Regio ‐ and Stereoselective Synthesis of a New Series of Spirooxindole Pyrrolidine Grafted Thiochromene Scaffolds as Potential Anticancer Agents

: A series of new spiro ‐ heterocycles engrafted spirooxindole/pyrrolidine/thiochromene scaffolds was synthesized by the three ‐ component 1,3 ‐ dipolar cycloaddition reactions in a fully controlled regio ‐ and stereo ‐ selective fashion. Condensation of several substituted isatin deriva ‐ tives with L ‐ proline generated the azomethine ylides which subsequently reacted with chalcones based thiochromene scaffold, and finally afforded the target spiro ‐ compounds. This simple pro ‐ tocol furnished a structurally complex, biologically relevant spiro ‐ heterocycles in good yields through a one ‐ pot process. All synthesized chalcone ‐ based thiochromene, along with the spiroox ‐ indole/pyrrolidine/thiochromene scaffolds, were tested for their anticancer activity against four cancer cell lines (PC3, HeLa, MCF ‐ 7, and MDA ‐ MB231). Toxicity of these compounds was also evaluated against human fibroblast BJ cell line, and they appeared to be not cytotoxic. For the prostate cancer (PC3) cell line, the most active hybrid, among synthesized series, was compound ( 7f ,IC 50 = 8.7 ± 0.7 μ M). The most potent spirooxindole/pyrrolidine/thiochromene hybrid against cervical (HeLa) cancer cells was compound ( 7k , IC 50 = 8.4 ± 0.5 μ M) having chlorine and p ‐ trifluoromethyl substituents attached to phenyl rings. Finally, against the MCF ‐ 7 and MDA ‐ MB231 breast cancer cell lines, compound ( 7d ) was the most active member of this series (IC 50 = 7.36 ± 0.37, and 9.44 ± 0.32 μ M, respectively).

There are many reports on different approaches in the literature on the construction of spirooxindole systems [44,45]. For example, Mannich reactions and related transformations were successfully employed in the construction of naturally occurring spirooxindole alkaloid, (±)-elacomine [46]. An oxidative rearrangement approach of tetrahydro-β-carbolines and related core structures [47], an intra-molecular Heck reaction and similar transformations approach were reported by L. Overman and M. Rosen for the total synthesis of spiro-trypro-statins B [48]. Transition metal-catalyzed synthesis of the spirooxindole scaffold [49] and 1,3-dipolar cycloaddition reactions are regarded as useful approaches for the construction of the spirooxindole scaffold [50]. However, successful approaches for the synthesis of thio-chromenyl/spirooxindole systems are still limited, although replacement of the oxygen atom by sulfur could be a rational strategy for improving pharmacological activity in drug discovery [51][52][53]. The design and synthesis of new compounds as potent and safe anti-cancer agents with low side effects is still a challenge.
Based on literature reports which highlight the biological importance of thiochromene sub-structure and spirooxindole/pyrrolidine scaffolds, our aim was to synthesize a new hybrids comprising three pharmacophores, spirooxindole/pyrrolidine/thiochromene employing three components 1,3-dipolar cycloaddition reaction. This protocol is based on multiple bond formations [54] and hence were able to generate compounds with structural complexity for possible use in agrochemical, drug discovery, and pharmaceutical industries.

General
"Thiochroman-3-one, acetophenone derivatives, substituted isatins and L-proline were purchased from Aldrich or TCI, and used as received. All solvents were used as received when experiments were conducted in air. Flash chromatography was performed on 100-200 mesh silica gel. The 1 H-and 13 C-NMR spectra of the synthesized compound were recorded on a JEOL 400-MHz spectrometer (JEOL, Ltd., Tokyo, Japan) at ambient temperature. The solvents used were DMSO-d6, and CDCl3; the chemical shifts (δ) were given in ppm. Single-crystal X-ray data of compound 7m were collected on a Rigaku Oxford Diffraction Supernova diffractometer at 120 K. Melting points were determined using Mel-Temp apparatus and are uncorrected. Thin Layer Chromatography (TLC) was conducted on silica gel (Kiesel gel G, E.Merck) and spots were detected under UV light at 254 nm. FT-IR spectra were measured on a Perkin Elmer, Spectrum 100 FT-IR spectrometer (FT-IR, Perkin Elmer, USA). Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 eV."

