Functionalised dihydroazo pyrimidine derivatives from Morita–Baylis–Hillman acetates: synthesis and studies against acetylcholinesterase as its inhibitors

Abstract

A wide array of dihydro[1,5]azo[1,2-a]pyrimidine 2-esters have been synthesised at room temperature in moderate to good yields through one-pot reaction between nitrostyrene derived MBH acetates and aminoazole derivatives with γ-α cyclisation. The reaction involves a cascade S_N2 reaction followed by intramolecular Michael addition–cyclisation with the generation of two new carbon–nitrogen bonds. The structure was further confirmed by single X-ray crystal analysis. Docking and in vitro studies of dihydrobenzimidazo pyrimidine derivatives against acetylcholinesterase (AChE) have been performed and the compounds 3d and 3e exhibit potent inhibitory activities with an IC₅₀ of 46.8 nM and 42.5 nM respectively.

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Introduction

Diversely functionalised heterocyclic rings exist in nature and in many biologically relevant compounds, such as nucleic acids, antibiotics and hormones. A wide range of pharmacological properties shown by heterocyclic units inspired synthetic organic and medicinal chemists to pursue the synthesis of novel molecules and study their biological properties. Among them, benzimidazoles, imidazoles, triazoles and tetrazoles have exhibited biological properties such as anticancer and antimicrobial, and serve as antitubercular agents, benzodiazepine receptor agonists, calcium channel blockers and are also present as core structures in several currently marketed drugs.^1,2 The various biological properties of imidazole and fused imidazole derivatives, such as antibacterial, antifungal, analgesic, antitubercular, anticancer, anti-HIV, antiarthritic and antitumor, have been extensively investigated.³ Imidazo[4,5-b]pyridines, which are structurally related to benzimidazoles have shown diverse biological activities such as cytotoxic activities depending on the substituents of the heterocyclic ring.⁴ Several anxiolytic drugs such as fasiplon, taniplon, and divaplon holding imidazopyrimidine fragments are currently used as drugs in market⁵ (Fig. 1).

Fig. 1 Imidazopyrimidine containing drugs.

The main concept for the synthesis of novel dihydroimidazo pyrimidine ester derivatives originated from biological importance of dihydropyrimidines and imidazole derivatives. They exhibit pharmacological properties like anticancer activity, antiplatelet activity,⁶ HIV-1 integrase inhibitors^7a and anti-Alzheimer's disease.^7b–d Moreover, these pyrimidines and imidazole/benzimidazole derivatives are reported to have AChE inhibitory properties.^7a,e–k Also the structure activity studies proved that pyrimidine moieties plays an important role in the inhibition of AChE^7b,h and the benzimidazole scaffold is the ring isoster of indanone moiety of donepezil, one of the most potent AChE inhibitor. Hence it was assumed that combining the two inhibitory moieties may further enhance the inhibitory potency towards AChE. More over expansion of aromatic plane may improve stacking and hydrophobic interaction with the enzyme as these interactions plays a crucial role in the binding of inhibitors to AChE.

Acetylcholinesterase inhibitors (AChEIs) temporarily restore native levels of the neurotransmitter acetylcholine (Ach) and prevents the loss of cholinergic transmission in brain areas. However, cholinergic deficit is one of the hallmarks of Alzheimer's disease (AD).^8a,b AD, is associated with selective loss of cholinergic neurons and reduce the levels of acetylcholine neurotransmitter and it is characterised by progressive loss of memory, attention and depression.^8c According to the Alzheimer's Association, AD is the one of the top 10 causes of death in high income countries. In recent decades, researchers focused on the increase of cholinergic neurotransmission by inhibiting the enzyme AChE as one of the main stream option for AD. Currently, the drugs launched in market for cholinergic approach for AD such as tacrine, donepezil, rivastigmine and galantamine (Fig. 2) increase neurotransmission at cholinergic synapses in the brain and improving cognition.

Fig. 2 AChE inhibitors clinically used for the treatment of AD.

