Synthesis and evaluation of small organic molecule as reactivator of organophosphorus inhibited acetylcholinesterase

Abstract A series of uncharged salicylaldehyde oximes were synthesized and evaluated for the reactivation of organophosphorus (OP) nerve agents simulants Diethylchlorophosphonate (DCP) & Diethylcyanophosphonate (DCNP) and pesticides (paraoxon & malaoxon) inhibited electric eel Acetylcholinesterase (AChE). The computational software Swiss ADME and molinspiration were used to unfold the probability of drug-likeness properties of the oximes derivatives. Substituted aromatic oximes with diethylamino or bromo group with free hydroxyl group ortho to oxime moiety were found efficient to regenerate the enzymatic activity in in-vitro AChE assay. The alkylation of the ortho hydroxyl group of derivatives led to the loss of reactivation potential. The derivatives with a hydroxyl group and without oxime group and vice versa did not show significant reactivation potency against tested OP toxicants. Further, we also evaluated the reactivation potential of these selected molecules on the rat brain homogenate against different OPs inhibited ChE and found maximum reactivation potency of oxime 2e. The in-vitro results were further validated by molecular docking and dynamic studies which showed that the hydroxyl group interacted with serine amino acids in the catalytic anionic site of AChE enzyme and was stable up to 200 ns consequently providing proper orientation to oxime moiety for reactivating the OP inhibited enzyme. It has thus been proved by the structure-activity relationship of oximes derivatives that hydroxyl group ortho to oxime is essential for reactivating OP inhibited electric eel AChE. Amongst the twenty-one oximes derivatives, 2e was found to be most active in regenerating the paraoxon, malaoxon, DCP and DCNP inhibited AChE enzyme. Graphical Abstract

the military and civilians as there is a threat of using them by the terrorist for spreading terrorism around the world, despite the fact that these agents had been banned by international agencies (Gori et al. 2021).Among the few deadly incidences reported in the past include alleged use of Tabun by the Iraqi army in 1984 against Iran (Mandel 1993), Sarin attack in Tokyo subway in 1995 (Tu 2014), in Syria on Khan Sheikhoun in 2017 and use of VX in Malaysia to kill Kim Jong Nam (Okumura et al. 2005).Extensive use of organophosphorus based pesticides in the agriculture sectors for enhancing crop yields has an adverse impact on flora and fauna including human health (Aktar et al. 2009, Thakur et al. 2022).Numerous epidemiological studies revealed that between 750 000 and 3 000 000 OP poisoning is being arisen every year globally (Jeyaratnam 1990, Srivastava et al. 2005).
AChE is a serine hydrolase type of neuromuscular enzyme, responsible for the breakdown of substrate acetylcholine (ACh) into two metabolic products acetate and choline, to terminate the cholinergic transmission at the cholinergic synaptic site (Figure 2) (Silman and Sussman 2005).OP could be able to inactivate the AChE enzyme by forming an irreversible covalent bond thus cripples its functional activity (Bajgar 2004).Crystallographic studies demonstrated that the AChE consisted of two main sites, one is a peripheral anionic site (PAS or P-site) which is located at the entrance of a narrow gorge while another one is the catalytic active site (CAS or A-site) existed at bottom of a narrow gorge that is mainly responsible for the termination of the action of ACh (Ashima et al. 2020) oxygen atom of serine residue of AChE is standard one till date.Monopyridinium (Pralidoxime 2-PAM) or bispyridinium oxime includes (Asoxime, Obidoxime, Trimedoxime, Methoxime) (Bajgar 2010) is the first-line treatment of choice for OP toxicity as a reactivator of AChE with the combination of atropine for the symptomatic relief, this well-known treatment is being used against the OP toxicity (Wilson 1951, Jokanovi c andStojiljkovi c 2006).However, there are several serious and major challenges associated with the current antidote oximes including poor penetration to the blood-brain barrier (BBB) due to the presence of charge on pyridinium and there is no universal antidote for enzyme reactivation at central nervous system (CNS) (Cavalcante et al. 2019, Worek et al. 2020).Therefore, poor reactivation and undesired toxic effects of the existing molecules urge a need for an effective and safer antidote against OP toxicity.
Nevertheless, several strategies have been developed to overcome these challenges such as the preparation of uncharged reactivators for example (monoisonitrosoacetone or MINA, diacetylmonooxime or DAM, amidine-oximes, pro-2-PAM, and RS194B) to enhance the BBB permeability (Wei et al. 2017).Despite crossing BBB the above-mentioned reactivators were not shown sufficient reactivation against the OP-inhibited AChE, due to the less binding affinity of uncharged molecules toward the A-site of AChE.Due to the lack of binding affinity of uncharged oximes, reactivators consist of a moiety that can bind to PAS, and an oxime as a reactivating part in the same molecule connected via linker have been designed and evaluated against OP inhibited AChE (Gorecki et al. 2020).Mostly pyridinium and salicylaldehyde oxime were used as a reactivating moiety in PAS-based reactivators (Jokanovi c and Prostran 2009, de Koning et al. 2011).As salicylaldehyde oxime was acted as reactivating moiety, therefore we planned to evaluate derivatives of salicylaldehyde oximes as reactivators against paraoxon, malaoxon, DCP and DCNP.Due to the extreme toxicity of these nerve agents their nontoxic (nitrophenyl ethyl methylphosphonate (NEMP or VX surrogate), nitrophenyl isopropyl methylphosphonate (NIMP or sarin surrogate) (Chambers et al. 2015, Chambers andMeek 2020) or less toxic simulants diethylcyanophosphonate (DCNP or tabun surrogate) and Diethylcholorophosphate (DCP or sarin surrogate) were used (Thakur et al. 2022, Thakur and Sharma 2022, Zhao et al. 2022).
Firstly, we performed the molecular docking of salicylaldehyde oximes within the active site of the AChE enzyme to evaluate its binding with the active site.We obtained moderate to good docking scores of all the oximes derivatives which compelled us for testing their potential as reactivators of OP inhibited AChE.Hence, we decided to synthesize a series of salicylaldehyde oxime with varying nature of substituents like electron-donating, electron-withdrawing, and lipophilic groups to find out the effect of different substituents on reactivation potency.

