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Chemico-Biological Interactions 119–120 (1999) 173–181

3-Hydroxyquinuclidinium derivatives: synthesis ofcompounds and inhibition of acetylcholinesterase

Elsa Reiner a,*, Mira S& krinjaric-S& poljar a, Sanja Dunaj a,Vera Simeon-Rudolf a, Ines Primoz' ic' b, Srðanka Tomic b

a Institute for Medical Research and Occupational Health, Ksa6erska c. 2, P.O. Box 291,10001 Zagreb, Croatia

b Department of Chemistry, Faculty of Science and Mathematics, Uni6ersity of Zagreb,Strossmayero6 trg 14, 10000 Zagreb, Croatia

Abstract

Four compounds were prepared: 3-hydroxy-1-methylquinuclidinium iodide (I), 3-(N,N-dimethylcarbamoyloxy)-1-methylquinuclidinum iodide (II), and two conjugates of I and IIwith 2-hydroxyiminomethyl-3-methylimidazole in which two parts of the molecule werelinked by –CH2–O–CH2− (III and IV). III and IV are new compounds and their synthesisand physical data were given. All compounds were tested as inhibitors of human erythrocyteacetylcholinesterase (EC 3.1.1.7, AChE). The enzyme activity was measured in 0.1 Mphosphate buffer (pH 7.4) at 10 and 37°C with acetylthiocholine (ATCh) as the substrate.The obtained enzyme/inhibitor dissociation constants were between 0.05 and 0.5 mM at 10°Cand between 0.2 and 0.6 mM at 37°C. At both temperatures compounds III and IV hadhigher affinities for the enzyme than compounds I and II and this difference was morepronounced at 10 than at 37°C. The carbamates II and IV were also progressive AChEinhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M−1

min−1 at 37 and 10°C, respectively. Compound IV was a very weak carbamoylating agentwith rate constants of inhibition of 100 and 63 M−1 min−1 at 37 and 10°C, respectively. Theoxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M−1 min−1

at 37 and 10°C, respectively. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Quinuclidinium-imidazolium compounds; Quinuclidinium carbamates; Synthesisof quinuclidinium derivatives; Reversible and progressive acetylcholinesterase inhibition;Reaction of acetylthiocholine with quinuclidinium oximes

* Corresponding author. Tel.: +385-1-4673188; fax: +385-1-4673303.

0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

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E. Reiner et al. / Chemico-Biological Interactions 119–120 (1999) 173–181174

1. Introduction

Four quinuclidinium derivatives were prepared (Table 1) for testing as in-hibitors of acetylcholinesterase (AChE, EC 3.1.1.7) and as antidotes againstorganophosphate toxicity. Compound I is a 3-hydroxyquinuclidinium derivativeand compound III a conjugate of I with an imidazolium oxime. Compounds IIand IV are carbamates of I and III, respectively.

Compound I was shown by Sterling et al. [1] to protect rats against thetoxicity of soman when administered in conjunction with atropine and pyri-dinium aldoxime methylchloride (2-PAM). It was further shown by Ringdahl etal. [2] that I is an inhibitor of the choline transport into cholinergic nerveendings. 3-Carbamoyloxyquinuclidinium derivative similar to II was shown to bea potent protector against soman intoxication [3]. Furthermore, some conjugatesof 3-substituted quinuclidinium derivatives with pyridinium oximes were shownto be antidotes against tabun and soman poisoning in dogs and monkeys [4,5].Finally, it was shown that 3-oxo-1-methylquinuclidinium and two conjugates ofthat compound with imidazolium oximes protect AChE in vitro against phos-phorylation by soman and VX, and protect mice against soman poisoning [6].

In this paper, the synthesis of compounds I–IV is described, their stability inaqueous solutions tested and the reversible and progressive inhibition of humanerythrocyte AChE studied. The antidotal effect of these compounds against so-man and tabun has been described earlier [7–9].