Therefore, the desirable protocols that would generate various heterocyclic compounds for the synthesis of natural and bio-active compounds is of great demand. Recently, cascade reactions gained tremendous potential in the area of organic synthesis to maximise the results. We envisioned that synthesis of dihydro[1,5]azo[1,2-a] pyrimidine 2-ester derivatives could be possible by reaction of nitrostyrene derived MBH acetates and aminoazole derivatives (Scheme 1).

Scheme 1 Strategy towards construction of dihydroazo pyrimidine derivatives.

The Morita–Baylis–Hillman (MBH) reaction⁹ has an attractive strategy for the synthesis of multifunctional heterocyclic molecules in recent years. The MBH reaction is a key step in the synthesis of bioactive and designed molecules.¹⁰ Earlier, Baylis and Hillman reported the synthesis of α-hydroxyethyl nitroethylenes from MBH reaction.¹¹ Later, Namboothiri and Chen made further investigation on this reaction and emphasized the use of electrophilic ethyl glyoxylate.¹² The MBH reaction of nitroalkenes have been utilised for the synthesis of several heterocycles and carbocycles.¹³ Further acetylation on hydroxyl group of these conjugated nitroalkene afforded MBH acetates as excellent Michael acceptors containing four potential electrophilic sites (α, β, γ, δ). All four electrophilic sites are highly reactive and used as efficient synthons to synthesise the diversified molecules (Scheme 2). The MBH adducts, can act as dielectrophilic reagents to undergo cascade reactions with various bifunctional nucleophiles.¹⁴

Scheme 2 Potential electrophilic sites of MBH acetates.

Recently Namboothiri and Chen demonstrated that the potential electrophilic site at α-position and further Michael addition at β-position in the synthesis of fused furans,¹⁵ imidazopyridines,¹⁶ and 2-substituted imidazoles.¹⁷ Li Liu demonstrated the formation of γ–α and γ–β cyclic products using 2-mercapto benzimidazoles for the synthesis of benzimidazo[2,1-b]-1,3-thiazine and thiazolo[3,2-a]benzimidazole respectively.¹⁸ Nair, reported the synthesis of fused pyrans using MBH acetates with cyclopentanedione as α–γ cyclic product.¹⁹ Recently, Shao reported the cascade reaction of MBH acetates with N-substituted hydrazine for the synthesis of pyrazoles, in which the reaction was initiated by a S_N2 reaction at the electrophilic γ-position followed by Michael addition at α-position.²⁰ Zou demonstrated the reactivity of all four subsequent potential electrophilic sites (α, β, γ, δ) of MBHAs to form imidazo[1,2-a]pyridines (α–α), indolizines (α–β), pyrroles (α–γ) and benzo[b][1,6]oxazocin-2-ones (α–δ).²¹ Herein, we firstly report a facile regioselective attack at γ position of MBH acetates using various bifunctional nucleophiles such as 2-aminobenzimidazole, 2-aminoimidazoles and 2-amino-1,3,5 triazole followed by another at α-position to give rise to functionalised dihydroazo pyrimidine derivatives with γ–α cyclisation.

Results and discussion

Chemistry

Our initial efforts were focused on optimising the reaction parameters with respect to base, solvent and temperature. Accordingly, the reaction was studied with MBH acetate of nitro alkene 1 and 2-amino benzimidazole 2 as a model substrate. The reaction was performed in THF at room temperature without base. Unfortunately no product formation was observed by LCMS even after heating at 70 °C (Table 1, entry 1 and 2). While adding organic bases like triethyl amine and DIPEA, we observed the formation of 4-phenyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-2-carboxylic ester 3 in lower yields (Table 1, entry 3 and 4). Our sub-sequent efforts were focused on use of bases such as pyridine, 2,6-lutidine, DABCO, DBU which delivered 3 in 40–50% yield (Table 1, entry 5–8) and inorganic bases like K₂CO₃, Cs₂CO₃ gave compound 3 in moderate to good yields (Table 1, entry 9 and 10). Among all the bases screened, Cs₂CO₃ was found to give a superior yield (80%) in THF over 12 h at RT. Among the solvent screens (CH₂Cl₂, CHCl₃, MeOH, EtOH, CH₃CN, and 1,4-dioxane) (Table 1, entry 11–16), CH₃CN and 1,4-dioxane shows the formation of compound 3 in good yield (∼81%). Finally we optimised the condition is Cs₂CO₃ (2.0 eq.) in acetonitrile/1,4-dioxane as a solvent of choice to yield compound 3 (82%) at RT.