Conventional method for the synthesis of salicylaldehyde based oxime (2a-2f and 4a-4n)
A solution of hydroxylamine hydrochloride (0.017 g, 2.48 mmol) and NaOH (0.05 g, 1.2 mmol) in 5 mL of ethanol was stirred at room temperature for 5 min.0.5 g, 2.4 mmol of their corresponding aldehydes (1a-1f, 3a-3n) was added to the above solution and heated at 65 C for 4-5 h.The product formation in the reaction mixture was monitored by TLC in 25% ethyl acetate:hexane.The resulting precipitates were filtered and washed with water.The final product was purified by column chromatography using Ethyl acetate and Hexane (2:8) as eluent.After the column, the product was washed with distilled hexane and dried (Scheme 1 and Table 1).
2.2.3.Microwave method for the synthesis of salicylaldehyde based oxime (2a-2f and 4a-4n) The starting materials hydroxylamine hydrochloride (0.017 g, 2.48 mmol), NaOH (0.05 g, 1.2 mmol) and corresponding aldehydes were mixed in a G30 vial with 2-3 mL of ethanol.The reaction mixture was stirred at room temperature and then irradiated at 65 C (160 W) for 5 min.The progress of the reaction was monitored by TLC in 25% ethyl acetate:hexane.The solid product was precipitated out from the ethanol solvent, concentrated, washed with water and later with distilled hexane then dried.

