Synthesis and molecular modeling studies of cholinesterase inhibitor dispiro[indoline-3,2′-pyrrolidine-3′,3′′-pyrrolidines]

A set of dispiro[indoline-3,2′-pyrrolidine-3′,3′′-pyrrolidines] 8a–l was regioselectively synthesized utilizing multi-component azomethine cycloaddition reaction of 3-(arylmethylidene)pyrrolidine-2,5-diones 5a–e, isatins 6a–c and sarcosine 7. Single crystal X-ray studies of 8c add conclusive support for the structure. Compounds 8e and 8g reveal cholinesterase inhibitory properties with promising efficacy against both AChE and BChE and were found to be more selective towards AChE than BChE as indicted by the selectivity index like Donepezil (a clinically used cholinesterase inhibitory drug). Molecular modeling studies assist in understanding the bio-observations and identifying the responsible parameters behind biological properties.


Introduction
Dementia is one of the most serious health problems for older people. About 50 million people are suffering from dementia globally with 10 million new cases yearly according to the WHO (World Health Organization). 1 Alzheimer's disease (AD) represents the most common cause of dementia (60-70%). 1 AD is a fatal chronic neurodegenerative disease associated with memory impairment and language decits besides high degeneration of cholinergic neurons of the central nervous system. 2,3 Although no cure has been discovered for AD, a few pharmacological targets have been rationalized. Reduction of formation and aggregation of pathological hallmarks of AD (insoluble amyloid-b oligomers and tau neurobrillary tangles) is a pharmacological target for AD. Other targets include modulation of neurotransmitter signals (cholinesterase inhibitors and N-methyl-D-aspartate receptor blockers). 4 Although the full mechanism of AD is not well elucidated yet, extensive studies explained that the brain of AD patients suffers from cholinergic neuron damage. This is why acetylcholine (AC) level is considered an important therapeutic target of AD. AC is an important brain neurotransmitter with major roles in memory and maintaining consciousness. 5 Acetylcholinesterase (AChE) is a catabolic enzyme capable for hydrolysis of AC. Butyrylcholinesterase (BChC) also regulates the AC levels. This is why inhibition of both cholinesterases is useful for AD patients. 2,6 Tacrine (Cognex) 1 was the rst cholinesterase inhibitor approved drug 7 (Fig. 1). However, due to many clinically adverse effects including elevated liver transaminase levels it has been discontinued in many countries. 8, 9 Meanwhile, many researchers are still interested in this compound for developing analogs of lesser side effects. [9][10][11] Currently, many cholinesterase inhibitors are clinically used as AD drugs of which Galantamine (Razadyne) 2 (approved by FDA "Food and Drug Administration" on 28 Feb. 2001) for mild and moderate AD. 4,12,13 Rivastigmine (Exelon) 3 (approved by FDA in 21 April 2000) is also useful for mild and moderate dementia patients caused by AD or Parkinson's disease. 4,14,15 Donepezil (Aricept) 4 is also a cholinesterase inhibitor approved by FDA on 25 Nov. 1996 for AD. 4,16,17 Currently, available drugs are used to manage and prevent progress of the disease over time but unfortunately not able to cure. Additionally, the drugs lack long term efficacy and also associated with severe side effects. This is why urgent need of effective anti-AD agents are still compelling. 18 The present study is focused on the construction of novel dispiro[indoline-3,2 0 -pyrrolidine-3 0 ,3 00 -pyrrolidines] and exploring their cholinesterase properties. Rational for the targeted chemical scaffold is based on the bio-isosteric form of the indanyl nucleus of Donepezil 4 and the indolyl heterocycle of the targeted agents. 19 Additionally many natural and synthetic indole containing-compounds show promising cholinesterase inhibitory properties 20-23 including mono-and bis-spiroindoles. [24][25][26][27] The biologically active spiro-indoles developed by our group also prompted the current study. [28][29][30] Results and discussion Chemistry Synthetic route towards the targeted dispiro[indoline-3,2 0 -pyrrolidine-3 0 ,3 00 -pyrrolidine]-2,2 00 ,5 00 -triones 8a-l is depicted in Scheme 1. Azomethine ylides generated from the reaction of reuxing isatins 6a-c and sarcosine 7 in ethanol reacted regioselectively with 3-(arylmethylidene)pyrrolidine-2,5-diones 5ae 31-33 affording solely the corresponding dispiro analogs 8a-l (TLC monitor). The non-stabilized azomethine ylide is formed in situ due to the applied reaction conditions (reuxing ethanol) through CO 2 elimination from spiro-oxazalidinone. The latter is formed via condensation of amino acid (sarcosine) with isatin(s). 34 The IR spectrum of compound 8a (example of the synthesized compounds) shows strong bands at n ¼ 1786, 1713 cm À1 assignable for the stretching vibration of carbonyl groups. 1 H-NMR spectrum of 8a reveals the diastereotopic methylene protons of pyrrolidinedionyl H 2 C-4 00 and pyrrolidinyl H 2 C-5 0 at d H ¼ 2.37, 2.71 and 3.49, 3.84, respectively. The methine pyrrolidinyl HC-4 0 is shown as a triplet signal at d H ¼ 4.39. 13 C-NMR spectrum of 8a reveals the pyrrolidinedionyl CH 2 (C-4 00 ) and pyrrolidinyl CH 2 (C-5 0 ) at d C ¼ 36.7, 58.4, respectively. The spiro carbons are shown at d C ¼ 61.0, 77.6 assignable for C-3 0 (C-3 00 ) and C-3 (C-2 0 ), respectively. The methyl and methine (C-4 0 ) carbons are revealed at d C ¼ 34.6, 48.4, respectively. Additionally the carbonyl carbons are exhibited at d C ¼ 173.0, 177.1 and 177.5. HSQC of compounds 8b and 8g support these interpretations (ESI Fig. S1-S38 † show the spectral charts of the synthesized compounds). Single crystal X-ray study of compound 8c supports the structure (Fig. 2).

