Synthesis of Oxazoloquinolone Derivatives and Evaluation of their Potency to Reactivate Rat Brain Acetylcholinesterase Inhibited by Chlorpyrifos

INTRODUCTION

Most of the widely used Organophosphorous (OP) insecticides have high degree of acute toxicity and thus has a threat of accidental poisoning. The OPs are widely used as pesticides in agriculture and for various purposes in the industry.¹ The OPs commonly used for agricultural purposes are parathion, chlorpyrifos, malathion etc. These compounds inhibit irreversibly, the enzyme acetylcholinesterase (AChE, EC3.1.1.7).Their toxic effect is based on phosphorylation of the enzyme active site, in which they are covalently bounded to the serine hydroxyl group.^2,3

To counter this effect, oximes catalyse the reactivation of AChE by exerting nucleophilic attack on phosphoryl group. Oximes theoretically work by removal of phosphoryl group from inhibited AChE enzyme, resulting in enzyme reactivation.⁴ Despite of all this, the treatment of OP poisoningis still limited due to the following reasons; that there is no versatile antidote capable of effectively restoring the activity of AChE inhibited by random organophosphates. Secondly Oxime reactivators are effective only when administered before the “aging” of AChE-OP complex and anticholinergics, like atropine, are effective only on muscarinic and not on nicotinic receptors (nAChRs).^5,6

The present communication describes the synthesis of oxazoloquinolone linked to secondary heterocyclic amine derivatives and also their subsequent evaluation for reactivation efficacy against OP inhibited AChE. The compounds 1a-1b were synthesized according to reported literature.⁷ The acetyl group of compounds 1a-1b were subjected for bromination to yield compounds 2a-2b as mentioned inprevious literature.⁸ Further, the bromoacetyl moiety was treated with secondary heterocyclic amine to achieve compounds 3a-3h. The titled compounds were obtained by refluxing the 3a-3h with hydroxylamine hydrochloride in presence of pyridine to yield 4a-4h. Through physical and spectral analysis, the structures of compounds were confirmed. The synthesized compounds were evaluated for their reactivation efficacy against chlorpyrifos inhibited rat brain AChE by Ellman’s method.⁹

MATERIALS AND METHODS

All the chemicals and solvents were supplied by Merck, S.D Fine-Chem. Limited, Mumbai and used without further purification. The reactions were monitored with the help of thin layer chromatography using pre-coated aluminum sheets with GF254 silica gel, 0.2mm layer thickness (E.Merck). The melting points were taken on the Veego (VMP-MP) melting point apparatus and were uncorrected. The IR spectra of the compounds were recorded using KBr on Shimadzu IR AFFINITY-1. The ¹ H NMR and 13C NMR spectra of the synthesized compounds were recorded on Bruker avance II 400 NMR spectrometer (with TMS as internal reference) at Sophisticated Analytical and Instrumentation Facility (SAIF), Punjab University (Chandigarh). The Mass spectra were recorded on Waters, Q-TOF Micromass (LC-MS).

DTNB [5, 5’-dithiobis-(2-nitrobenzoic acid)] and acetylthiocholine iodide were purchased from Sigma-Aldrich, USA and used without further purification. Potassium dihydrogen phosphate and dipotassium-hydrogen phosphate were obtained from E. Merck (India) and used without further purification. Organophosphate Chlorpyrifos gift sample was obtained from Indian Institute of Horticulture, Bangalore, Karnataka, India. 2-PAM was prepared according to the reported method.¹⁰

General procedure for synthesis of 4-Hydroxy-1-methyl (3a-3d)/phenyl (3e-3h)-3-(2- (secondary amino)acetyl) quinoline-2(1H)-one:

To a solution of 3-(bromoacetyl)-4-hydroxy-1-methylquinolin-2(1H)-one (0.002 mole) in dry benzene, the secondary amine (0.004 mole) was added and heated under reflux for 2 to 3 h. The solvent was removed by distillation and the solid residue obtained was suspended in water, heated to boiling, filtered, and residue was collected. The base thus obtained was purified by crystallisation from ethanol and water (90:10).

4-Hydroxy-1-methyl-3- (2-morpholinoacetyl) quinolin-2(1H)-one (3a): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3445.96 (-OH), 1725.36 (-C=O acetyl); 1628.63 (-C=O amide); ¹ H NMR (CDCl3 , δ ppm): 16.58 (s, 1H, -OH), 7.10-8.17 (m, 4H, Ar-H), 4.10 (s, 2H, COCH₂ ), 3.63 (t, 4H, 3,5 morphonyl CH₂ ), 3.51 (s, 3H, N-CH₃ ), 2.49 (t, 4H,2,6-morphonyl CH₂ ).