Table 1 Screening of reaction conditions^a

S. no	Base	Solvent	Temp	Yield^b (%)
a All reactions were performed with 0.34 mmol of 1 and 2 with 4 mL of solvent for 12 h.b Isolated yield by column chromatography.
1	—	THF	RT	0
2	—	THF	70 °C	0
3	Et3N	THF	RT	20
4	DIPEA	THF	RT	22
5	Pyridine	THF	RT	40
6	2,6-Lutidine	THF	RT	40
7	DABCO	THF	RT	44
8	DBU	THF	RT	49
9	K2CO3	THF	RT	60
10	Cs2CO3	THF	RT	80
11	Cs2CO3	CH2Cl2	RT	45
12	Cs2CO3	CHCl3	RT	48
13	Cs2CO3	MeOH	RT	60
14	Cs2CO3	EtOH	RT	68
15	Cs2CO3	CH3CN	RT	82
16	Cs2CO3	1,4-Dioxane	RT	81

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Having these optimised conditions in hand, we explored the scope of 2 with different MBH acetates 1. All the reactions were completed in 12 h at room temperature to give the desired product in moderate to good yields. The yields were good with MBH acetates possessing electron donating groups (3b, 3c, 3h and 3l) to afford the corresponding products in excellent yields (73–82%). On other hand, MBH acetates bearing hindered aryl groups at ortho position (3d, 3e, 3i) delivered moderate yields (60–70%). The yields were low in case of MBH acetates having strong electron withdrawing groups (3j, 3k) and pyridine substrates (3f, 3g). We next carried the scope of this optimised conditions with 2-aminoimidazoles for the synthesis of 5-phenyl-5,8-dihydroimidazo[1,2-a]pyrimidine-7-carboxilic ester derivatives (3m–3r). It was observed that, compared to 2-aminobenzimidazole, 2-aminoimidazole required 15 h to obtain the desired product in moderate yields (45–66%) (Scheme 3).

Scheme 3 Substrate scope of MBHAs towards reaction with 2-aminobenzimidazoles and 2-aminoimidazoles.

Our studies were extended to use of 3-amino 1,2,4-triazoles for the synthesis of 7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-5-carboxylicesters with MBHAs using the optimised conditions. In case of 3-amino 1,2,4-triazoles, we observed two regio isomers in the ratio of 7 : 3 by LCMS and NMR. To improve the regioselectivity, different solvents (MeOH, THF, CH₃CN, 1,4-dioxane and DMF) were tried. To our delight, DMF provided the excellent regioselectivity (9 : 1) at 60 °C by using Cs₂CO₃ as a base (Scheme 4). The yields are good in case of MBHAs possessing substituents at meta and para position (4b–4f) compared to MBHAs having substitutions at ortho positions (4g and 4h).

Scheme 4 Synthesis of dihydro triazopyrimidine derivatives.

After the successful demonstration of the reactivity of different MBHAs with 3-amino-1,2,4-triazole, we focused our attention for the synthesis of 7-phenyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-5-carboxylic esters (5a and 5b) by reacting 5-amino tetrazoles with different MBHAs and are summarised in Scheme 5. The above conditions were, not suitable for reacting MBHAs with guanidine hydrochloride. Thus, there is no reaction was observed even after 12 h reflux when guanidine hydrochloride was treated with MBH acetates.

Scheme 5 Synthesis of dihydro tetrazolopyrimidine derivatives follows.

The structures of all compounds were confirmed by LCMS, ¹H NMR and ¹³C NMR spectral data. The compounds 3e (CCDC 1446736) and 4b (CCDC 1456725) were confirmed by single crystal X-ray diffraction studies and were shown in Fig. 3.

Fig. 3 ORTEP diagram of (a) compound 3e and (b) compound 4b.

