Development of spiro-3-indolin-2-one containing compounds of antiproliferative and anti-SARS-CoV-2 properties

A series of 1″-(alkylsulfonyl)-dispiro[indoline-3,2′-pyrrolidine-3′,3″-piperidine]-2,4″-diones 6a‒o has been synthesized through regioselective multi-component azomethine dipolar cycloaddition reaction of 1-(alkylsulfonyl)-3,5-bis(ylidene)-piperidin-4-ones 3a‒h. X-ray diffraction studies (6b‒d,h) confirmed the structures. The majority of the synthesized analogs reveal promising antiproliferation properties against a variety of human cancer cell lines (MCF7, HCT116, A431 and PaCa2) with good selectivity index towards normal cell (RPE1). Some of the synthesized agents exhibit potent inhibitory properties against the tested cell lines with higher efficacies than the standard references (sunitinib and 5-fluorouracil). Compound 6m is the most potent. Multi-targeted inhibitory properties against EGFR and VEGFR-2 have been observed for the synthesized agents. Flow cytometry supports the antiproliferation properties and shows the tested agents as apoptosis and necrosis forming. Vero cell viral infection model demonstrates the anti-SARS-CoV-2 properties of the synthesized agents. Compound 6f is the most promising (about 3.3 and 4.8 times the potency of the standard references, chloroquine and hydroxychloroquine). QSAR models explain and support the observed biological properties.

www.nature.com/scientificreports/ flagged as attractive bio-active targets long ago (Gerhard Domagk, 1935) due to their antibacterial properties 31 . The sulfonamide motif can tether the bioactive agent in the targeted receptor/protein by forming hydrogen bonding with the neighboring amino acid functions. It is usually recognized as the bioisostere of the carboxylic group with limited drawbacks relative to the latter (metabolic toxicity, instability and capability for diffusion through bio-membranes) 31,32 . Many sulfonamides exert high efficacy as anticancer agents and have been approved as therapeutics. Belinostat [approved by Food and Drug Administration (FDA) in 2014] is used for treatment of peripheral T-cell lymphoma as a histone deacetylase inhibitor 33,34 . Vemurafenib (Zelboraf, approved by FDA in 2011 and 2017) treats BRAF V600 mutated late-stage skin cancer and Erdheim-Chester disease 35,36 . Dabrafenib (Tafinlar, approved in 2013 and 2018) is used for treatment of advanced melanoma and Mekinist also treats BRAF-positive cancer 37,38 (Fig. 2). Many sulfonamide candidates have been reported as potential antitumor agents due to their aromatase, topoisomerase or carbonic anhydrase inhibitory properties 31,[39][40][41] . Cancer is one of the most severe diseases threatening human life. Although many methodologies and techniques in addition to numerous drugs have been discovered and approved for cancer treatment, millions of people continue to suffer from the illness every year. The off-target effect is one of the major drawbacks of cancer therapeutics 42 . Consequently, recent focus in cancer chemotherapy is on the development of highly selective anticancer agents devoid of off-target effects, thereby producing enhanced potency/efficacy towards the cancer cell with reduced side effects 31 .
Owing to the anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) properties of indole [43][44][45][46][47][48] and sulfonamide-containing compounds 49 the targeted agents within the current study are also considered for anti-SARS-CoV-2 investigation. In the beginning of 2020, a health-socio-economic disaster emerged globally due to the highly infectious disease. Severe acute respiratory syndrome due to pathogenic viral infection of  www.nature.com/scientificreports/ SARS-CoV-2 leads to COVID-19 (coronavirus disease 2019). It is postulated that viral zoonotic jump (probably bat) to human was firstly recognized in Wuhan, China. The infection then spread worldwide causing a universal pandemic, according to WHO (World Health Organization) in March 2020 50,51 . Lack of an effective therapeutic was a major factor in the ensuing threat to human life. Several symptoms are associated with infection of which a cough, running noise, loss of smell and sometimes fever were highly publicized. In severe cases, blood clotting disorders and stroke are observed 52 . Drug repurposing was a wise and rapid strategy for urgent identification of potential therapeutics capable of controlling the disaster and saving lives. Usually, identification of novel drugs (de novo approach) with successive testing at pre-clinical and clinical phases takes several years. On the other hand, drug re-purposing allows faster adoption and utilization of well-studied existing and accessible therapeutics for treatment of infected patients 53 . Recently, Paxlovid (combination of Nirmatrelvir and Ritonavir) and Molnupiravir (Lagevrio) were approved (Dec. 2021) by the FDA emergency use authorization 54-57 (Fig. 3). Recent publications have mentioned the effective treatment of COVID-19 patients with anticancer drugs 58,59 . The successful clinical trials of colon cancer patients with antiviral drugs alone or in combination with anticancer drugs 60 , also inspired the biological studies considered in the current work. Reported are the results of investigation of antiproliferation (against cancer cell lines) and antiviral (anti-SARS-CoV-2) properties of the targeted spiro-heterocycles with particular focus on safety-related effects on normal cells. Arbidol which is an indolyl scaffold (Fig. 3) and been used as anti-influenza drug, was recently re-purposed against SARS-CoV-2 [61][62][63][64][65] . This is encouraging regarding the prospect of using the compounds from the current investigation against SARS-CoV-2 in addition to anti-tumor properties based on the mentioned bio-properties of the chemical scaffold considered. with structure determination presented in Supplementary Table S1. A possible factor in the ability of the molecules to interact with biological systems is molecular flexibility, in which the rotational freedom of the alkanesulfonyl and phenyl groups would play a role. The geometry of the pyrrolidine-piperidine systems in all the crystals of 6b, 6c and 6d is very similar, as indicated by the torsion angles (Supplementary Table S2). The molecule in the crystal structure of 6h also has generally similar geometry but significant deviation, of over 20° from the nearest value for the other molecules, for angle 4-5-6-1. This indicates that some flexibility is possible for the pyrrolidine-piperidine ring system. It is notable that the crystal of 6h contains a solvate molecule (ethanol).
The anti-A431 results support the SAR observation regarding the role of chlorine substitution to the indolinyl heterocycle in enhancing the antiproliferation properties relative to the unsubstituted analogs (compounds 6l and 6o are exceptions). The -I effect of the halogen atom attached to the phenyl group is also a contributory factor in the development of anti-A431 properties. Compounds with a fluorophenyl ring have lower anti-A431 proliferation values than the corresponding agents with a chlorophenyl ring. The bromophenyl-containing compound (6m) is superior to the other halogenophenyl-containing analogs. This is supported by the anti-A431 observations of compounds 6m/6i/6e (IC 50 = 2.434, 4.403, 16.064 µM, respectively), 6j/6f (IC 50 = 4.083, 4.764 µM, respectively), 6k/6g (IC 50 = 2.966, 6.167 µM, respectively) and 6l/6h (IC 50 = 3.694, 6.042 µM, respectively). The SARs are comparable to those previously discussed for anti-MCF7 and anti-HCT116 properties.