4-Hydroxy-1-methyl-3-(2-(piperdin-1-yl) acetyl) quinolin-2(1H)-one (3d): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3438.67 (aromatic -OH), 2937.76(aliphatic –CH str), 1721.51 (-C=O acetyl); 1627.04 (-C=O amide); ¹ H NMR (CDCl₃ , δ ppm): 16.87 (s, 1H, -OH), 7.22- 8.17 (m, 4H, Ar-H), 4.10 (s, 2H, COCH2 ), 3.66 (s, 3H, -N-CH₃ ), 2.42(t, 4H, 2,6-piperdinyl CH₂ ), 1.40 (t, 6H, 3,4,5- piperdinyl CH₂ ). LCMS: C₁₇H₂₀N₂O₃ (M+1) m/z 300.15; calcd. 301.

4-Hydroxy-1-phenyl-3-(2-morpholinoacetyl) quinolin-2(1H)-one (3e): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm-1): 3449.91 (-OH), 1732.21 (-C=O acetyl); 1643.23 (-C=O amide); 1 H NMR (CDCl₃ , δ ppm): 16.18 (s, 1H, -OH), 7.10-8.48 (m, 9H, Ar-H), 4.25 (s, 2H, COCH₂ ), 3.68 (t, 4H, 3,5 morphonyl CH₂ ), 2.42(t, 4H,2,6-morphonyl CH₂ ).

4-Hydroxy-1-phenyl-3-(2-(piperdin-1-yl) acetyl)quinolin-2(1H)-one (3h): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3428.22 (aromatic -OH), 2968.72 (aliphatic –CH stre), 1735.51 (-C=O acetyl); 1645.14 (-C=O amide); ¹ H NMR (CDCl₃, δ ppm): 16.68 (s, 1H, -OH), 7.22-8.57 (m, 9H, Ar-H), 4.28 (s, 2H, COCH₂ ), 2.65 (t, 4H, 2,6- piperdinyl CH₂ ), 1.68 (t, 6H, 3,4,5- piperdinyl CH₂ ). LCMS: C₁₇H₂₀N₂ O₃ (M+1) m/z362.16; calcd. 363.

General procedure for synthesis of 3- (substituted methyl)-1-phenyl / methylisoxazolo- [3,4-c]-quinolin-2-ones: Equimolar mixture of 4a-4h, hydroxylamine hydrochloride, 5mL of dimethylformamide and 5mL of pyridine wererefluxed for 8-16 h, progress of the reaction was monitored by TLC (ethyl acetate: n-hexane 1:1). Solution obtained was cooled and poured on to the crushed ice while stirring and allowed to stand overnight. Precipitate thus obtained was filtered and recrystallized using a suitable solvent.

5-Methyl-3-(morpholinomethyl) isoxazolo [4,5-c] quinolin-4 (5H)-one (4a): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 2916.37 (asymmetry of CH₂ ), 2848.86 (symmetry of CH₂ ), 1681.93 (-C=O amide), 1556.55 (-N-H); ¹ H NMR (CDCl₃ , δ ppm): 7.5-8.1 (m, 4H, Ar-H), 3.65 (t, 4H, 3,5- morphonyl CH₂ ), 3.61 (s, 2H, CH₂ ), 3.15 (s, 3H, N-CH₃ ), 2.5 (t, 4H, 2,6- morphonyl CH₂ ). Mass: Molecular formulaC₁₆H₁₇N₃ O₃ ; (m/z) =300(M+1).

5-Methyl-3-((piperidin-1-yl) methyl) isoxazolo [4,5-c] quinolin-4 (5H)-one (4d): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3456.52 (aromatic –C-H), 2945.39 (asymmetry of CH₂ ), 2835.80 (symmetry of CH₂ ), 1675.23 (-C=O amide); 1 H NMR (CDCl₃ , δ ppm): 7.45-8.24 (m, 4H, Ar-H), 3.76 (s, 2H, CH₂ ), 3.66 (s, 3H, -N-CH₃), 2.84 (t, 4H, 2,6- piperdinyl CH₂ ), 1.64 (t, 6H, 3,4,5- piperdinyl CH₂ ). Mass: Molecular formula C₁₇H₁₉N₃ O₂ ; (m/z) = 298(M+1).

3-(Morpholinomethyl)-5-phenylisoxazolo [4,5-c] quinolin-4(5H)-one (4e): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3462.22 (aromatic –C-H), 1687.71 (-C=O amide), 1556.55 (-N-H); ¹ H NMR (CDCl₃ , δ ppm): 6.5-8.2 (m, 9H, Ar-H), 3.7 (t, 4H, 3,5- morphonyl CH₂ ), 3.6 (s, 2H, CH₂ ), 2.5 (t, 4H, 2,6- morphonyl CH₂ ). Mass: Molecular formula C₂1H₁₅N₃ O₃ ; (m/z) =357 (M+1).