2. Experimental

2.1. Synthesis of compounds

3-Hydroxy-1-methylquinuclidinium iodide (I) and 3-(N,N-dimethylcarbamoy-loxy)-1-methylquinuclidinium iodide (II) were prepared by quaternization of thecommercially available 3-hydroxyquinuclidine (Sigma) or its N,N-dimethylcar-bamoyloxy derivative [10]. 3-Hydroxy-1-[3-(2-hydroxyiminomethyl-3-methyl-1-imi-dazolio)-2-oxapropyl]quinuclidinium dichloride (III) and 3-(N,N-dimethylcarb-amoyloxy) - 1 - [3 - (2 - hydroxyiminomethyl - 3 - methyl - 1 - imidazolio)-2-oxapropyl]-quinuclidinium dichloride (IV) were synthesized by the reaction of 3-hydroxy-1-(3-chloro-2-oxapropyl)quinuclidinium chloride or 3-(N,N-dimethylcarbamoyloxy)-1-(3-chloro-2-oxapropyl)quinuclidinium chloride which were prepared by amodified method of Amitai et al. [4], with one equivalent of 1-methylimidazole-2-aldoxime [11,12] in dry DMF at room temperature (24 h). The precipitate wasfiltered, washed with dry ether and vacuum dried. The structures of compoundswere deduced from IR, one- (1H, 13C broadband decoupling and APT) andtwo-dimensional NMR (H,H-COSY, NOESY and HETCOR) spectra. Recrys-tallisation solvents, physical and spectral data are given in Table 1.

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Table 1Structures and physical data of prepared compoundsa

Structure M.p.a (°C) Yield (%) IRb (cm−1) 1H NMRc (ppm)Compound 13C NMRc

17.4, 21.1, 26.1, 51.1, 55.1,1.74–2.15 (m, 5H); 2.94 (s, 3H)327–328 76I 3500–3100, 3001,56.1, 63.5, 64.83.05–3.04 (m, 1H); 3.24–3.40 (m,2940, 1464, 1120

4H); 3.63–3.71 (m, 1H); 4.04–4.07 (m, 1H); 5.53 (d, J=3.66Hz, 1H)

2951, 2880, 1707,II 18.1, 20.8, 23.6, 35.7, 36.1,1.80–2.18 (m, 4H); 2.25–2.28 (m,173–175 651H); 2.83 (s, 3H); 2.89 (s, 3H); 50.9, 55.0, 55.7, 62.3, 67.9,11832.97 (s, 3H); 3.28–3.60 (m, 5H); 155.03.81 (dd, J=13.28, 8.09 Hz,1H); 4.86 (m, 1H)

3400–3300, 3046, 16.9, 20.5, 26.9, 36.4, 50.3,182–184 (decom- 1.75–2.20 (m, 5H); 3.09–3.20 (m,52III2967, 2797, 1624position) 51.3, 59.9, 63.1, 78.1, 87.9,1H); 3.30–3.60 (m, 4H); 3.72–

123.9, 125.0, 135.1, 138.33.78 (m, 1H); 3.97 (s, 3H); 4.10(m, 1H); 5.05 (dd, J=16.17, 8.55Hz, 2H); 5.89 (s, 1H); 6.06 (s,2H); 7.98 9d, J=1.52 Hz, 1H);8.26 (d, J=1.52 Hz, 1H); 8.61(s, 1H); 13.58 9s, 1H)

IV 17.5, 20.1, 24.4, 35.7, 36.1,1.90–2.18 (m, 4H); 2.30–2.34 (m,3400–3500, 3039,162–163 (decom- 47position) 2969, 2836, 1710 1H); 2.84 (s, 3H); 2.91 (s, 3H); 36.5, 50.3, 51.0, 57.6, 67.4,

78.2, 87.9, 123.7, 124.9, 135.1,3.33–3.70 (m, 5H); 3.85–4.03 (m,1H); 3.97 (s, 3H); 4.94 (m, 1H); 138.3, 155.05.10 (dd, J=19.08, 8.40 Hz,2H); 6.05 (s, 2H); 7.96 (s, 1H);8.25 (d, J=1.53 Hz, 1H); 8.62(s, 1H); 13.61 (s, 1H)

a Melting points are uncorrected. I was recrystallised from ethanol, II, III and IV from methanol-ether.b Infrared spectra (KBr pellets) were obtained on a Perkin-Elmer FT-IR 1725× spectrometer.c 1H and 13C NMR spectra were recorded on a Varian XL-GEM 300 spectrometer (in DMSO-d6, internal Me4Si).


2.2. Stability of compounds in aqueous solutions

Compounds I–IV were dissolved in redistilled water at room temperature. Thestability was monitored by TLC (Merck, DC-alufolien, aluminium oxide 60 F254,neutral, type E) after 1-, 2-, 6-, 24- and 96-h periods. The solvent was then removedunder reduced pressure, and IR and NMR spectra of the residues were recorded.Furthermore, compounds I–IV were dissolved in phosphate buffer (0.1 M, pH 7.4)at 37°C and the stability was monitored as described above after 1-, 2-, 6- and 24-hperiods. Solvent systems used in TLC studies were acetonitrile:chloroform (5:2) forI and II (iodine detection) and acetonitrile:acetic acid:acetone (9:1:1) for III and IV(iodine and UV detection).

