Regio- and Stereoselective Synthesis of Dispiropyrrolidineoxindole Derivatives by Using Classical and Microwave Conditions, and Molecular Docking Studies of Them as Potential Cyclin Dependent Kinases (CDK5) Inhibitors

Abstract A Solvent-free microwave-assisted green approach has been presented the synthesis of dispiropyrrolidineoxindole compounds with regio- and stereoselectivity and the excellent yields in short reaction times. A comparative study of the synthesis of dispiropyrrolidineoxindole derivatives by conventional heating and microwave condition has been described by the cycloaddition of azomethine ylides generated from sarcosine and isatin with the dipolarophile aurones. Molecular docking studies were carried out to explore the synthesized bispirooxindoles as inhibitors of CDK5. One of these compounds exhibited excellent binding affinity for cyclin dependent kinases (CDK5). Also, the structures were established by spectroscopic techniques as well as single crystal X-ray analysis. Graphical Abstract


Introduction
Multicomponent reactions are powerful synthetic tools for the efficient creation of complex organic molecules in a one-pot fashion. Furthermore, the amount of needed solvents and energy for separating and purifying of intermediates is significantly reduced that is beneficial from the green chemistry issues point of view. These strategies have acquired extensive consideration in pharmaceutical science and combinatorial chemistry. 1 Moreover, Microwave irradiation, the nonconventional energy source, has emerged as a valuable synthetic methodology for scientific experts and medication designers in multicomponent reactions because of offering an environmentally friendly approach, enhancing the yields, diminishing the reaction times and a few byproducts forming. 2-4 Also, temperature sensitive reactions to be better controlled in the milder conditions created by microwave irradiation. Under these particular reaction conditions, dielectric properties, entrance profundity and energy transfer are unique in relation to those in conventional heating, so an improvement in the chemo-, regio-, and stereoselectivity can be noticed. 5,6 The utilization of microwave irradiation in organic reaction, and specifically in 1,3-dipolar cycloaddition has recently acquired a lot of consideration and extraordinary advancement has been accomplished in this area. 7 Literature survey reveals that the spiropyrrolidineoxindole scaffold of natural or synthetic is enriched with a wide scope of pharmacological activities. For example, Horsfiline, isolated from Horsfieldia superba, utilize as a native medication. 8 Elacomine, naturally occurring oxindole alkaloids, isolated from Eleagnus commutate. 9 Rhynchophylline and Isorhynchophylline have been utilized as antihypertensive, antipyretic and anticonvulsant medications for the treatment of dizziness, epilepsy and headache. 10 (Figure 1) Recently, some complex molecules with bispirooxindole frameworks have been synthesized, which has caused important notice in the organic synthetic and medicinal chemistry community. [11][12][13][14][15][16][17] Due to the extraordinary biological properties of spiropyrrolidineoxindole derivatives, the design and study of manufacturing methods for these compounds possess extremely importance. The progressively massive numbers of these scaffolds and unlimited quantity of techniques used in their preparation have created them great candidates for the microwave assisted approach. 18

Results and discussion
The three-component azomethine ylide cycloaddition reaction of isatin, sarcosine and Aurone was investigated to compare conventional method 19 whit green reaction conditions under microwave irradiation. Initially, a model reaction with isatin (1a), sarcosine (2a) and 6-methoxy-2-(3nitrobenzylidene) benzofuran-3(2H)-one (3a) was performed in methanol. As shown in Table 1, by the reaction in methanol after 12 h, the product of bis-spirooxindole (4aa) was obtained in 47% yield (Table 1, entry 1). In order to increase the reaction yield some factors such as different solvents, temperature and time were examined under microwave irradiation as shown in Table 1.
A range of solvents with different polarities like MeOH, EtOH, THF, DMF, Toluene, 1,4dioxane and water were screened. The non-polar (Toluene, 1,4-dioxane) solvents were found to be incompetent for the desired transformation (Table 1, entries 3, 4). In addition, the reaction yield was comparatively less in polar aprotic solvents (THF, DMF) than polar-protic solvents such as MeOH, EtOH and water (Table 1, entries 2, 5). The reaction under reflux condition in MeOH was proceeded for 12 hours to obtain the desired product 4aa that was 47% yield. Also, the same reaction was performed under MW irradiation at different temperatures and power in Methanol for 15 min to furnish the desired 4aa and the best reaction result related to entry 8 with 91% yield. Reduction of irradiation time and power led to reduced efficiency. (Table 1, entries 8-10).
Interestingly, we found that changing the conditions from conventional heating to solvent-free microwave irradiation led to a reduction in reaction time with improvement in the yields from 47 to 94%. (Table 1, entry 11). Also, reduction of irradiation time and power led to reduced efficiency. (Table 1, entries 12, 13) Thus, microwave irradiation (300 w) at 100 C in Methanol and solvent-free condition were the optimized reaction conditions to compare conventional method.
MW mediated reactions in MeOH is considered a sufficient method for the synthesis of intermediate 4 without using any base and metal. In this method, it takes only few minutes to complete the reaction and it offers good yields (77-91%). In our final series of experiments, we examined the equivalent solvent-free reaction, simply by gentle grinding of the three components and irradiation of the mixture under microwave conditions. This afforded the anticipated cycloadducts in excellent yields (80-94%) with high regio and stereoselectivity and also sufficient purity with easy work up, when it compared with conventional heating method ( Table 2).
The structures of products were established by spectroscopic techniques as well as single crystal X-ray analysis. In the proton NMR spectrum of 4ea ( Figure 2) a sharp singlet appeared at d 2.17 for -NCH 3 protons of the pyrrolidine ring. The -NCH 2 protons and benzylic proton of pyrrolidine ring appeared as triplet at d 3.62 and 4.20, 4.33 which explained the regiochemistry of the cycloaddition. In contrast, if the other regioisomer had been formed, the benzylic proton would have appeared as a singlet instead of triplet or doublet of doublet in the 1 H NMR spectrum. For the 13 C spectrum of 4ea, the two spiro quaternary carbons resonated at 76.34, 109.45 ppm and the oxindole and tetralone carbonyl carbons resonated at 173.80 and 197.13 ppm and the -NCH 3 carbon demonstrated a signal at 42.81 ppm.
In Scheme 1, a reasonable pathway for the synthesis of dispiropyrrolidine oxindole compounds is given. In the reaction isatin (1) condenses with sarcosine (2) to product of 5. Because of oxazolidone 5 is composed exclusively with trans stereochemistry, the exit of group CO 2 from this specie proceeds through a stereospecific 1,3-cycloreversion to lead the formation of anti-1,3dipolar 8. Then, 1,3-dipolar addition of anti-azomethine ylide 8 and dipolarophile (3) generates dispiroheterocycle 4.
Also, the dispiroheterocyclic structure was confirmed by single-crystal X-ray diffraction analysis of 4ec (CCDC 2143257) ( Figure 3). The relative configuration of these dispiroheterocyclic products was also assigned according to the single-crystal X-ray diffraction analysis.