Synthesis of (4a-e) and (7a-m)
Three steps synthesis of the target compounds are presented in Schemes 1 and 2. The first step was to synthesize 4-chloro-2H-thiochromene-3-carbaldehyde (2) which is required to prepare chalcone based thiochromene scaffolds (4a-e) (Scheme 1). The second step is a three-component reaction in which the 1,3-dipolar cycloaddition key-reaction between thiochromene based-chalcone and the azomethine ylides, generated the substituted isatins (6a-e) (Isatin (6a), 5-chloroisatin (6b), 6-chloroisatin (6c), 5-fluoroisatin (6d), and 5-nitroisatin (6e) and amino acid L-proline (5) (Scheme 2). Thus, the diversity points in our library are the substituents at the isatin ring, and the aromatic substituents in the thiochromene based-chalcone. All three component reactions were carried out by heating an equimolar mixture of the thiochromene based-chalcone, isatin derivative, and L-proline in MeOH under reflux conditions for 3-6 h. After completion of the reaction as checked by TLC, the solvent was evaporated and the cyclized spiro-compounds were purified by column chromatography to afford pure cycloadducts in a very good yield. The following aromatic substituents in the thiochromene based-chalcone in the para-position (Cl, F, NO2, Br, and CF3) were employed to explore the substrate scope, and to establish generality of this approach. The structures of the spirooxindoles (7a-m) were assigned based on spectrophotometric tools including 1 H-& 13 C-NMR, MS and IR spectral analyses which have been found with a high constituency with the proposed chemical structures. Additionally, compound (7m) was obtained in crystalline form suited for single crystal X-ray diffraction analysis. The final cycloadducts were obtained in excellent regio-selectivity and diastereo-selectivity. The reaction mechanism assumed to proceed via two steps shown in Scheme 3. The first step is to generate the azomethine yields by the reaction of isatins with L-proline to afford the cyclic lactone, followed by decarboxylation. The cycloaddition step-2 occurs in a such way as to make the two carbonyls of the oxindole ring and the chalcone moiety trans to each other to minimize the steric repulsion in the final product. Besides, the stereochemistry of the stereo-genic centers is unambiguously confirmed by single crystal X-ray analysis for compound (7m) as a representative example. 1 H-NMR spectrum showed the assigned protons and matched with the proposed structure. A singlet at δ 8.32 was assigned to the NH proton. The signals occurred at δ 7.74 -6.66 were assigned for the aromatic protons while the protons of the fused pyrrolidine ring were assigned at δ 4.73 -1.91. The two protons of the methine group adjacent to the sulfur atom of the thiochromene ring were observed at δ 3.64 and 3.56. 13 C-NMR spectrum showed the characteristic carbon signals of the proposed compound. IR spectrum showed the desired final compound's functionalities and the functional groups disappeared as active carbon in isatin derivatives, as well as the olefin functional group in the chalcones.

X-Ray Structure Description
The X-ray structure of (7m), crystallized in the monoclinic crystal system and of centrosymmetric C2/c space group, with eight molecular units per unit cell was elucidated. The unit cell parameters are a = 36.6524(7) Å, b = 8.27830(10) Å, c = 17.1963(3) Å, 102.216(2)° and V = 5099.55(15) Å 3 . The X-ray structure was in agreement with the spectral characterization and the formation of the spiro system of the compound (Figure 2). The two spiro-moieties (oxindole and pyrolizine) are linked together via the C15 asymmetric center. The thiochromene moiety was also found to be connected to the pyrolizine via the C10 chiral carbon. The molecular units of this compound form a dimer via noncovalent interactions (Figure 3). In this figure two molecules of (7m) are connected via N-H…O hydrogen bonding interactions. Crystal data and structure refinement for compound (7m) are listed in