Neither in distilled water nor in phosphate buffer were any visible changesnoticeable on TLC, IR or NMR spectra of the residues obtained after the removalof reaction solvents. The spectra were identical to those registered for parentcompounds I–IV before they were dissolved.

2.3. Inhibition of acetylcholinesterase

The enzyme source was native human erythrocytes; the final dilution duringenzyme assay was 400-fold. The experiments were done in 0.1 M phosphate buffer(pH 7.4) at 10 and 37°C. The substrate was acetylthiocholine iodide (ATCh). Theenzyme activity was measured spectrophotometrically [13] with the thiol reagentDTNB (final concentration 0.33 mM). The total volume of the reaction mediumwas 3.0 ml. The increase in absorbance was read at 412 nm at 15-s intervals againsta blank which contained the erythrocyte suspension and DTNB in buffer. Theactivities were corrected for spontaneous substrate hydrolysis and for substratehydrolysis due to the reaction of ATCh with the oxime (oximolysis).

2.3.1. Re6ersible inhibitionThe studied compound was added to a medium containing the erythrocytes

suspended in buffer, substrate and DTNB. Recording of the absorbance began 30s after addition of the compound; control samples contained no studied compound.

2.3.2. Progressi6e inhibitionErythrocytes suspended in buffer, DTNB and the studied compound were

incubated for a given time (3 min for II; up to 90 min for IV) before the substratewas added. Recording of the absorbance began 30 s after addition of the substrate.Inhibition at zero-time was measured as described under reversible inhibition.

2.3.3. OximolysisOximolysis was measured in a medium containing buffer, DTNB, substrate and

the studied compound. Spontaneous substrate hydrolysis was measured in amedium containing buffer, DTNB and substrate. The blank contained buffer andDTNB.

E. Reiner et al. / Chemico-Biological Interactions 119–120 (1999) 173–181 177

3. Results and discussion

Four quinuclidinium derivatives (I–IV) were prepared (Table 1) of which III andIV were new compounds. The stability of all four compounds was checked underconditions used in in vitro and in vivo studies; TLC, IR and NMR spectra wereused in order to detect possible structural changes with time. In all experiments thecompounds remained unchanged, indicating that they underwent no structuralchanges in media used in biological studies.

3.1. Reaction of acetylthiocholine with the oximes

The two oximes, III and IV, reacted with ATCh (Table 2). The second order rateconstant of the reaction (k %) was calculated from Eq. (1):

c/t=k %·s ·i (1)

where c is the released thiocholine concentration after time t, and s and i are theinitial substrate and oxime concentrations. Initial concentrations were used becausethe time of reaction was short and the decrease in concentrations was thereforeneglected.

At 10°C the reaction is about sevenfold slower than at 37°C, which means thatenzyme activities measured at 10°C in the presence of compounds III or IV undergosmaller corrections than those measured at 37°C and this in turn means thatactivities at 10°C can be measured at higher oxime and substrate concentrationsthan at 37°C.

The rates of oximolysis of compounds III and IV at 37°C were about the sameas rates measured for the reaction of ATCh with other imidazolium or pyridiniumoximes [6,14].

3.2. Re6ersible and progressi6e inhibition of acetylcholinesterase

All four compounds were reversible inhibitors of AChE. The enzyme/inhibitordissociation constants Ka were calculated from Eq. (2) [15,16]:

Table 2Second order rate constants for the reaction of the substrate acetylthiocholine with the indicatedcompoundsa

Substrate (mM) k %9S.D. (M−1 min−1)Compound, mM

37°C 10°C

3.190.2III, 0.25–1.00.05–10 25933.390.50.10–1.0 2191IV, 0.10–1.0

a Constants (k %) were calculated according to Eq. (1). Each constant was obtained from measurementsdone with seven to ten substrate/compound concentration pairs, and each measurement was repeatedfour times.