Molecular docking studies
The docking of synthesized ligands with cyclin dependent kinases (CDK5) shows that all the compounds have acceptable interactions with reasonable matching within the binding site of receptor. It is involved in biological pathways; the postmitotic CDK5 plays important roles for neuronal migration and differentiation. CDK5 is a notable pharmacological target because its deregulation is implicated in widely neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. 20 In this study, it has been used the B3LYP/6-311G level of density functional theory (DFT) to get the most stable structure for the ligands. The geometry of the compounds was optimized in gas phase using the GAUSSIAN-09 package program. 21 The protein structure file (PDB code: 1UNH) was downloaded from PDB (www.rcsb.org/pdb). Docking scores for all compounds showed in Table 3 that 4ea ligand has the best interactions with protein. 3D interactions of 4ea ligand with CDK5 exhibited in Figure 4. The bonding interactions with amino acids in the active pockets were displaced in 2D view ( Figure 5) these studies were simulated using AutoDock vina program. 22

Conclusion
In summary, in our research, it has developed a green and economical protocol for intramolecular 1,3-dipolar cycloadditions of azomethine ylides leading to dispirooxindoles skeletons with sterically congested two vicinal spiroquaternary stereocenters in a single step. The developed methodology  yielded dispiropyrrolidineoxindole with excellent yields and diastereoselectivities under mild reaction conditions. The reactions were investigated under three different conditions, in the solvent under reflux and microwave conditions, as well as solvent-free under microwave irradiation which illustrates that microwave irradiation is an efficient methodology to promote 1,3-dipolar cycloaddition reactions, leading to the enhancement of yields and shorter reaction times.
In addition, in silico molecular docking studies, which were carried out to explore the inhibitory activity of novel compounds, revealed compound 4ea exhibits the highest binding affinity for cyclin dependent kinases (CDK5) and thus, it could be a promising lead starting molecule for CDK5 inhibitor.

Chemistry
All the compounds evaluated in this work were synthesized in one-pot sequences using a singlemode microwave cavity (Ethos-MR, 2.45 GHz, maximum power 1000 W). All materials used are commercially available and were purchased from Merck and used without any additional purification. 1 H NMR and 13 C NMR of products were recorded on a Bruker (Avance DRX-500) spectrometer using DMSO-d6 as solvent at room temperature. Chemical shifts d were reported in ppm relative to tetramethylsilane as an internal standard. Monitoring of the reactions was carried out by thin-layer chromatography using Merck TLC silica gel 60 F254 plates. Elemental analyses were conducted with a Perkin-Elmer 2004 Series II CHN analyzer.
Dispiro products (4aa-4cc); typical experimental procedure for the synthesis of them    reflux for the appropriate time. The reaction progress was evidenced by TLC analysis. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by recrystallization from MeOH to afford the desired product (4aa) with 47% yield (221.4 mg). Method B: A mixture of isatin 1a (1 mmol, 147.1 mg), sarcosine 2 (1 mmol, 89.2 mg), 6methoxy-2-(3-nitrobenzylidene)benzofuran-3(2H)-one 3a (1 mmol, 297.1 mg) in methanol was irradiated under microwave conditions in a teflon flask at 100 C for 15 mins. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by recrystallization from MeOH to afford the desired product (4aa) with 91% yield (428.7 mg).

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
We gratefully acknowledge the funding support received for this project from the Sharif University of Technology (SUT), Islamic Republic of Iran.