Biological Activity
All chalcones-based-thiochromenes (4a-e), and the spirooxindole/pyrrolidine/thiochromenes (7a-m) were initially examined for their toxicity against the human fibroblast BJ normal cell line. The results depicted in Table 1 indicated that all the synthesized compounds were non-toxic, except compounds (7d) and (7k) which appeared to be slightly toxic at 30 μM concentration. The toxicities of these two compounds may be due to the presence of F, CF3 and Cl substituents at the phenyl ring-based chalcone, and the chlorine atom attached to C-5 incorporated isatin moiety.
The antiproliferative activity against four human cancer lines, including prostate PC-3, cervical HeLa, and breast (MCF-7 and MDA-MB231) were evaluated by MTT assay [55][56][57][58][59] and the results were compared with the standard anti-cancer drug doxorubicin as a reference. MCF-7 is a human breast cancer cell line with glucocorticoid, progesterone and estrogen receptors it is widely used worldwide for in vitro anti-cancer assay. The cell line is known to retain mammary epithelial characteristics, particularly estrogen (the first hormone to respond to breast cancer) processing via estrogen receptors [60,61]. In contrast the MDA-MB-231 cell line is particularly used to model late stage cancer, and is considered to be a good triple negative model because of the lack of growth factor receptor HER2 and absence of ER, PR, and E-cadherin [62].
The results of antiproliferative activity against the prostate cancer PC3 cell line by the spiro-oxindole/pyrrolidine/thiochromene series (7a-m) showed that the most active hybrid in these series was compound (7f) (IC50 = 8.7 ± 0.7 μM), having para-NO2 aromatic substituent, and the fluorine at C-5 of the isatin ring, whereas complete loss of activity was observed for compound (7g) having a chlorine atom at C-5 of the isatin ring. The replacement of para-NO2 with para-bromo at the aromatic ring also contributed towards a decrease in activity, as observed for compounds (7h) (IC50 = 16 ± 0.7 μM). Further decrease in activity was observed for compounds with 4-chloro (7i, IC50 = 27.7 ± 0.9 μM) and 5-nitro (7j, IC50 = 22.5 ± 0.4 μM) groups, attached to the isatin ring. Present of a chlorine atom at C-6 of the isatin ring, in combination with the para-fluoro-substituted benzene ring, decreased the activity of compounds (7e, IC50 = 27.5 ± 0.5 μM), and (7i, IC50 = 27.7 ± 0.9 μM) against PC-3 cells. On the other hand, compound (7k) with a chlorine atom at C-5 at the isatin ring and p-trifluoromethyl benzene appeared to be the second most active member of the series (IC50 = 15.6 ± 0.3 μM), whereas replacement of the C-5 chlorine isatin ring with C-5 nitro isatin moiety resulted in a complete loss of activity, as observed in compound 7l. Compounds (7a-d, 7g, 7l, and 7m) were not active.

Conclusions
During this study we have successfully achieved the synthesis of a new library of hybrid based spirooxindole/pyrrolidine/thiochromenes (7a-m) by three-component 1,3-dipolar cycloaddition reactions in complete regio-and stereo-selective fashion. The anti-cancer assay showed promising results as good candidates for further studies. Compounds (7f, IC50 = 8.7 ± 0.7 μM) exhibited more potent activity against PC3, whereas hybrid (7k) was most active against cervical cancer HeLa (IC50 8.4 ± 0.5 μM) and for breast cancer MCF-7 cell lines (7d, IC50 = 7.36 ± 0.37 μM), whereas (7d, IC50 = 9.44 ± 0.32 μM) also appeared more active against MDA-MB231 breast cancer cell line. The mechanism of action and in vivo study will be considered in the near future to further validate results of in vitro assays. Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in Supplementary Material.