -CHz-CHz-CHa-) ; 4.41 (t, 2 x -0-CHZ-) ; 7.33-7.56 (m, 4H); 8.36-8.43 (m, 2H); 10.28 (s, 2 x CHO); IR(CDC1a): 1709 cm-l (C=O). Anal. (CrsH14Nz04) C, H, N.

3,3’- (Tetramethylelzedioxy) bis(2-pyridinecarboxaldehyde) 4c Yellow prisms (yield: 75%) mp : 155OC ; lH NMR (CDCla) 6 ppm : 2.00-2.35 (m, 2 x CHZ); 4.10-4.40 (m, 2 x -0-CHa-); 7.35- 7.55 (m, 4H); 8.35-8.42 (m, 2H); 10.30 (s, 2 x CHO); IR (CDC!a): 1709 cm-l (C=O). Anal. (CXHXNZO~) C, H, N.

3,3’- (Pentamethylertedioxy) bis(2-pyridinecarboxaldehyde) 4d Buff-colored crystals (yield: 92%) mp : 62oC; 1H NMR (CDCls) 6 ppm: 1.60-2.00 (m, 3 x CHa); 3.984.30 (m, 2 x -0-CHZ-) ; 7.36-7.50 (m, 4H) ; 8.33-8.45 (m, 2H); 10.40 (s, 2 x CHO); IR (CDCla): 1710 cm-l (C=O). Anal. (C17HrsNa04) C, H, N.

3,3’- (Hexamzthylenedioxy) bis(2-pyridinecarboxaldehyde) 4e Brown crystals (yield : 80%) mp : 74C; lH NMR (CDCIa) 6 ppm : 1.50-2.20 (m, 4 x CHZ) ; 4.004.35 (m, 2 x -0-CHz-) ; 7.35- 7.50 (m, 4H) ; 8.35-8.45 (m, 2H) ; 10.50 (s, 2 x CHO) ; IR (CDCls) : 1709 cm-l (CEO). Anal. (c18&0N204) C, H, N.

3,3’-(Polymethylenedioxy) bi3 (2-pyridinecarboxaldehyde) dioximes 5a-e General procedure. To a solution of the appropriate dialdehyde 4 (0.05 mul) in SO-90% ethanol (150 ml) a concentrated aqueous solution of hydroxylamine is added (0.11 mol, liberated in situ from its hydro- chloride by potassium carbonate). After 4 h of refluxing with stirring, the solution is cooled to O°C and the solid oximes 5a-e are seoarated by filtration, washed with three portions of water (30 ml):

3,3’- (Ethylenedioxy) bis (2-pyridinecarboxalhyde) dioxime 5a Buff-colored crystals (yield : 90 %) mp > 280X! ; lH NMR (DMSO-da) 6 ppm: 4.39 (s, 2 x CH2); 7.16-7.35 (m, 2H); 7.43-7.60 (m. 2H); 8.10-8.20 (m, 2H); 8.25 (s, 2 x -CH=N); 11.30 (bs, 2 x OH). JIShT-CH = 2.5 Hz. Anal. (Cr4H~N404) C, H, N.

3.3’- f Trimethvlenedioxv 1 his f2-uvridinecarboxaldehvde ) dioxime 56 Buff~colored crystals (yield :‘96%) mp : 251OC ; rH*NMR (DMSO-ds + CDCla, 7/3) 6 ppm: 2.33 (t, -CHZ-CHZ-CHZ); 4.28 (t, 2 x -O- CHa-); 7.15-7.50 (m, 4H); 8.16 (dd, 2H, J = 4.5 Hz and 1.5 Hz); 8.35 (s, 2 x CH=N); 11.50 (bs, 2 x OH). Jr~~-cn = 2.5 Hz. Anal. (C~H16N404) C, H, N.

67

3,3’-(Tetvamethylenedioxy) bis(2-pyridinecavboxaldehyde) dioxim? SC Buff-colored crystals (yield : 92%) mp : 225oC; lH NMR (DMSO- de) 6 ppm : 1 J-l.93 (m, 2 x CHZ) ; 4.14 (bs, 2 x 7.27-7.40 (m, 2H); 7.52 (dd,

-0-CHz-) ; 2H. J = 7.5 Hz and 1.5 Hz): 8.16 (dd.

2H, J = 4.25 Hz) ; 8133 (s, 2 x. CH-N) ; 11.50 (bs, 2 x OH).‘&& : 2.5 Hz. Anal. (C16HisN404) C, H, N.

