An ultrasound assisted synthesis of spirooxindolo-1,2,4-oxadiazoles via [3+2] cycloaddition reaction and their anti-cancer activity

A facile and ultrasound assisted green methodology was adopted to achieve spirooxindolo-1,2,4-oxadiazoles via a [3+2] cycloaddition of aryl nitrile oxides and isatin Schiff bases at room temperature with good yields and lesser reaction times. The proposed procedure avoids traditional column chromatography resulting in good to excellent yields. The synthesized compounds were evaluated for their anti-cancer activity against SK-OV-3, HeLa, HCT-116, DU-145, A549 and HEK-293 cell lines. Two of them showed significant anti-cancer activity against HeLa with IC 50 value 10.75±0.39 µM and 12.43±0.77 µM respectively


Introduction
1,2,4-oxadiazole ring system has become a popular scaffold in the field of drug discovery and development. 1,2,3he oxadiazole rings, which were an essential part of the pharmacophore for many different treatments present in the lead compounds to treat a wide range of disorders, including diabetes, obesity, inflammation, cancer, and infection. 4,5,6Additionally, 1,2,4-oxadiazoles are being actively employed to create novel materials with beneficial features, including solar cells, fluorogenic chemosensory polymers, liquid crystals, organic light emitting diodes (OLEDs), and high energy materials (HEDM). 7,8Due to these wide range of applications, synthetic organic chemists are particularly interested in developing efficient and effective methods for their production. 9,10,11In the field of medicinal chemistry, heterocyclic compounds, notably spirooxindoles, have played a significant role.The [3+2] cycloaddition process is a highly regio-selective approach for the synthesis of spiroxindoles. 12,13,14,15Spirooxindole alkaloids found in nature, such as horsfiline, extracted from Horsfieldia superba are used in traditional medicine (Figure 1). 16,17However, the spiroxindoles having 1,2,4-oxadiazole ring systems are rare in the literature.
On the other hand, cancer is the second biggest cause of death after cardiovascular illnesses, despite the extensive research that has been done. 18This remains a serious health threat and is one of the leading causes of mortality around the world. 19Chemotherapy is the most cost-effective treatment option offered to patients across the world, but, can have severe side effects, and malignancies can develop resistance to some of the most regularly used medications leaving the patients with limited treatment alternatives. 20As a result, more anti-cancer medications are needed with improved cancer cell selectivity.
Modern synthetic chemistry has recently paid increasing attention to ultrasonic irradiation (USI). 21The use of mechanical and thermal energy for chemical reactions without significantly changing the reaction media is made possible by the acoustic cavitation that is caused by ultrasonication. 22At room temperature, the energy produced by the cavity effect causes solubility, mass transfer, diffusivity, speeds up reactions, and shorten reaction time. 23As far as we are aware, there were no reports on the synthesis of spirooxindolo-1,2,4oxadiazoles using ultrasonic irradiation.Therefore, in this paper, we describe the use of ultrasound to aid in the [3+2] cycloaddition process to produce spirooxindolo-1,2,4-oxadiazoles, which were then examined for their anti-cancer potential as part of our continuing research into green chemistry and bio-active spirooxindoles. 24,25,26

Chemistry
To optimize the reaction parameters, we used 3-(phenylimino)indolin-2-one 1a and N-hydroxycarbimidoyl chloride 2 27 as model reactants (Table 1).Initially, a reaction was carried out in methanol at room temperature, yielding a trace amount of the desired product 3a (Table 1, entry 1).The reaction was then carried out in the presence of Et3N in MeOH.To our pleasure, the required product 3a was obtained with 40% yield (Table 1, entry 2).Solvents such as CH3CN, DCM and CHCl3 were tested to improve the yield.And the findings revealed that CHCl3 is the optimum solvent to obtain the desired compound 3a in 75% yield (Table 1, entries 3-5).Various bases such as DMAP, DABCO, DBU, and K2CO3 were investigated in order to determine the reaction efficiency (Table 1, entries 7-10), and Et3N was discovered to be an efficient base for the formation of target compound 3a.However, as it is a regular practice in our laboratory towards green methodologies, we tried the reaction in ultrasonication using CHCl3, DCM and CH3CN solvents (Table 1, entries 11-14), and found that CHCl3 is the best solvent for the generation of title compound 3a with 85% yield in 20 min (Table 1, entry11).Further when the reaction was proceeded beyond 20 min no improvement of the yield was observed (Table 1, entry 12).Further, we carried out the reaction at 60 °C in conventional as well as © AUTHOR(S) ultrasonication but there was no significant effect on reaction yield (Table 1, entry 6 & 15).Variation of the base equivalents from 2.0 to 1.5 and then 2.5 was resulting a negative impact on product yield (Table 1, entries 16, 17).As a result, we decided that 1 mmol of 1a, 1.1 mmol of 2, and 2 equiv. of Et3N in CHCl3 (Table 1, entry11) at room temperature under ultrasound irradiation is the best reaction condition for the generation of target compounds.Spectral methods IR, 1 H, 13 C NMR and mass spectrometry were used to characterize the structures of synthesized compounds 3a-r.The structures were also confirmed by single crystal X-ray diffraction method (3r).For instance, the peak at 1736 cm -1 in the IR spectrum of the compound 3o corresponds to the stretching AUTHOR(S) Scheme 2. Fragmentation pathway of the compound 3k.
Furthermore, the regiochemistry of the produced compounds was demonstrated by single crystal X-ray diffraction method of compound 3r (Figure 2) (CCDC: 2222697).Scheme 3 depicts the reaction process for the formation of target molecules 3a-r.When N-hydroxycarbimidoyl chloride 2 reacts with the base, it produces N-oxide 4. 28,29 The target product 3a was obtained when this in situ produced N-oxide undergoes a [3+2] cycloaddition reaction with isatin Schiff base 1a. 30,31heme 3. Plausible reaction pathway for the generation of target compound 3a.