A possible mechanism for the synthesis is described in Scheme 6. The primary amine of 2-amino benzimidazole attacks the γ electrophilic site of MBH acetate through S_N2 reaction to afford intermediate I. Intramolecular Michael addition occurs at α electrophilic site of MBH acetate to generate intermediate II via 6-endo trig process. The intermediate II results in formation of III by loss of hydrogen atom, which on tautomerisation gives IV. The excess base facilitates the elimination of HNO₂ to give dihydro-benzo[4,5]imidazo[1,2-a]pyrimidine-2-carboxylic ester as 3. Thus the reaction is completed by formal γ–α cyclisation on MBH acetate.^18,20 Reaction of N-methyl 2-amino-benzimidazole with MBH acetate shows very low conversion for the desired product with nearly 10% product formation. Reaction with N-benzyl and N-Boc derivative of amino-benzimidazole resulted in recovery of the starting material, with no trace of product formation even at 70 °C. This can be easily anticipated due to steric hindrance by the nucleophilic nitrogen to attack at the elctrophilic sp³ carbon atom of MBH acetate.

Scheme 6 Proposed mechanism.

Biology

The in vitro inhibitory effect of newly synthesized ligands were assessed by Ellman's method²² using AMPLITE™ AChE assay kit (AAT Bioquest, Inc., Sunnyvale, CA). In order to check the inhibitory activity of the compounds towards the AChE an initial screening has been carried out with a ligand concentration of 208 nM. Enzyme and ligands were incubated for 15 minutes and checked the enzyme activity towards the substrate. A control experiment has also been carried out without ligand. Finally the optical density was plotted against time (velocity time graph) (Fig. 4) and deduced the percentage of inhibition of the compounds (Table 2). Since all the compounds were displayed strong inhibitory activity, the half maximal inhibitory activity (IC₅₀) value for all compounds were deduced. Enzyme inhibition studies revealed that the newly synthesized compounds exhibit inhibitory activity against AChE in nM range. All of these compounds displayed enhanced inhibitory profile than tacrine and reported IC₅₀ of galantamine (two known AD drugs).²³ The maximum and minimum IC₅₀ values obtained are 91.8 and 42.52 nM respectively. The compound 3a exhibited an IC₅₀ value 70.78 nM. It was also noticed that the phenyl group baring any substitutions are found to have lower IC₅₀ value except 3c. The presence of methyl group in the phenyl ring of the compound 3c′ has drastically increased the IC₅₀ value to 91.8 nM. With the substitution of polar groups such as –OH (3h) and –NO₂ (3j), the AChE inhibitory effects was better as compared with 3a. Introduction of halogens in phenyl moiety exerted prominent AChE inhibition (IC₅₀ values in the range of 42–53 nM) of the compounds such as 3b, 3d, 3e and 3i. It was already reported that the substitution of halogen is an important method in drug design since it can increase the binding affinity of the compounds through halogen bonding and alters the physico chemical properties.²⁴ Substitution of an additional halogen in the phenyl ring of the compounds 3d and 3e make it more effective against AChE with IC₅₀ values 46.86 and 42.52 nM respectively (Fig. 5). Whereas the substitution of phenyl ring with pyridine ring (3g) has markedly decreased the inhibitory effect towards AChE. The IC₅₀ values of all compounds are shown in Table 2.

Fig. 4 Velocity time graph obtained for native enzyme and in presence of compounds (208 nM).

Table 2 The in vitro AChE inhibitory profile of compounds 3a–3j

Compound	% of inhibition at 208 nM	hAChE IC50 (nM) + SD
3a	53	70.78 ± 10.01
3b	70	52.64 ± 1.07
3c	62	91.8 ± 3.6
3d	76	46.86 ± 1.16
3e	74	42.52 ± 5.17
3g	69	71.48 ± 5.04
3h	61	67.32 ± 4.94
3i	67	52.58 ± 15.65
3j	65	68.4 ± 7.94
Tacrine	66	551.58 ± 19.17
Galanthamine		360 ± 10

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Fig. 5 Relative residual activity of AChE plotted against the different concentrations of the most potent compounds 3d (a) and 3e (b).