AChE assay & AChE inhibition by OPs
The in-vitro enzymatic assay method was used to evaluate the reactivation potential of the newly synthesized oximes.We followed Ellman's (Worek et al., 2012, Patwa et al. 2020) colorimetric method and detailed protocol described in our previous publication (Thakur et al. 2022).In brief, the assay was carried out in Tris-HCl buffer (50 mM, pH 7.4 at 37 C) in 96-well plate in triplicates.AChE stock solution was prepared in Tris-HCl buffer pH 7.4 (50 mM), and finally, 0.0245 U/mL concentration was used for reactivation studies.We have used paraoxon for AChE inhibition, and it was dissolved in the Tris-HCl buffer.1-10 mM concentrations of the paraoxon were tested for the inhibition of AChE and the result of the study suggested that 5 mM of paraoxon showed 82 ± 3% AChE inhibition in 20 min.We found that malaoxon showed maximum inhibition at 5 lM concentrations however, DCP and DCNP showed maximum inhibition at 10 lM concentrations.The stock concentration of the tested compounds was prepared in methanol and further dissolved in the Tris-HCl buffer.ATCI (2.7 mM) and DTNB (5 mM) were prepared in the Tris-HCl buffer with pH 8.The reaction mixture contained OP inhibited enzyme (40 mL), reactivators (40 mL), DTNB (100 mL), and ATCI (40 mL).The incubation and absorbance was recorded at 412 nm on the kinetic mode for 20 minutes.The reactivation potential was calculated using the following equation: whereas Ec is the control enzyme activity, Ei is the inhibited enzyme activity, and Er is the activity of the reactivated enzyme after incubation with the salicylaldehyde oximes.The Sprague Dawley rat brain was used to investigate the reactivation potential of the synthesized molecules.All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) of the National Institute of Pharmaceutical Education and Research, Raebareli (NIPER-R), Lucknow, Uttar Pradesh, India.The IAEC approval number is (NIPER/RBL/ IAEC/10/MARCH 2018).Male Sprague-Dawley rats (80-100 g) were procured from the CSIR-Central Drug Research Institute, Lucknow, India.The brain homogenate (5%) was prepared in Table 1.Derivatives of aromatic oximes.
phosphate buffer (pH 7.4, 0.1 M).It was centrifuged at 7000 rpm for 10 min at 4 C, and the resulting supernatant was used for AChE estimation.The brain homogenate was incubated with paraoxon, malaoxon, DCP and DCNP for 20 min at 37 C. Finally, the reaction mixture contained OP inhibited brain homogenate (40 mL), reactivators (40 mL), DTNB (100 mL), and ATCI (40 mL).The absorbance was recorded at 412 nm on the kinetic mode for 20 min.

Protein preparation
In the last three decades, several crystal structures were being solved for AChE.However, none of the crystal structures can show the exact mechanism of how 2-PAM is reversing the binding of serine.Several quantum mechanics/ molecular mechanics (QM/MM) studies were being done to understand the molecular mechanism of this reaction (Vidossich and Magistrato 2014).In the present work, we have shown the possible binding modes of newly synthesized compounds which are showing excellent activity in reversing the serine binding.PDB ID: 5HFA was selected from the protein data bank for this study because of two potential reasons, first the resolution of the structure (2.20 Å), and second most important it was crystallized with 2-PAM (Franklin et al. 2016, Paula et al. 2018).
Protein Preparation Wizard in Maestro 2021-1 was used to prepare input structures (PDB ID: 5HFA).As the crystallized pH was not available, pH 7.4 was considered to set the correct ionization states of protein residues using ProPka.Finally, the complete system was minimized using the OPLS4 force field.

Ligand preparation and molecular docking
All the synthesized compounds along with 2-PAM were sketched in ChemDraw 20 and taken to maestro interface.The Ligprep module was utilized to minimize the 3D geometry of input structures using the OPLS4 force field.While generating the grid for molecular docking we found that 2-PAM can be found at two different places in the binding site.However, none of the crystallized poses was close to the diethyl phosphonate fragment bound covalently to the Ser203.This shows the complex was crystallized before any chemical reaction that is why we have utilized both sites for our study.We generated the grid at both 2-PAM molecules and performed the molecular docking.

Molecular dynamics
To gain deeper insights into the binding modes of the six compounds keeping 2-PAM as reference standard we performed a 200 ns simulation for each complex.As the crystal structure provided shows two different positions for 2-PAM and molecular docking keeps protein rigid, it is necessary to identify the suitable site for further docking experiments for test compounds.The molecular dynamics simulations were performed in Desmond 2021-1 package of the Schrodinger.The detailed protocol used for the simulations is the same as employed earlier (Gahtori et al. 2020, Jena et al. 2021).All the complexes were solvated using SPC (Berendsen et al. 1987) water model by placing them in a box keeping a buffer size of 10 Å. OPLS4 force field (Roos et al. 2019) was used to model the protein-ligand complexes was used.To neutralize the system counter ions were added.Each of these complexes was energy minimized for 100 ps before going for an actual production run.Further, each minimized complex was equilibrated in five steps, where the temperature was gradually increased to 300 kelvin (K) using the Nose-Hoover thermostatic algorithm (Posch et al. 1986).In step one system was simulated for 100 ps at 10 K to reduce steric clashes between atoms.In step two systems were simulated at NVT ensemble for 12 ps, the temperature was still kept the same as step one.In step three each system was simulated at NPT ensemble, where 1 atm pressure was maintained using Langevin barostat (Martyna et al. 1994).In step four temperatures were gradually increased to 100 K for 12 ps using an NPT ensemble with restraint on solute heavy atoms.In step five all the restraints were released and a short molecular dynamics for 24 ps was done.After equilibration, a full 200 ns of MD run was performed on each system The 200 ns time scale was chosen as we observed huge conformational changes in ligand conformations.So the complexes were simulating until the system attain equilibrium.Further analysis of each system was done using the Schrodinger 2021-1package (Bowers et al. 2006).