3,3’- (Petitamethylenedioxy) bis(2-pyridinecauboxaldehyde) dioxim? 5d Buff-colored crystals (yield : 93 %) mp : 234%; iH NMR (DMSO- ds) 6 ppm: 1.50-2.05 (m, 3 x CHZ); 4.10 (bs, 2 x OCHZ) ; 7.22- 7.50 (m, 4H); 8.16 (dd, J = 4.5 Hz and 1.5 Hz); 8.35 (s, 2 x CH=N); 11.50 (bs, 2 x OH). Jl+-cn = 2.5 Hz. Anal. (C~~HZON~O~) C, H, N.

3,3’- (Hexamethylenedioxy) bis(2-pyuidinecavboxalhyde) dioxime 5e Buff-colored crystals (yield: 91%) mp : 204OC; iH NMR (DMSO- ds) S ppm : 1.40-1.93 (m, 4 x CHZ) ; 4.06 (bs, 2 x -0-CHa-) ; 7.23-7.56 (m, 4H); 8.13 (m, 2H); 8.33 (s, 2 x CH=N); 11.50 (bs, 2 x OH). Jlt~+cn = 2.5 Hz. Anal. (ClaHanN404) C, H, N.

3,3’- (Polymethylenedioxy) bis [2- [ (hydvoxyimino) methyl] - I - methylpyyri- dinium] diiodides la-e Geneva1 procedure. A suspension of dioximes 5a-e (0.1 mol) in a solution of iadomethane (1 mol) in anhydrous THF (250 ml) was heated to reflux (nitrogen atmosphere) under stirring for 140 h in an oil bath thermostated at 70°C (complete quaternarization was controlled by lH NMR analysis of aliquots). After cooling, the yellow crystals were separated by filtration then washed 4 times with 50 ml portions of anhydrous diethyl ether.

3,3’- (Ethylertedioxy)bis[2- [ (hydvoxyimino)m?thyl] -I-methylpyridinium] diiodide la Yellow crystals (yield : 98 %) mp : 222OC; lH NMR (CDCX-DMSO- de, l/l) 6 ppm: 4.46 (s, 2 x CHZ); 4.85 (s, 2 x CHs); 8.00-8.16 (m, 2H); 8.43 (s, 2 x CH=N); 8.42-8.66 (m, 2 x H); 8.83 (bd, 2 x H, J = 6 Hz) ; 12.80 (s, 2 x OH). .Ji~-cn = 2 Hz. Anal. (CXHZO- IzN404) C, H, I, N.

3,3’- (Trimethylenedioxy) bis [2- [ (hydvoxyimino)m~thyl] - I -methylpyridi- Hiurn] diiodide lb Yellow crystals (yield : 100 %) mp : 210°C; iH NMR (CDCls-DMSO- ds, l/l) 6 ppm : 2.45 (t, -CHZ-CH2-CHZ-); 4.46 (s, 2 x CHa); 4.56 (t, 2 x -O-CH2-); 7.96-8.15 (m, 2H); 8.36-8.53 (m, 2H); 8.48 (s, 2 x CH=N); 8.80 (bd, 2H, J = 6 Hz); 12.80 (s, 2 x OH). JisN-cH = 2 Hz. Anal. (C~~HZZIZN~O~) C, H, I, N.

3,3’- (Tetramethylenedioxy) bis [2-[ (hyduoxyimino) methyl] - I- mcthylpyui- dinium] diiodide lc Yellow crystals (yield : 99 %) mp : 210% ; ‘H NMR (CDCX-DMSO- d6, l/l) 6 ppm: 1.85-2.10 (m, 2 x CHs); 4.164.50 (m, 2 x -O- CHZ + 2 x CHs); 8.00-8.16 (m, 2H); 8.30-8.45 (m, 2H); 8.41 (s, 2 x CH=N); 8.76 (bd, 2H, J = 6 Hz) ; 12.80 (bs, 2 x OH). Jnx-cH = 2 Hz. Anal. (Ci~H&N404) C, H, I, N.

3,3’- (Pentamethylemedioxy) bis[2-[ (hydroxyimino) methyl] -I- methylpyui- dinium] diiodide Id Yellow crystals (yield: 98 %) mp : 184oC; lH NMR (CDCX-DMSO- ds, l/l) 6 ppm: 1.50-2.10 (m, 3 x CHZ); 4.36 (t, 2 x -0-CHa); 4.41 (s, 2 x CHa); 7.95-8.20 (m, 2H); 8.33-8.50 (m, 2 x H); 8.43 (s, 2 x CH=N); 8.80 (bd., 2H, J = 6 Hz); 12.80 (s, 2 x OH). Jij,-c, = 2 Hz. Anal. (Ci&t~&N404) C, H, I, N.