The newly synthesized compounds were evaluated for their drug like behavior through in silico predictions. The ADME profiles of the compounds are given in Table 3. Some of the important parameters [molecular weight (MW) and volume (V), CNS activity, partition coefficient (log P), number of hydrogen bond donors and acceptors (HBD and HBA), polar surface area (PSA), number of rotatable groups (rotor), number of N and O (N and O), BBB permeability (log BB), permeability across Mandin–Darby canine kidney, (MDCK) and intestinal epithelial cells (Caco-2), solvent accessible surface area (SASA) and % of human oral absorption (% HOA)] that are crucial for CNS activity were identified and displayed in Table 3 along with the reference values.^25,26

Table 3 ADME profile of the compounds predicted by QikProp program

Compound	MW (≤450 Da)	HBD (≤3)	HBA (≤7)	log P (≤5)	CNS (≥0)	Rotor (≤7)	N and O (≤7)
3a	319	1	3	4.35	0	2	5
3b	398	1	3	4.92	0	2	5
3c	333	1	3	4.66	0	2	5
3d	416	1	3	5.01	0	2	5
3e	433	1	3	5.32	0	2	5
3g	417	1	4	4.41	0	2	6
3h	335	2	4	3.58	−2	3	6
3i	337	1	3	4.53	0	2	5
3j	348	1	5	3.51	−2	3	7

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Compound	BBB (−1.2 to 1.2)	SASA (320–735 Å^2)	Volume (≤1250 Å^3)	PSA (60–70 Å^2)	Caco (≥500 nm s^−1)	MDCK (≥500 nm s^−1)	% HOA
3a	−0.51	605	1052	64	1171	587	100
3b	−0.35	634	1105	64	1171	1554	100
3c	−0.54	637	1112	64	1171	587	100
3d	−0.27	641	1118	64	1170	2391	100
3e	−0.22	649	1141	63	1174	3126	100
3g	−0.51	634	1106	76	675	1362	100
3h	−1.12	618	1077	86	355	161	94
3i	−0.43	612	1065	64	1169	899	100
3j	−1.16	620	1090	96	319	144	92

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Using these physicochemical properties it is possible to differentiate the CNS drugs from a group of molecules. It was found that the values for MW, volume, N and O, rotor, HBD and HBA were in the preferred range for all the compounds. The lipophilicity (derived from log P) of molecules are in the allowed limits except 3d and 3e. It was already reported that CNS acting agents may have slightly higher log P value.⁸ PSA is always linked with BBB penetration and normally for CNS active molecules the PSA values will be low compared to other therapeutics.²⁷ For majority of the compounds, the PSA value is optimal except 3g, 3h and 3j. The upper limit of PSA for the BBB permeability was reported as 90 Ų, and hence the two compounds such as 3g and 3h can also be taken into consideration.²⁸ The SASA of all compounds are seems to be in the range of CNS acting agents. The polar groups containing compounds such as 3h and 3j has very poor BBB, MDCK cell permeability, Caco cell permeability and CNS activity. MDCK and Caco cell lines are used as in vitro models to study the BBB permeability.²⁹ The online BBB prediction tool also gave the similar results. It showed all compounds except 3h and 3j have the ability to cross BBB.

The conclusion made from the enzyme kinetics studies was further explored with the help of molecular docking followed by binding energy calculations. The close analysis of the AChE–ligand complexes revealed a common binding pattern for all the newly synthesized ligands. They binds at the bottom of the active site, spanning along the acyl binding pocket and makes contacts with peripheral anionic site residues also. In all complexes, the aromatic residues such as F297, F295, F338, Y337, Y341 and H447 are oriented perpendicular to the dihydrobenzimidazo pyrimidine ring and arrests the rotational and translational movement of the ligands in the active site. Apart from these, hydroxyl group of Y337 and Y124 are acting as an anchor to hold the compounds at its current positions through a series of hydrogen bonds with keto group and ring nitrogens. A classical π–π stacking interaction was also observed between the phenyl ring of the compounds and the indole ring of W86. Furthermore, π–π stacking between dihydrobenzimidazo pyrimidine ring system and H447 an F338 was also observed. Docking studies also showed that the compounds 3d and 3e were more potent inhibitors than other compounds with binding energies −87.03 and −92.26 kcal mol⁻¹ respectively. These compounds forms additional hydrogen bonds with keto group and Y341. They also exhibit halogen bonds with the polar atoms of the following residues such as Y119, G120, G121, S125 and Y133. Apart from that a series of van der Waals interactions and hydrophobic interactions are also found to stabilize the interactions. The residue such as Y72, V73, W86, Y124, Y133, F295, F297, Y337, F338, Y341 and I451 seems to be involved in the hydrophobic interactions. Whereas in case of tacrine, the interactions are mainly favoured by stacking formed against Y337 and W86. A hydrogen bond with N atom and main chain carbonyl oxygen of H447 is also seen. It also forms a series of hydrophobic interactions like the compounds 3d/3e. But it lacks halogen bonding and other hydrogen bonding interactions as seen in case of 3d and 3e. These interactions may be partly contributing to the highest inhibitory potency of the compounds over tacrine. The binding mode of the top scored ligands (3d and 3e) was displayed in Fig. 6. The binding energies of all the compounds are mentioned in Table 4.