Results and discussion
Tj and his coworkers found that initially phosphorylation by nerve agents takes place at hydroxyl group of salicylaldoximes followed by the formation of nontoxic phosphonic acid and isoxazole product.The later product was obtained through the intramolecular attack of phenol onto the nitrogen atom.This step blocks the reinhibition of AChE by the active phosphorylated oximes and makes the regeneration of AChE irreversible (Dale and Rebek 2009, Soukup et al., 2018, Cavalcante et al. 2019).Mercey et al. and other researchers described nonquaternary 3-hydroxypyridinaldoxime for the reactivation of OP inhibited acetylcholinesterase enzyme (Mercey et al. 2011, 2012, Kliachyna et al. 2014, Renou et al. 2014, 2016, Santoni et al. 2018, Zorbaz et al. 2018, 2020, Da Silva et al. 2022).In addition, Wei et al. synthesized the salicylaldoxime conjugates and evaluated for reactivation potency of OP inhibited AChE (Wei et al. 2017).The design of reported conjugates of salicylaldoximes was based on a dual binding strategy with the rationale of incorporating the structure fragment which is capable of binding with PAS and connecting oxime as a reactivating fragment via a linker (Wei et al. 2017).Therefore, we thought to test the various derivatives of salicylaldoximes alone as it is responsible for reactivating the OP inhibited AChE in conjugates (Wei et al. 2017, Kitagawa andCavalcante 2019) in order to rationalize the reactivation activity toward various pesticides and nerve agents mimics with the structure of oximes and particularly the significance of hydroxyl group in the oximes.

Synthesis
The two series of oxime derivatives 2a-2f and 4a-4n were synthesized as illustrated in Scheme 1.The series of compounds 2a-2f were synthesized directly by treating corresponding various substituted benzaldehyde with hydroxylamine hydrochloride.The 4a-4n series of compounds were obtained by O-alkylation of the free hydroxyl group with different derivatives of benzyl halide and alkyl halide via nucleophilic substitution to give the alkylated product (3a-3n, Scheme 1), then reacted with hydroxylamine hydrochloride to form corresponding oximes derivatives.The reaction was monitored on pre-coated silica gel TLC in 25% ethyl acetate: hexane as eluent and visualized under the ultra-violet lamp at wavelength 254 nm.The R f value of the product was found lower than corresponding aldehydes.The yields of products were obtained in the range of 70% to 95% after column chromatography.The conventional method of synthesis of oximes took time for the completion of the reaction in the time range of 5-6 h.Then the same protocol was used in microwave synthesizer, drastically dropped in reaction time and got better yields as well.We got the solid product in both methods which were collected by filtration and washed with distilled hexane.The color of the compound is creamish to brownish solid for most of the compounds except 2a and 4e are yellow color liquid products at room temperature.The purity of the compound was determined by HPLC in eluent ACN:Water, Method A (see supplementary file) for compounds 2a-2d and 4o and Method B (see supplementary file) for compounds 2e-4n.The structure of synthesized oximes derivatives was confirmed by 1 H-NMR, 13 C-NMR, HRMS spectroscopic techniques (spectra given in supplementary material from Figures S1-S42).In 1 H NMR spectra of compound 2e showed a signal at d 10.71 indicated hydroxyl group attached to nitrogen of oxime functional group, 9.95 showed the phenolic hydroxyl proton, 8.10 indicated CH ¼ N-proton, 7.71-6.05aromatic protons, 3.29-3.25 for the methylene protons and 1.04 for the methyl protons.The various substituted oximes derivatives are tabulated in Table 1.