RPE1 cell line.
The safety of the synthesized agents against non-cancer/normal RPE1 (retinal pigment epithelium) cell line was assessed ( Table 1, Supplementary Fig. S52). The SI (selectivity index) for the tested compounds against RPE1 cell line confirms the safety towards non-cancer cells. Compound 6m (the most promising analog synthesized against MCF7, HCT116 and A431 cell lines) shows high SI values (SI = 4.1-6.1). Compound 6k (potent agent against PaCa2) reveals considerable SI value (SI = 1.7). Compound 6d and 6h which are also of high anti-PaCa2 show remarkable SI values (SI = > 3.8, > 3.6, respectively).
Illumination of the cells by lasers can identify the stained propidium iodide (PI) DNA cell content in stoichiometric population value. This is a commonly accessible methodology for studying cancer cell antiproliferation and cell cycle phase suppression 75 Supplementary Fig. S48). It is noteworthy that the percentage (%) of DNA content for compound 6l was higher in G1 phase than the control experiment (% DNA = 66.13, 57.12 for compound 6l and control experiment at G1 phase, respectively). This is conclusive evidence for the ability of compound 6l to suppress the tested cell line (MCF7) by arresting the cell cycle progress at G1 phase. Meanwhile, accumulation of % DNA content was observed by compound 6m at both G1 and S phases relative to the control experiment (% DNA = 62.51, 35.11; 57.12, 29.61 for compound 6m and control experiment at G1 and S phases, respectively). This is an indication for the ability of compound 6m to affect the MCF7 cell cycle progression due to its antiproliferation properties by arresting it at G1/S phases. Decrease of G2/M phase by both the tested compounds relative to the control experiment also supports the ability of the agents for suppression cell cycle progress due to their antiproliferation properties (% DNA = 10.13, 2.38, 13.27 for compounds 6l, 6m and control experiment at G2/M phase, respectively) ( It is also notable that both the tested compounds increase the apoptosis of the tested cancer cells 77 . Moreover, the total amount of apoptosis for the tested cell line is higher for compound 6m than that for 6l (total number of apoptosis = 38.41, 42.55 for compounds 6l and 6m, respectively). The late stage apoptosis of cells was observed in higher amount for compound 6l than for 6m (late stage apoptosis = 21.01, 13.71 for compounds 6l and 6m, respectively). On the other hand, necrosis observed for compound 6m is relatively higher than that of compound 6l (necrosis = 3.01, 3.96 for compounds 6l and 6m, respectively). In conclusion, it can be stated that both the tested compounds 6l and 6m are apoptosis and necrosis forming to the tested cell line due to their antiproliferation properties with compound 6m seeming more effective than 6l. These observations agree with the antiproliferation properties observed through MTT assay (Table 3, Fig. 8).
EGFR/VEGFR-2 inhibitory properties. EGFR (epidermal growth factor receptor) is a transmembrane protein tyrosine kinase involved in proliferation and differentiation in human cells (either normal or malignant). Increased EGFR activity due to overexpression is involved in many cancer types including non-small cell lung, breast, head and neck cancers. Therefore, agents targeting EGFR can serve as anticancer therapeutics with minimal off-target side effects [78][79][80] .
VEGFR (vascular endothelial growth factor receptor) is a cell surface tyrosine kinase receptor. Many members of the VEGFR family have been identified, indicating VEGFR-2 as the most important one. VEGFR-2 has attracted a lot of attention due to its important role against tumor-associated angiogenesis. Angiogenesis is the formation of new blood capillaries from pre-existing blood vessels. This is an essential process for many cellular functions such as proliferation, migration, and survival necessary for embryonic and adult development. Abnormal angiogenesis is associated with many diseases (such as inflammation, rheumatoid arthritis and cancer). This is the reason inhibition of VEGFR is a compelling approach to starve tumor cells and arrest solid tumor proliferation and metastasis [81][82][83][84] . Many small molecules have been discovered to possess VEGFR-2 inhibitory properties, including sunitinib 81 (which is an indolyl scaffold with structural resemblance to the targeted synthesized agents).
The synthesized agents were considered for inhibition of EGFR and VEGFR-2 based on their solid tumor proliferation properties and chemical structural resemblance to sunitinib. The western blot technique was applied utilizing the IC 50 observed for each respective agent synthesized during MTT assay 85,86 . Consideration of multitargeted inhibitory properties investigation is based in the fact that cancer initiation and proliferation sometimes utilizes many receptors or signaling pathways. Clinical effectiveness reveals that single-target drugs usually suffer from cancer cell resistance due to heterogeneity of tumor cells. This is why multi-targeted agents are preferable over single-target or multi-component drug cocktails 87,88 .
Evident SARs from the results indicate that the chloroindolyl-containing compounds have higher efficacy against SARS-CoV-2 than the unsubstituted analogs (compounds 6d, 6l and 6o are exceptions). The ethylpiperidone-containing heterocycles (compounds 6g and 6k are exceptions) have lower potency against SARS-CoV-2 than the methyl-containing analogs as shown by pairs 6a/6c, 6b/6d, 6f/6h and 6j/6l (IC 50  Acceptable safety indexes (SI) were observed for the synthesized agents. The chloroindolyl-containing heterocycles have higher SI than the unsubstituted indolyl-containing analogs (compounds 6b and 6o are exceptions).

