Ons because it had 5 coupling signals with H3 , H9,ten , and H11,13 (Figure S18, Supplementary Materials). HMQC Spectra explain the 1 and 1 interaction of H1 with C2 , C3 , C7 , C11 , and CO which further confirm the position of H1 proton (Supplementary Supplies Figure S19). two.two. Biological Activity All synthesized di-spirooxindole analogs 4a , attached with MNITMT Inhibitor substituted cyclohexanone moiety, had been initially examined for toxicity against human fibroblast BJ cell line and appeared to be non-toxic except compound 4g which was slightly toxic (IC50 = 21.7 0.2) at 30 concentration. The antiproliferative activity against four cancer cell lines, which includes prostate PC3, cervical HeLa, and triple-negative breast cancer (MCF-7 and MDA-MB231) was evaluated by MTT assay, when normal drug doxorubicin was used as a reference for comparison (Table 1).Molecules 2021, 26,7 ofTable 1. Results of anticancer assay against BJ, PC3, HeLa, MCF-7, and MDA-MB231 cells.Chemical Structure 4a-n Cancer Type/Cell Line (IC50 , ) a,b Human Fibroblast BJ Prostate PC3 Cervical HeLa Breast MCF-7 Breast MDA-MB=4aNA24.1 1.7.1 0.25.04 0.19.50 0.4bNA3.7 1.NA27.72 0.24.08 0.4cNA17.9 0.NA27.82 1.20.62 2.4dNA29.8 0.NANANA4eNA19.6 1.26.five 0.NANA4fNANANANS cNS4g21.7 0.14.three 1.NANANSMolecules 2021, 26,8 ofTable 1. Cont.Chemical Structure 4a-n Cancer Type/Cell Line (IC50 , ) a,b Human Fibroblast BJ Prostate PC3 Cervical HeLa Breast MCF-7 Breast MDA-MB=4hNANANANA14.43 0.4iNANANANA7.63 0.4jNANA11.9 0.NA10.49 0.4kNANANANANA4lNANA7.2 0.NA14.45 0.4mNANA24.six 0.JPH203 In Vivo NANA4nNANANANANASTD.aDoxorubicinNA1.9 0.0.9 0.0.79 0.b0.32 0.IC50 was evaluated applying MTT assay and will be the standard deviation from three independent experiments. tested compound didn’t show anticancer activity at 30 . c NS: Not soluble.NA indicates that theMolecules 2021, 26,9 ofAmong synthesized di-spirooxindole analogs 4a , compound 4b (IC50 = three.7 1.0 ) was identified to be the most active candidate against prostate cancer PC3 cell line in comparison to typical drug doxorubicin (IC50 = 1.9 0.4 ) and non-substituted spirooxindole analogue 4a (IC50 = 24.1 1.1 ). Structurally, in comparison to 4a, compound 4b consisted of 6-chloro substituted isatin moiety attached to non-substituted phenyl rings containing (2E,6E)-2,6-dibenzylidenecyclohexanone. The change of 6-chloro phenyl substituents of isatin moiety with 5-flouro, 5-methoxy, and five nitro phenyl rings contributed towards a gradual decrease in activity as in compounds 4c (IC50 = 17.9 0.2 ), 4e (IC50 = 19.6 1.2 ), and 4d (IC50 = 29.eight 0.1 ), respectively. Having said that, a major boost in activity was observed in compound 4g (IC50 = 14.three 1.0 ) getting a parabromo-substituted benzene rings attached to five nitro isatin moiety rather of five nitro isatin moiety containing compound 4d (IC50 was 29.8 0.1 ). All other compounds, i.e., 4f, and 4h appeared to be inactive against prostate cancer cell line PC3. The anticancer potential of the spirooxindole analogs 4a , attached with cyclohexanone moiety, was also evaluated against cervical cancer HeLa cell line in comparison for the regular drug doxorubicin (IC50 = 0.9 0.14 ). Probably the most active spirooxindole analog appeared to become compound 4a (IC50 = 7.1 0.2 ), possessing un-substituted isatin and aromatic ring of chlacones moieties. No adjust in activity was observed for compound 4l incorporated with 6-choloro isatin and p-fluoro-substituted aromatic ring of chlacones moieties (IC50 = 7.two 0.five ). However, substitution of p-fluoro atom of aromatic.