In-vitro reactivation study and its correlation with structure
After the synthesis of oximes, we determined the optimum concentration of paraoxon, malaoxon, DCP and DCNP to be required for maximum inhibition of AChE in Ellman's assay which further would be used for in-vitro reactivation experiments.The various concentrations of paraoxon ranging from 1 mM to 10 mM in Tris-HCl buffer were tested for maximum inhibition of AChE.The incubation time was kept for 20 min and absorbance was recorded at each minute interval time up to 20 min instantaneously after adding ATCI.The maximum inhibition of 82 ± 3% was achieved at 5 lM concentration of paraoxon and above 5 lM concentration almost the same % of inhibition was obtained so we decided to perform a reactivation study at this concentration (see supplementary file Figure S43).Then we incubated all the synthesized oxime derivatives with paraoxon inhibited eel AChE enzyme to find out the reactivation strength.The minimum threshold reactivation value for tested oximes was set at 10 ± 1% for the selection of oximes for further modification (Kitagawa and Cavalcante 2019).Twenty-one variations were explored on the aromatic ring to generate the structure-activity relationship.The in-vitro reactivation profile of all the synthesized oximes along with 2-PAM against the paraoxon-inhibited AChE is shown in Table 2.
The only oxime 2e showed 10.28 ± 0.16% reactivation of paraoxon inhibited enzyme at 100 mM concentration in our experimental condition which was fallen in the set threshold value.However, one of the study reported in the literature has shown to some extent less reactivation ability (Kitagawa and Cavalcante 2019).Afterward, we determined reactivation at 1000 mM concentration and found the following oximes derivatives 2b, 2e, 2f, 4d, 4e, 4f, 4j and 4k displayed reactivation potency of more than 10%.The percentage reactivation potential of 2b, 2e, and 2f was found 42.25 ± 0.19, 53.50 ± 0.42 and 48.75 ± 1.15% while standard antidote 2-PAM showed 70.5 ± 0.78% of reactivation at 1000 mM concentration.Among these three oximes derivatives, 2e contains a functional group N, N-diethyl amino group opposite to oxime group.The structural motif present in the 2b, 2e, and 2f oximes derivatives (Table 2: Entries 2, 5 & 6) is hydroxyl group ortho to reactivating moiety oxime which indicated that hydroxyl group helps in providing proper orientation for binding with AChE and may have some contribution in the formation of oximate anion (Saint-Andr e et al. 2011).To confirm the role of ortho hydroxyl group, then we tested the oximes derivatives in which hydroxyl group was placed meta (Table 2: Entry 3) and para (Table 2: Entry 4) with respect to oxime functional group, found drastically loss of reactivation ability.After this, we did the alkylation of the hydroxyl group of oxime derivative 2f with various types of alkyl halide (Table 2: Entry 6) led to the formation of various derivatives from 4a to 4n (Table 2: Entries 7 to 20) which were found less capable of reactivating paraoxon inhibited eel AChE.The O-alkyl chain with bromo group attached to ortho to oxime moiety gave varied reactivation activity.Further O-benzyl substituted group with electron-withdrawing atoms showed no trend in a particular direction for reactivation activity.We tested compound 4o without oxime functional group as well but found a large dropped in reactivation ability.The above study showed that the hydroxyl group and oxime group both are required to be ortho to each other for good reactivation ability.Then, these three lead molecules (2b, 2e & 2f) were tested against the different OP inhibited eel AChE enzymes such as malaoxon, DCP and DCNP.The minimum concentration of malaoxon, DCP and DCNP required for the maximum inactivation of the enzyme was determined in the same way as determined for paraoxon.We found that paraoxon & malaoxon showed maximum inhibition (80-85%) at 5 lM concentrations however, DCP and DCNP showed maximum inhibition (80-85%) at 10 lM concentration.Our result suggests that all three molecules were effectively restored the AChE activity.However, we found that 2e showed maximum reactivation against paraoxon, malaoxon, DCP, and DCNP was 53.50 ± 0.42%, 57.34 ± 1.49%, 44.93 ± 1.62%, and 46.26 ± 1.47% at 1000 mM concentration (Figure 3).When compared to the 2-PAM our molecules is less potent, however, it is reported that 5-10% of reactivation is enough for the survival of the OP poisoning cases (Kuca et al. 2007).The reactivation potential of 2e could be correlated with the in-silico results.Insilico docking results revealed that 2e oxime derivative takes proper orientation in the AChE pockets for the interaction with the groups present in the AChE.We found a À7.5 docking score for pocketed to B site which is near to the 2-PAM docking score.Based on our findings we could conclude that molecule 2e could be a potential lead oxime derivative that takes forward for further modification in the structure intending to obtain a more pharmacologically efficient antidote that could be used in the treatment of cases of OP toxicity.Percentage AChE reactivation against the paraoxon inhibited enzyme is shown in Table 2.All the values are expressed in percentage AChE reactivation results in mean ± SD (n ¼ 3).
Outcomes of these studies suggest that the 2e molecule showed a satisfactory effect on the electric eel AChE.Then our interest was rise to see their effect on the rat brain ChE.Therefore, we used rat brain homogenate to evaluate the reactivation potential of the selected molecules.The results suggested that 2e molecule showed 50.5 ± 3.0%, 51.8 ± 1.6%, 46.9 ± 1.3%, 53.0 ± 0.69% reactivation against the paraoxon, malaoxon, DCP & DCNP, respectively (Figure 4).Thus our outcomes suggest that 2e could be a potential reactivator against the wide OP toxicant.However, more detailed studies are required to broaden its therapeutic use.