Cholinesterase inhibitory properties. Alzheimer's disease is a progressive neurodegenerative illness
and causes the majority of dementia cases in the elderly. Progression of the disease affects the areas of the brain responsible for memory, thoughts and language skills of the patient. In the advanced stage of the disease, the patient is rendered unable to meet their basic needs of life. Most of the medications available act to slow the progress of the symptoms. Restoring the level of cholinesterases [acetylcholinesterase (AChE), butyrylcholinesterase (BChE)] is one of the most important approaches in Alzheimer's disease treatment [90][91][92] . Acetylcholine is a brain neurotransmitter with a major role in the maintenance of memory and consciousness 93 . AChE is the enzyme that hydrolyses acetylcholine. BChE can also regulate its level. Therefore, inhibition of both AChE and BChE is one of the main approaches for treating Alzheimer's 94,95 . The synthesized agents in the current study were assessed for AChE and BChE inhibitory properties based on the fact that many indolyl-containing compounds inhibit cholinesterases [96][97][98][99] . Numerous reports have also referred to the cholinesterase inhibitory properties of spiro-indole-containing compounds [25][26][27][28]100 .
The results (Table 6) reveal that some of the synthesized spiro-3-indolin-2-ones have promising inhibitory properties against both AChE and BChE. However, none of the synthesized agents shows potency comparable  www.nature.com/scientificreports/ It is evident that the fluorophenyl-containing compounds are more inhibitory against AChE than the unsubstituted phenyl-containing analogs as shown by pairs 6e/6a, 6f/6b, 6g/6c and 6h/6d (IC 50  The SI (selectivity index due to IC 50 against AChE relative to the IC 50 against BChE) of compound 6e is higher than that of the standard reference used (SI = 0.7, 0.8 for compound 6e and donepezil, respectively). This is due to its selective inhibitory properties against BChE relative to the AChE (IC 50 = 15.12, 22.17 μM against AChE and BChE, respectively). It is also noted that the SI values of compounds 6g and 6h are similar to that of the standard reference (SI = 0.8). Compounds 6b and 6n have SI comparable to that of the standard reference (SI = 0.9 for compounds 6b and 6n).

