3-hydroxyquinuclidinium derivatives: synthesis of compounds and inhibition of acetylcholinesterase

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  • Chemico-Biological Interactions 119120 (1999) 173181

    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, Sranka 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


    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 CH2OCH2 (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, AChE). The enzyme activity was measured in 0.1 Mphosphate buffer (pH 7.4) at 10 and 37C with acetylthiocholine (ATCh) as the substrate.The obtained enzyme:inhibitor dissociation constants were between 0.05 and 0.5 mM at 10Cand between 0.2 and 0.6 mM at 37C. 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 37C. The carbamates II and IV were also progressive AChEinhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M1

    min1 at 37 and 10C, respectively. Compound IV was a very weak carbamoylating agentwith rate constants of inhibition of 100 and 63 M1 min1 at 37 and 10C, respectively. Theoxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M1 min1

    at 37 and 10C, 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.

    PII: S0009 -2797 (99 )00026 -5

  • E. Reiner et al. : Chemico-Biological Interactions 119120 (1999) 173181174

    1. Introduction

    Four quinuclidinium derivatives were prepared (Table 1) for testing as in-hibitors of acetylcholinesterase (AChE, EC 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 IIV 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 [79].

    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.

  • E.










    Table 1Structures and physical data of prepared compoundsa

    Structure M.p.a (C) Yield (%) IRb (cm1) 1H NMRc (ppm)Compound 13C NMRc

    17.4, 21.1, 26.1, 51.1, 55.1,1.742.15 (m, 5H); 2.94 (s, 3H)327328 76I 35003100, 3001,56.1, 63.5, (m, 1H); 3.243.40 (m,2940, 1464, 1120

    4H); 3.633.71 (m, 1H); 4.044.07 (m, 1H); 5.53 (d, J3.66Hz, 1H)

    2951, 2880, 1707,II 18.1, 20.8, 23.6, 35.7, 36.1,1.802.18 (m, 4H); 2.252.28 (m,173175 651H); 2.83 (s, 3H); 2.89 (s, 3H); 50.9, 55.0, 55.7, 62.3, 67.9,11832.97 (s, 3H); 3.283.60 (m, 5H); 155.03.81 (dd, J13.28, 8.09 Hz,1H); 4.86 (m, 1H)

    34003300, 3046, 16.9, 20.5, 26.9, 36.4, 50.3,182184 (decom- 1.752.20 (m, 5H); 3.093.20 (m,52III2967, 2797, 1624position) 51.3, 59.9, 63.1, 78.1, 87.9,1H); 3.303.60 (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, J16.17, 8.55Hz, 2H); 5.89 (s, 1H); 6.06 (s,2H); 7.98 9d, J1.52 Hz, 1H);8.26 (d, J1.52 Hz, 1H); 8.61(s, 1H); 13.58 9s, 1H)

    IV 17.5, 20.1, 24.4, 35.7, 36.1,1.902.18 (m, 4H); 2.302.34 (m,34003500, 3039,162163 (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, 135.1,3.333.70 (m, 5H); 3.854.03 (m,1H); 3.97 (s, 3H); 4.94 (m, 1H); 138.3, 155.05.10 (dd, J19.08, 8.40 Hz,2H); 6.05 (s, 2H); 7.96 (s, 1H);8.25 (d, J1.53 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).

  • E. Reiner et al. : Chemico-Biological Interactions 119120 (1999) 173181176

    2.2. Stability of compounds in aqueous solutions

    Compounds IIV 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 IIV were dissolved in phosphate buffer (0.1 M, pH 7.4)at 37C 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 IIV 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 37C. 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 119120 (1999) 173181 177

    3. Results and discussion

    Four quinuclidinium derivatives (IIV) were prepared (Table 1) of which III andIV were new compounds. The stability of all four c


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