In-silico studies
The oximes derivatives were evaluated for various parameters of pharmacokinetics, drug-likeness, toxicity, molecular docking and dynamics using computational software and techniques.

Evaluation of ADME parameters
Swiss ADME tool provides broad information regarding the ADME and physicochemical properties of the compounds.Various parameters were evaluated with the help of these tools to find that these oximes were able to cross the BBB and reactivate the OP-inhibited AChE in the brain.Lipinski et al. has set the basic rules for an orally administered drug candidate and later Reichel modified the rules for CNS drug candidate (Lipinski 2004, Pajouhesh andLenz 2005).
The predicted values are listed in Table 3. Almost all the synthesized oximes have fulfilled the criteria to cross the BBB.The pKa plays an important role in the reactivating OP inhibited AChE.Compound 2e shows the highest reactivation activity for OP inhibited AChE might be due to their optimal pKa value lies within the range of 7.5-8.5 which makes the oxime to dissociate sufficiently to form oximate and act as reactivator (Acharya et al. 2011).In-vivo studies suggested that 10% of 2-PAM reaches the corpus striatum of rats (Sakurada et al. 2003).In addition to 2-PAM, obidoxime reaches the CNS up to 3-5% in both rats and mice (Demar et al. 2010, Karasov a et al. 2010).Based on the parameters, values indicated that these designed compounds lie within the desirable range.Therefore, these compounds are expected to penetrate the BBB up to more extent than 2-PAM.
Figure 5 shows the boiled egg diagram and ADME parameters for compound 2e calculated by Swiss ADME online software.The boiled egg graph consists of 2 regions (a) yellow region (b) white region.The yellow region is also known as Boiled egg yolk indicates the molecules that lie in the region can cross the BBB while the white region called Boiled egg which predicts that the molecule to be absorbed by the Gastrointestinal tract (GIT) route (Daina et al. 2017).It has been found that our best molecule 2e lies in the yellow region (Boiled egg yolk) suggested that compound 2e can permeate the BBB.

Estimation of toxicity parameters of compound 2e
Protox-II online server is used for the toxicity prediction of Compound 2e.Its main objective is to predict the toxic or harmful effects of chemical compounds in form of lethal dose (LD 50 ) in mg/kg weight, toxicity class and reduce the time, cost, and need of animal models for experiments.It also helps in experimental follow-up studies, enhances hit selection and lead optimization (Banerjee et al. 2018).Figure 6 shows the calculated toxicity values of our best molecule 2e having an LD 50 of 1131 mg/kg and falls under the category of toxicity class IV.The results revealed that Compound 2e is inactive in the case of hepatotoxicity, carcinogenicity, mutagenicity, cytotoxicity, nuclear receptor signaling pathways and stress response pathways.This tool suggests that our molecule 2e can be considered a nontoxic or safer molecule.

Molecular modeling studies
The crystallized structure of AChE and 2-PAM shows there could be two possible sites for the attack of 2-PAM to diethyl phosphonate covalently attached with Ser203.The distance  (Lipinski et al. 2001, Pajouhesh andLenz 2005).
DRUG AND CHEMICAL TOXICOLOGY of the 2-PAM reactive site from phosphonate is 8.7 Å and 13.8 Å respectively.In the earlier studies, it was hypothesized that quaternary nitrogen of 2-PAM forms an ionic bond with the AChE protein and sits well into the cavity, and then the whole reaction proceeds.However, the crystallized pose of 2-PAM did not provide any such information, which shows that the complex was crystallized before the reaction starts (Souza et al. 2020). 2 D and 3 D interaction diagrams of 2-PAM was shown in Figures 7 and 8.