Fig. 6 Binding pattern of compounds 3e (A) and compound 3d (B) at the active site of hAChE. The protein residues are shown in lines and the compounds are shown in stick. The hydrogen bonds are indicated by dotted lines.

Table 4 The binding energetics of the compounds at the active site of hAChE

Comp.	Glide score	Binding energy	Interacting residues
3a	−8.24	−74.38	F338, H447, Y337 and W86
3b	−8.62	−75.55	F338, H447, Y337, Y124 and W86
3c	−8.09	−72.60	F338, H447, Y124, Y337 and W86
3d	−9.15	−87.03	F338, H447, Y341, Y124, Y337 andW86
3e	−9.79	−92.26	F338, H447, Y341, Y337, Y124 and W86
3g	−8.70	−85.55	F338, Y124, Y337 and W86
3h	−9.11	−75.07	F338, Y124, Y133, G121 and W86
3i	−8.90	−76.65	F338, H447, Y124, Y337 and W86
3j	−9.04	−74.13	F338, H447, Y124, Y337 and W86
Galanthamine	−9.69	−81.51	F338, S203, E202 and Y337

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In general, the effective AChE inhibition of the compounds are achieved by interaction with the residues in the peripheral anionic site (Y72, Y124 and Y341), anionic site (W86 and Y337), acyl binding site (F295 and F297) and active site (H447). Also the orientation of these compounds in the ligand binding site is in such a manner that it completely mask the entry of the substrate in to the active site. Also it can hinder H447 and make it unavailable for the catalytic activities. Stability of the compounds (3d and 3e) at their bound positions was investigated through 20 ns MD simulation. During the simulation, the intermediate structures were saved and superimposed to the native structure with respect to the ligand position. The RMSD indicates that none of the complex deviates much from their respective binding positions. The maximum deviation observed for ligand was 1.4 Å and the minimum was 0.6 Å. The studies indicate that the ligands are stable in their binding position. The RMSD of two top scored compounds 3d and 3e were displayed in Fig. 7.

Fig. 7 The RMSD graph of Cα (blue in colour), ligand displacement (pink in colour) of compound 3d (A) & compound 3e (B).

Conclusions

In summary, we have carried out a regioselective (γ–α) attack of bifunctional nucleophiles to MBH acetates. Various bifunctional nucleophiles such as 2-aminobenzimidaole, 2-aminoimidazole and 3-amino 1,2,4 triazole on reaction with MBH acetate gave moderate to excellent yields (45–82%) of dihydroazo pyrimidine derivatives. Interestingly, of all the potential elctrophilic sites (α, β, γ, δ) the first Michael addition occurs at γ position of MBH acetates in a S_N2 followed by another nucleophilic attack at α position with a 6-endo trig cyclisation to give rise to functionalised dihydroazo pyrimidine derivatives. The in vitro enzyme inhibition studies of these derivatives indicate that they exhibits much higher potency than the known drugs such as tacrine and galantamine. The above results describes the importance of MBHAs in the synthesis of dihydroazo pyrimidine derivatives and their possible utilization against AD treatment as AChE inhibitors.