5-phenyl-3-((piperidin-1-yl) methyl) isoxazolo [4,5-c] quinolin-4 (5H)-one (4h): This was prepared and purified as per the above mentioned procedure: IR (KBr, cm^-1): 3488.21 (aromatic –C-H), 1669.51 (-C=O amide), 1572.55 (-N-H); ¹ H NMR (CDCl3 , δ ppm): 7.12-8.68 (m, 9H, Ar-H), 3.92 (s, 2H, CH₂ ), 2.92 (t, 4H, 2,6- piperdinyl CH₂ ), 1.85(t, 6H, 3,4,5- piperdinyl CH₂ ). Mass: Molecular formula C₂₂H₂₁N₃ O₂ ; (m/z) = 360(M+1).

In Vitro Experiments

The in vitro reactivation of chlorpyrifos-inhibited AChE was carried out using test compounds 4a-4h in triplicate using phosphate buffer solution (0.1 M, pH 8.0 at 37ºC) following Ellman method. Values depicted in figures are average of triplicate runs with a standard deviation. A freshly prepared stock solution of chlorpyrifos (1.4 X 10-3 M) in isopropanol was stored under refrigeration. All test stock solutions were prepared in dimethylformamide. DTNB stock solution (0.01 M) was prepared in phosphate buffer solution (pH 8.0, 0.1 M). The substrate stock (acetylthiocholine iodide, 0.075 M) was prepared in distilled water. The incubation mixture was prepared by the addition of 50 µL of chlorpyrifos compounds (1.4 X 10-3 M) to a mixture of 50 µL enzyme in 350 µL phosphate buffer solution (0.1 M, pH 8.0). The mixture was allowed to stand for 15 min at ambient temperature to give 96 ± 1% inhibition of enzyme activity. No further increase in the inhibition of enzyme activity was observed even after 1 h of the incubation with Chlorpyrifos and Methyl parathion at this concentration. It was then followed by addition of 50 µL of test solution (0.001 M) to start reactivation. The final volume of the reactivation cocktail was 500 µL. After 10 min of reactivation, the enzyme activity was assayed by Ellman’s method.

Twenty microliters of reactivation cocktail was transferred to a cuvette containing 50 µL DTNB in phosphate buffer solution (pH 8.0, 0.1 M). The enzyme activity was then assayed by addition of 50 µL of substrate to the cuvette against a blank containing reactivation cocktail without substrate. The final volume of the assay mixture was adjusted to 3 mL. The reactivation of inhibited enzyme was then studied at an interval of 10 min and followed up to 1 h.

Percentage reactivation was calculated using the following equation:

% Reactivation = 𝐸𝑟 − 𝐸𝑖 / 𝐸𝑜 − 𝐸𝑖 × 100

where, Eo is the control enzyme activity at 0 min (without inhibitor and reactivator), Ei is the inhibited enzyme activity (without reactivator) determined in the similar manner as described above, and Er is the activity of reactivated enzyme after incubation with the test compounds.

RESULTS AND DISCUSSION

The acetyl group of the compounds 1a-1b was subjected for bromination to yield the compounds 2a-2b. Further, the bromoacetyl moiety treated with secondary heterocyclic amine to achieve the compounds 3a-3h. The titled compounds were obtained by refluxing the 3a-3h with hydroxylamine hydrochloride in presence of pyridine to yield the 4a-4h. Through physical and spectral analysis, the structures of compounds wereconfirmed. The synthesis of compounds 4a-4h was represented in Scheme 1. Physicochemical data of the final derivatives abridged in Table 1.

The compounds 4a-4h were assayed for their in vitro reactivation efficacy against chlorpyrifos inhibited AChE. All results obtained are summarized in Table 2. As resulted, 2-PAM was the most potent reactivator in the treatment of OP-inhibited AChE at concentration tested (0.001 M). In this case, the compounds 4b (45.1%, 60 min), 4f (44.8%, 60 min), 4c (34.2%, 60 min), and 4g (34.6%, 60 min) achieved promising reactivation as compared to 2-PAM (68.2%, 60 min) against chlorpyrifos inhibited AChE.

CONCLUSION

Compounds 4b, 4c, 4f, and 4g having oxazoloquinolone linked substituted piperzine derivatives gave good activity against chlorpyrifos inhibited AChE. Moreover, these oxazoloquinolone derivatives appear to be very promising because of their sufficient reactivation strength at lower concentration (10-3M).

ACKNOWLEDGMENTS

The work was supported by the grant of Rajiv Gandhi University of Health Sciences, Karnataka, No. P-160:2015-16 dated: 06-01-2016. The authors are thankful to Principal, Bapuji Pharmacy College, Davangere, for providing necessary facilities to carry out this research work. The authors are also thankful to Indian Institute of Horticulture, Bangalore for providing chlorpyrifos.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.