AUTHOR(S)
Figure 2. ORTEP representation of the compound 3r.The thermal ellipsoids are drawn at 50% probability level.

Conclusions
An efficient green methodology using ultrasonication was developed for the synthesis of spirooxindolo-1,2,4oxadiazole derivatives (3a-r) characterized by spectral methods and the structures were authenticated by SXRD.The methodology is advantageous to synthesize the target molecules with good yields and lesser reaction times.The in-vitro anti-cancer activities revealed that the compound 3i and 3j showed significant activity against cervical cancer HeLa cell lines.The compounds 3d, 3h and 3p showed moderate activity against HeLa cell lines.The compound 3i also exhibited a significant activity against ovarian cancer SK-OV-3 cell lines.The SAR analysis revealed that the compounds containing chlorine atom at R 3 position (3i, 3j) showed significant activity whereas the compounds containing fluorine atom at R 1 (3d, 3h, 3p) showed moderate activity.The in silico anti-cancer molecular docking studies with the compounds 3m, 3r and 3o showed least binding energy against histone deacetylases enzyme (PDB ID: 1VKG protein).These in vitro and in silico studies suggest that these compounds are promising hits for anti-cancer activity.

Experimental Section
General.All the chemicals and solvents were purchased from Aldrich/Spectrochem.All melting points were checked by using Stuart SMP30 melting point apparatus (Bibby Scientific Ltd.United Kingdom) and were uncorrected.Ultrasonication irradiation was performed on MAXSELL MX 200SH-6LQ Ultrasonic power 200 W.The reaction progress was checked with TLC plates (E.Merck, Mumbai, India).IR spectra were recorded on KBr disc by using Perkin-Elmer 100S spectrophotometer (Perkin-Elmer Ltd.United Kingdom) from 4000-400 cm -1 . 1 H and 13 C NMR spectra were recorded on Avance-III Bruker-400 MHz spectrometer (Bruker Corporation Ltd., Germany) using DMSO-d6 as solvent and TMS as an internal standard and chemical shifts are expressed as ppm.Mass spectra were recorded on a Jeol JMSD-300 spectrometer (Jeol Ltd., Tokyo, Japan) in acetonitrile solvent.The SXRD data of the compound 3r was collected and solved by using Bruker Kappa Apex II CCD diffractometer and ShelXT software.
General procedure for the synthesis of isatin Schiff bases (1a-r) 37,38,39,40 An oven dried 50 mL reaction flask with a magnetic stirring bar was charged with isatin (10 mmol), aniline (10  mmol) in EtOH (15 mL).At 60 °C, the mixture was agitated, and the catalytic quantity of glacial CH3COOH was added.After the reaction was completed (as monitored by TLC), ice cold water was added, and the solid was filtered under vacuum, washed with cold methanol, and dried.
General procedure for the generation of target compounds 3a-r Et3N (2.0 equiv) was added dropwise during 10 minutes to a solution of isatin Schiff bases 1a-r (1.0 mmol) and N-hydroxycarbimidoyl chloride 2 (1.1 mmol) in CHCl3 (3 mL).For the proper period of time, the reaction was left at room temperature under ultrasound irradiation.The reaction mixture was concentrated under reduced pressure after completion of starting materials (TLC, EtOAc/n-hexane 1:10-1:8), and the residue was recrystallized from methanol to get the required products 3a-r.