Table 3Reversible inhibition of acetylcholinesterase by the indicated compounds measured with acetylthio-choline as substratea

Substrate (mM)Compound (mM) Ka9S.D. (mM)

10°C37°C

0.5590.03I, 0.50–1.0 0.10–1.0 0.3490.050.3790.03 0.4690.201.0–10II, 0.25–1.0

0.07890.018III, 0.25 and 0.50 0.05–0.50 0.3190.010.05190.0060.19790.0030.10–1.0IV, 0.05–1.0

a Ka is the enzyme/inhibitor dissociation constant calculated according to Eq. (2). Each constant wasderived from measurements done with four to nine substrate/inhibitor concentration pairs, and eachmeasurement was repeated three to four times.

Kapp= (6i ·i)/(60−6i)=Ka+ (Ka/Km)·s (2)

where Kapp is the apparent enzyme/inhibitor dissociation constant at a givensubstrate concentration (s), 60 and 6i are the enzyme activities measured at a givensubstrate concentration in the absence and in the presence of a given inhibitorconcentration (i ) and Km is the Michaelis constant for the substrate.

The obtained Ka constants are given in Table 3. At both temperatures com-pounds III and IV had higher affinities for the enzyme than compounds I and II.The difference was more pronounced at 10 than at 37°C. Carbamoylation of I orIII had either no effect or slightly increased the affinity of the compounds for theenzyme.

Inhibition by I deviated from Eq. (2) at substrate concentrations above 1 mMindicating that this compound binds to more than one site on the enzyme [15,16].The same was found for the corresponding 3-oxoquinuclidinium compound [6].Compound I had a higher affinity for the human erythrocyte AChE than itscorresponding 3-oxoquinuclidinium derivative, while compound III had the sameaffinity as its corresponding 3-oxo derivative [6].

The carbamates II and IV were also progressive AChE inhibitors. The kinetics ofprogressive inhibition followed Eq. (3):

ln (6i/6t)=k2·i ·t/(i+Ka) (3)

where 6i and 6t are the enzyme activities in the presence of a given inhibitorconcentration (i ) after zero-time and after time t of inhibition; k2 is the maximumfirst order rate constant of inhibition and Ka the enzyme/inhibitor dissociationconstant. The ratio

k2/Ka=ka (4)

is the second order rate constant of carbamoylation (ka). The evaluated constantsare given in Table 4.

Compound IV is a very poor carbamoylating agent. At 10°C the first order rateconstants of carbamoylation were the same at three different inhibitor concentra-


Table 4Progressive inhibition of acetylcholinesterase by the indicated compoundsa

kaCompound (mM) Temperature (°C) k2 (min−1) Ka (mM)(M−1min−1)

II0.400.25–1.0 37 2.5 6300

20201.30.25–1.0 0.6610

IV1000.570.10–1.0 0.05737

�0.0032b 0.051c0.05–0.25 �6310

a At each inhibitor concentration the time course of inhibition was measured three to four times at10°C and five to seven times at 37°C. The enzyme/inhibitor dissociation constants Ka, and the rateconstants of carbamoylation k2 and ka were calculated according to Eq. (3) and Eq. (4).

b Mean value of the first order rate constants of carbamoylation measured with three differentconcentrations of IV.

c Ka from Table 3.

tions. Assuming that these rates correspond to the k2 value, the second orderrate constant ka was assessed by using Ka derived from reversible inhibition.Compound II is a more potent progressive inhibitor; k2 values are two to threeorders of magnitude higher than those of IV. Its Ka constants derived fromprogressive inhibition agreed with those derived from reversible inhibition.

All four compounds were shown to protect mice against soman and tabunwhen applied with atropine (i.p.) immediately after the organophosphate (s.c.)[7–9]. Protection was about the same against soman and tabun: compounds IIand IV protected all animals against 2 and 4 LD50, respectively, while com-pounds I and III protected against 1.3 LD50 of the organophosphorus com-pounds. The two carbamates (II and IV) were better protectors than thecorresponding non-carbamoylated compounds (I and III). The effect of carba-mates against organophosphate poisoning is at least partially attributed to car-bamoylation of AChE which protects the enzyme against phosphorylation[17–21].

Acknowledgements

The authors thank Anðelka Buntic (Institute for Medical Research and Occu-pational Health) for her skillful technical assistance in the kinetic studies, and B.Metelko, Z& . Marinic and B. Sokac' (Institute ‘Ruðer Bos' kovic’, Zagreb) for theNMR measurements. This work was supported in part by the Ministry of Sci-ence and Technology of the Republic of Croatia (Grant Nos. 00220104 and0119401).


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