X-ray studies
The ORTEP view of compound 8c is shown in Fig. 2. The compound is in the monoclinic system and space group P2 1 /c with four molecules in the unit cell and one molecule in the asymmetric unit of the crystallized form. Two spiro linkages exist in 8c attaching the central pyrrolidine ring to the pyrrolidinedione at C12 and to the indolyl heterocycle at C23. In general, the geometric parameters including both bond lengths Scheme 1 Synthetic route towards the targeted dispiro[indoline-3,2 0 -pyrrolidine-3 0 ,3 00 -pyrrolidine]-2,2 00 ,5 00 -triones 8a-l. and angles (ESI Tables S1-S3 †) are in good agreement with the pre-determined structures having similar rings and moieties. 35,36 The two phenyl rings (C4 / C9 and C15 / C20) as well as the indolyl heterocycle are planar conformations. The two pyrrolidinyl rings (C2-N3-C10-C12-C13) and (C12-C14-C21-N22-C23) are envelope conformations with the ap atoms being C12, lies 0.223 (3) A out of the mean plane of the remaining four atoms, and N22, lies 0.662 (4) A out of the mean plane of the remaining atoms, respectively. The sum of angles around the N22 atom is approximately 330 conrming its sp 3 hybridization. The C14 and C23 atoms occupy axial and equatorial positions with respect to the pyrrolidinedione. In the crystal, molecules are linked together by set of intermolecular C-H/O hydrogen-bonding interactions forming supramolecular assemblies ( Fig. 3 and Table 1).
Antiproliferative properties. Antiproliferative properties of the synthesized agents 8a-l were considered against RPE1 (human immortalized retinal pigment epithelial cell line) normal cell line utilizing the standard MTT (tetrazolium salt) technique. 37 The adopted technique is useful for exploring the cytotoxicity properties of the synthesized agents. From the obtained results (ESI Fig. S39 †), it has been noticed that none of the synthesized agents reveal any cytotoxicological properties against the tested cell line (IC 50 ¼ >100.0 mM).
Acute toxicological bio-assay. The most effective agents synthesized with cholinesterase properties (8e, 8g and 8h) were subjected for acute toxicological bio-assay in mice utilizing the standard technique. 38 None of the tested agents reveal any mortality rates or toxicological symptoms (including animal body, legs, hair or tail) at the tested doses (50, 100 and 250 mg kg À1 mice body weight) supporting the safe utility of the tested agents at the mentioned doses.