3,3’- (Hexamethylenedioxy) bis [2- [ (hydvoxyimino) methyl] -l-methylpyvi- dinium] diiodide le Yellow crystals (yield : 99 %) mp : 17OOC ; lH NMR (CDC13-DMSO- dg, l/l) 6 ppm: 1.33-2.10 (m, 4 x CHZ); 4.36 (t, 2 x OCHz); 4.50 (s, 2 x CHa); 8.00-8.16 (m, 2H); 8.26-8.41 (m, 2H); 8.46 (s, 2 x CH=N) ; 8.76 (bd, 2H, J = 6 Hz) ; 12.80 (s, 2 x OH). Ji~~-cn = 2 Hz. Anal. (CzoHasIzN404) C, H, I, N.

Biochemistry

Apparatus The reactor which contains the immobilized enzyme is a support for the filter disk (swinny type, Millipore). The same apparatus has

been used for rat brain slices [2]. The temperature of the reaction was kept at 25oC using a thermostated jacket.

Reagents Electric eel AChE type VI-§, 5-5’-dithiobis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine bromide were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. The buffer was 25 mM Tris-HCl (pH 7.8) and DTNB was 0.2 mM in Tris-HC! buffer. Organophosphorous agents (OP) : S-2-(diisopropylamino)ethyl o-ethyl methylphosphonothioate (Vx), isopropylmethylphosphonofluoridate (sarin), 1,2,2-trimethylprop- ylmethylphosphonofluoridate (soman) and ethyl-N,N-dimethyl-phos- phoramidocyanidate (tabun) were prepared in the laboratory.

Pveparatiolt of immobilized AChE The enzyme was immobilized by adsorption onto a glass fiber filter (10 mm diameter). Each disk received 20 ~1 of a fresh solution of enzyme in distilled water (usually 0.65 mg of protein/ml). The enzyme disk was doubled with a disk void of enzyme inside a toric joint and inserted into the reactor between two stainless steel grids and two flat joints. A new enzyme disk was used for each test.

Activity determination The acetylthiocho!ine hydrolysis was continuously monitored by the optical density at 412 nm. At a constant flow rate at 25OC, the activity of the immobilized enzym e is directly proportional to the optical density: Activity = Flow rate x OD/(E x I), where E is the molar absorptivity (13 600 cm-1 . M-l) and I the path length of the cell (1 cm). The flow rate, usually 72 ml/h, was measured for each test.

In the apparatus, substrate, inhibitor or reactivator were diluted three times by the other inputs; this was taken into account in the calculations. The substrate concentration routinely used in the test was 1.67 mM (5 mM before dilution).

Inhibition by ovganophosphorous compounds Inhibition of immobilized AChE was measured after a fixed reaction time (usua!ly 5 min) with a solution of inhibitor. The inhibition was performed without substrate. The observed first order rate constants ki(obs) were calculated according to: Ln(AJAo) = -ki(obs) x t; where A,, is the activity before inhibition and Ai is the activity after inhibition.

The inhibition was also continuously monitored in the presence of the substrate. With all the tested organophosphates, the activity decreas- ed exponentially, indicating that the inhibition of immobilized AChE, at a fixed inhibitor concentration, was actually a first order process.

Reactivation After inhibition and washing with buffer, the oxime solution was introduced for a fixed time (usually 5 min). The observed first order reactivation constant kobs was calculated according to : Ln(Ao - Ar) = - (kobs X t) + Ln(Ao - Ai) ; where Ar is the activity after reactivation. The spontaneous reactivation of inhibited enzyme was negligible. The reactivation tests were generally conducted in the presence of the substrate at 1.67 mM.

Kinetic parameters were expressed by equation: 1 1 Kr 1 -=-

k ohs kz+ kz’[Rl i 1 Reversible inhibition The reversible inhibition induced by oximes was evaluated by the ratio between initial activity and activity measured under continuous injection of oxime. At the concentrations used in these measurements, the oxime-induced acetylthiocholine hydrolysis was negligible. The fraction of inhibited activity was a hyperbolic function of the oxime concentration. Apparent inhibition constants for 1.67 mM substrate concentration were calculated by double-reciprocal plot method and linear regression.

The plot of vi/vi - ris as a function of l/[R] (with vis = initial rate without oxime, vi = initial rate with oxime concentration [RI, expressed by VQ = ( &x.s)/(S + &a) gives a straight line, whose slope allows an evaluation of Ki for [R] < 7 x 10-T.

Acknowledgments

Financial support from the ‘Direction des Recherches, Etudes et Techniques’ is gratefully acknowledged.

68

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