Molecular docking
A docking experiment was done on two (A and B) sites of AChE.All test compounds along with 2-PAM as reference were taken for molecular docking study.From a mechanistic point of view, the quaternary nitrogen of 2-PAM should interact with the Trp286 and Tyr124 which forms a hinge-like cavity to settle 2-PAM and could help 2-PAM to re-orient for the chemical reaction to remove diethyl phosphonate from AChE.However, in the crystal structure, 2-PAM was observed at two  different sites (Figure 7).Hence, both the sites were utilized for molecular docking.
After docking, we found that initially, 2-PAM was facing opposite to diethyl phosphonate in both active sites, Figure 7. Later on, site B docking was able to rotate the reactive center of 2-PAM.The same was not observed in the A site of AChE, this was due to the possible steric clashes between AChE residues and ligand.There is very little space to rotate the molecule and face the reactive center toward diethyl phosphonate at site A. However, all the molecules were not able to dock on site A, due to steric clashes between the protein residues.The docking score for compound 2e was found close to the 2-PAM score.Molecular docking results of our top hits were provided in Table 4 and all the compounds at both sites A and B are provided in Table S1 & S2 in the supplementary file.Further to understand the stability of these complexes we performed the molecular dynamics simulation of 2-PAM (as reference standard) at sites A and B and other five compounds (2a, 2b, 2e, 2f, 4o) at site B.

Molecular dynamics
We have selected five test molecules and 2-PAM at two different sites for molecular dynamics simulation.As observed in the crystal structure (PDB ID: 5HFA) 2-PAM was found at two places but was not accessible to react with diethyl phosphonate.However, the docking experiment gave a suitable pose at site B which was close to diethyl phosphonate and can initiate the reaction.On the other hand, 2-PAM conformation at site A was not found to be suitable, although we simulated that complex too for 200 ns.However, the simulation at site A was not successful and after 50 ns we found that the ligand was dragged into the solvent but at site B the protein-ligand contacts were stable.Quaternary ammonium group in 2-PAM at site B was allowed to stabilize the complex by making a strong interaction with Tyr341 and Tyr337 which lasts for more than 60%.RMSD and RMSF for both the sites were similar and dragging out of ligand did not make any remarkable change in protein conformation (Figure 9).
All the test molecules except compound 2a were able to stay in the binding site during the 200 ns simulation.Compared to 2-PAM all these molecules move slightly away and orient's OH group toward the diethyl phosphonate.This was due to the absence of quaternary ammonium which sits in the hinge region of ACh like 2-PAM.Our biological assay also supports our simulation data, Figure 8 Compounds 2b, 2e and 2f were able to stay at the binding site throughout the simulation and most of the interactions were last for more than 50%.RMSD and RMSF of all the stable complexes were found to be stable and within the acceptable range of 3Å.Although in Figure 9 it can be observed that even 2-PAM was dragged out into the solvent from site A there were no such changes in protein RMSD as well as RMSF.Site B RMSD was slightly lower than site A. RMSD and RMSF of 2b, 2e, 4o, and 2f was shown in Figure 10.4o was also seemed to be stable during the 200 ns MD run but the interaction was  quite weaker considering the other three molecules, hence can be dragged away from the binding site in a longer run.
3.4.2.1.Superimposition poses of docking and simulation results analysis.As mentioned in the protein preparation section, there are two different binding modes of 2-PAM were observed in the crystal structure.Theoretically, the OH group of 2-PAM should face the phosphate group of diethyl phosphonate to make a stable covalent bond.Docking studies on both the sites of 2-PAM (A and B) were done and we found that at site B there is a possibility of a stable contact with the phosphate group of diethyl phosphonate.Further, we have explored the interaction of 2-PAM at both sites by 200 ns of molecular dynamics simulation.On-site A 2-PAM was dragged out from the binding site and in site B it was able to stay there throughout 200 ns simulation run.
Figure 11 shows the change in ligand conformation before and after molecular dynamics simulation.
2-PAM, 2b and 2f did not show much change in their conformation and seems to be stable within the binding site.On the other hand, if we see the docked of 2e there is p-p stacking between ligand phenyl ring and Tyr124 and Trp286 and OH group was placed in parallel facing diethyl phosphonate group.However, during the simulation ligand rotated 180 degrees this is because the p-p stacking between Tyr124 and ligand phenyl ring was not very strong and was lost during the start of the simulation.Further, the rotation of the ligand favored an H-bond between Tyr124 and phenyl OH of ligand and three p-p stacking between Tyr341, Phe297, Trp286 (Figure 11).
Ligand 2b shows better interaction during the docking but during the simulation small reorientation of ligand occurs due to its smaller size.Most of the interactions were lost except p-p stacking between Tyr124, Figure 11 shows the movement of Tyr124 little toward the ligand.The stable Hbond interaction between ligand reactive OH and guanidium group of Arg296 holds the ligand conformation and did not allow it to move toward the diethyl phosphonate.However, extending the simulation may allow us to observe its movement toward the diethyl phosphonate group.Ligand 2f did not show much change due to an intramolecular H-bond and moved little upward in the binding site, which cost the ligand to lose one H-bond with Ser298 and another H-bond with Glu285.This upward movement was caused due to insertion of a water molecule between Trp286 and ligand hydroxy group, Figures 9 and 11.This upward movement of the ligand was much favored and allowed the ligand to stabilize within the binding site.
Molecular docking results showed that 2b, 2e and 2f were oriented toward the active site of the AChE-diethyl phosphonate complex.Similarly, molecular dynamics data also suggest all the three molecules were able to stay in the binding site and try to make their way toward diethyl phosphonate.