Molecular modeling studies
Molecular modeling is an efficient technique useful for validating biological properties and identifying the parameters necessary for bio-properties beside its unique importance for developing/estimating novel hits/leads.
2D-QSAR. The cholinesterase properties observed were undertaken by CODESSA-Pro soware accessible for optimizing 2D-QSAR models. [39][40][41] This is useful for well understanding the bio-observations and identifying the parameters necessary for bio-potency. Three descriptor QSAR models were developed due  to the AChE and BChE inhibitory properties of the synthesized dispiro-compounds 8a-l (ESI Tables S4-S9, Fig. S40 and S41, details of the QSAR descriptors are also mentioned in the ESI). † Goodness of the QSAR models was supported by the comparative values of the correlation coefficient (R 2 ) with their cross validation of leave one-out (R 2 cvOO) and leave many-out Additionally, the correlations of the observed and predicted values "specially the high potent synthesized compounds" add good support for the attained models.
3D-pharmacophore. 3D-pharmacophore modelling of the synthesized agents 8a-l was utilized by Discovery Studio 2.5 soware (ESI Fig. S42-S45, Tables S10 and S11 †). It has been noticed that, 3D-pharmacophoric modelling of the tested compounds as AChE inhibitors comprises four chemical features (two hydrophobics "H-1, H-2", one hydrogen bonding acceptor "HBA" and one positive ionisable "PosIon"). The tested compounds were tted with variable affinity with the mentioned chemical features giving rise to different estimated properties. Aryl groups attached to C-4 0 and N-1 are mapped with the hydrophobics H-1 and H-2, respectively. Meanwhile, the pyrrolidinyl carbonyl at C-2 00 and pyrrolidinyl N-1 0 are mapped with the HBA and PosIon, respectively. Mapping of the aryl rings at H-1 and H-2 explains their necessity in optimizing the AChE inhibitory properties. This observation supports the mentioned SAR due to the experimentally obtained results.
The 3D-pharmacophore modelling of the synthesized agents as BChE inhibitors exhibits three chemical features (hydrophobic "H", hydrogen bonding acceptor "HBA" and hydrogen bonding donor "HBD"). The aryl ring attached to the pyrrolidinyl N-1 00 is mapped with the hydrophobic "H". Meanwhile, the pyrrolidinyl carbonyl at C-2 00 and indolyl N-1 are mapped with the HBA and HBD, receptively. Again, mapping of the aryl ring at N-1 00 supported its importance for the revealed BChE inhibitory properties.

Conclusion
In conclusion it can be stated that, the synthesized dispirocompounds are good cholinesterase inhibitors especially compounds 8e and 8g which show good potency and selectivity index towards AChE over BChE similar to Donepezil (clinically used cholinesterase inhibitory drug). The attained QSAR and 3D-pharmacophore models are good enough to be considered for developing novel effective hits/leads with enhanced potency/ efficacy considering the elements controlling bio-observations (mainly the aryl rings attached to the pyrrolidinedione). Additionally, the multi-component azomethine cycloaddition procedure is an accessible technique for developing the targeted dispiro[indoline-3,2 0 -pyrrolidine-3 0 ,3 00 -pyrrolidines] in good yield (66-82%) and high regioselectivity.

Experimental
Melting points were determined on a capillary point apparatus (Stuart SMP3) equipped with a digital thermometer. IR spectra (KBr) were recorded on a Shimadzu FT-IR 8400S spectrophotometer. Reactions were monitored using thin layer chromatography (TLC) on 0.2 mm silica gel F254 plates (Merck) utilizing various solvents for elution. The chemical structures of the synthesized compounds were characterized by nuclear magnetic resonance spectra ( 1 H-NMR, 13 C-NMR) and determined on a Bruker NMR spectrometer (500 MHz, 125 MHz for 1 H and 13 C, respectively). 13 C-NMR spectra are fully decoupled.
Chemical shis were reported in parts per million (ppm) using the deuterated solvent peak or tetramethylsilane as an internal standard. Colorimetric enzyme inhibitory assays were performed in 96-well plates and the absorbance was recorded utilizing a microplate reader (Innite F50, Tecan, Switzerland).
Synthesis of dispiro[indoline-3,2 0 -pyrrolidine-3 0 ,3 00pyrrolidines] 8a-l (general procedure) A mixture of equimolar amount of the appropriate 5a-e (5 mmol), isatin 6a-c and sarcosine 7 in absolute ethanol (25 mL) was boiled under reux for the specic time. The separated solid while boiling, was collected and crystallized from a suitable solvent affording the corresponding 8a-g,i-k. In case of 8h,l the clear reaction mixture was stored at room temperature (20-25 C) overnight. So, the separated solid was collected and puried by crystallization from a suitable solvent.

Molecular modeling studies
The experimental procedures in details were mentioned in the ESI. †

Conflicts of interest
There is no conict to declare.