Experimental section

Experimental procedure for synthesis of compound (3a–3l)

To a solution of 2-aminobenzimidazole (0.15 mmol) in CH₃CN (1.0 mL) was added MBH acetate of nitro alkene (0.18 mmol) and Cs₂CO₃ (0.3 mmol) at RT. The reaction mixture was stirred for 12 h at RT. After completion of reaction (monitored by LCMS), the reaction mixture was quenched with water (5 mL). The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated to give the crude product. To this crude product was added methanol (4 mL) and stirred for 30 min at RT. A white precipitate was obtained which was filtered to give the desired product (60–82%).

Enzyme inhibition studies

The in vitro inhibitory effect of newly synthesized ligands were assessed by Ellman's method using AMPLITE™ AChE assay kit (AAT Bioquest, Inc., Sunnyvale, CA). The assay system consists of AChE from electric eel (EC 3.1.1.7), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB, known as Ellman's reagent) and acetylcholine. The assay procedure is as follows, an aliquots (total volume 100 μL) was prepared by mixing 5.7 nM AChE (prepared in 0.1% of BSA containing distilled water) with acetylcholine (500 μM) and DTNB in a reaction buffer of pH 7.4. As a result of enzyme substrate interaction, the yellow colour formation was monitored and measured at 405 nm using a spectrophotometer at a time interval 2 minutes each for 20 minutes. Later the optical density was plotted against time. The same experiment (in triplicate) was repeated by incubating (15 minutes) the enzyme with different ligands. The relative activities of all ligands were expressed and compared with that of native enzyme activity. Finally the half maximal inhibitory concentration (IC₅₀) was determined for each ligands and compared with a known inhibitor tacrine.

Molecular modelling studies

The physico chemical properties, atomic level of interactions and binding stability of newly synthesized compounds were predicted using different molecular modelling experiments. Initially the ligands (drawn using Chemsketch program) were prepared by correcting the bond lengths and bond angles followed by energy minimization using LigPrep module with OPLS-3 force field. Different tautomers of ligands were also generated and used for different studies. By using the module QikProp 4.7, the physico chemical, ADME (Absorption, Distribution, Metabolism and Excretion), lipinski rule of five, in silico BBB penetrability and CNS activity of newly synthesized ligands were predicted. The BBB permeability of the ligands were reasserted using a bayesian statistical model implemented in an online tool ‘B3PP 09’ (http://www.b3pp.lasige.di.fc.ul.pt). As a part of investigating the atomic level of interaction of newly synthesized ligands with human AChE a molecular modeling studies was carried out. Crystal structure of hAChE complex with galanthamine (4EY6) was downloaded from protein databank (PDB) and prepared by deleting all the crystallographic water molecules and adding hydrogen. The protonation states and partial charges were assigned for the charged residues during preparation. Finally the prepared structure was optimized and minimized. Later the ligand binding site was mapped by taking galanthamine as an axis point. Further the docking studies were performed between the prepared ligands and protein using extra precision (XP) mode of glide. The binding free energies of the ligand enzyme complex were also deduced using Prime MM-GBSA method.

The correlation between experimental IC₅₀ values and theoretical binding energies were identified and highly active complex were selected for molecular dynamics (MD) simulation studies to predict the stability of the complex. The enzyme ligand complexes were prepared for MD simulation by soaking the complex in to an orthorhombic solvent box (volume 508 093 ų) containing 13 100 TIP3P water molecules. The whole system was neutralized by adding 7 Na⁺ ions. Total simulation time lasts around 20 ns. The intermediate structures were saved at every 10 ps and superimposed to the native structure and deduced the RMS deviation. Finally a graph was plotted with RMS deviation against time.

Acknowledgements

SA thanks DST-SERB for financial support under young scientist scheme (SB/FT/CS-079/2014). EKR and AMS thanks Syngene. RC thanks Indian Council of Medical Research for the Senior Research Fellowship and KVD thanks IISER for the institute post-doctoral fellowship. The authors thanks IISc for providing single crystal X-ray diffraction data, and the authors gratefully acknowledge the ‘Bioinformatics Infrastructure Facility’ (supported by DBT, Govt. of India) located at the department of Biotechnology and Microbiology, Kannur University for providing the computational works. Authors RC and S.C acknowledge the financial support of Indian Council of Medical Research in the form of project (No. BIC/12(23)/2012).

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1446736 and 1456725. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra12507g

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