Conclusion
In this paper, we have reported a microwave-assisted synthesis of two series of salicylaldehyde oximes and evaluated its physicochemical parameters, performed molecular docking, molecular simulation using computational techniques and invitro reactivation potential against paraoxon, malaoxon, DCP and DCNP inhibited eel AChE.The study evaluated the significance of the hydroxyl group of the compounds on the reactivation ability with its structure.The in-vitro results demonstrated that the free hydroxyl group ortho to the oxime is essential to act as an efficient reactivator with substituents electron-donating as well as electron-withdrawing groups while masking the hydroxyl group with different alkyl halides or aryl halides exhibited poor reactivation ability.It has been proven that hydroxyl group ortho to oxime is essential for the improvement in reactivation potency.Meanwhile, the molecular docking studies were performed for all the synthesized compounds and shows good binding affinity with the AChE enzyme.The molecular simulation study has shown that the active oximes were oriented toward OP comparably inhibited AChE as 2PAM.Moreover, these oximes were easily synthesized in the microwave, purified, economical which demonstrates an advantage over the previously synthesized oximes, and were expected to cross the BBB due to their uncharged nature and result in CNS reactivation.In the future, the in-vivo assays were required to evaluate their CNS permeability, and maybe further changes are required for the development of efficient reactivators.
versions of the manuscript.All authors read and approved the final manuscript.
OP class of compounds like pesticides (parathion, paraoxon, malathion, malaoxon, chlorpyrifos, diazinon and dichlorvos) and chemical warfare nerve agents (Tabun, Soman, Sarin, VX) act as irreversible inhibitors of AChE (Colovi c et al. 2013) Figure 1.The nerve agents pose a serious threat to

Figure 3 .
Figure 3. Percentage reactivations of lead molecules against paraoxon (A) malaoxon (B), DCP (C) and DCNP (D) inhibited eel AChE are shown in the figure at 100 and 1000 mM concentration.All the values are expressed in the mean ± SEM (n ¼ 3).

Figure 4 .
Figure 4.The percentage reactivations of lead molecules against paraoxon (A) malaoxon (B), DCP (C) and DCNP (D) inhibited rat brain homogenate ChE are shown in the figure at 100 and 1000 mM concentration.All the values are expressed in the mean ± SEM (n ¼ 3).

Figure 5 .
Figure 5. (A) Represents the BOILED egg picture between the WlogP vs. TPSA of compound 2e.(B) In-silico ADME parameters of 2e calculated by Swiss ADME software safer molecule.

Figure 7 .
Figure 7. Co-crystallized pose of 2-PAM (PDB ID: 5HFA).The green circle shows site A and the red circle shows site B.

Figure 8 .
Figure 8. RMSD and RMSF of 2-PAM at (A) site A and (B) site B after 200 ns of molecular dynamics simulation.

Figure 9 .Figure 10 .
Figure 9. Protein-ligand interaction diagram during the 200 ns of simulation.2-PAM was considered as control.
1 H NMR and 13 C NMR spectroscopy on Jeol NMR spectrometer (Japan) at 500 MHz and having ROYAL HFX FGSQ probe in DMSO-d 6 and Acetone-d 6 as the solvent.Chemical shifts in the 1 H NMR and 13 C NMR are expressed in delta (d) or parts per million (ppm) from DMSO-d 6 (d H ¼2.5 ppm, d C ¼39.52 ppm, Acetoned 6 (d H ¼ 2.05 ppm, d C ¼ 29.84, 206.24 ppm) and tetramethylsilane (TMS) as an internal reference.Coupling constant (J) values are expressed in hertz.High-resolution mass spectra were recorded on Agilent LC/Q-TOF mass spectrometer (Agilent G6530, Germany) with electron spray ionization (ESI) source.

Table 4 .
Docking scores of top selected compounds.