Molecular modeling studies. Molecular modeling techniques are useful tools in medicinal chemical
studies. Various techniques can be utilized in predicating new hits/leads, identifying the parameters necessary for bio-properties and understanding the exhibited bio-observations 25,101 . QSAR (quantitative structure-activity relationship) is one of the available approaches capable of generating mathematical models for connecting the bio-properties with physico-chemical (descriptor) parameters. Three main steps of QSAR modeling are; chemical structure optimization, descriptor calculation and validated modeling identification 102 . The current studies were undertaken by the CODESSA-Pro software 25  where the ε HOMO , C iHOMO are the highest occupied molecular orbital energy and its coefficient, respectively.
The square root of partial surface area for atom C is a charge-related descriptor also with a high coefficient value = 936.267. Again, the compound with low mathematical coefficient value reveals potent antiproliferation properties against the tested cell lines. This is obvious in compounds 6a and 6l (descriptor value = 0.07704, 0.06474 corresponding to estimated IC 50 = 19.024, 1.387 µM, respectively). Partial positively/negatively charged surface area can be calculated by Eq. (2). 103 where S D stands for the solvent accessible surface area for the hydrogen bonding donor of the hydrogen atoms selected by the threshold charge. The q D is partial charge on the hydrogen bonding donor of the hydrogen atoms selected by the threshold charge. S tot is total solvent accessible molecular surface area. where the PPSA2 stands for the total positively partial charged molecular surface area. TMSA stands for the total surface area of the molecule.

A431 QSAR model.
Average information content is a topological descriptor with a negative coefficient value (− 2.14585). For this reason, the synthesized agent with a high mathematical value represents a potent antiproliferation agent as shown by compounds 6a and 6h (descriptor value = 4.49045, 5.02123 corresponding to estimated IC 50 = 28.534, 3.930 µM, respectively). The mean information content index can be calculated by Eq. (6). 103 where n i stands for the atom number in the i th class. The n is the total number of molecular atoms. k is the atomic layer numbers in the coordination sphere surrounding a specific atom.  S12-S14, Fig. S57). Rotational entropy is a thermodynamic descriptor with a negative coefficient (− 9.48172). This explains the high antiproliferation efficacy of an agent with high a mathematical descriptor value as exhibited in compounds 6l over 6a (descriptor value = 37.155, 38.725 corresponding to estimated IC 50 = 47.054, 7.824 µM for compounds 6a and 6l, respectively). The rotational entropy of a molecule can be calculated by Eq. (7). 103

PaCa-2 QSAR model. The two-descriptor QSAR model describes the antiproliferation properties of the
where I j stands for the principal moment of molecular inertia. The σ stands for molecular symmetry number with, handk standing for Planck's and Boltzmann's constants. T is the absolute temperature (K). www.nature.com/scientificreports/ Maximum population of an electronic atomic orbital is a semi-empirical descriptor with a negative coefficient value (− 510.133). Therefore, a compound with a low mathematical value indicates low antiproliferation properties as shown in compound 6n over 6j (descriptor value = 1.98534, 1.93405 corresponding to estimated IC 50 = 7.488, 33.729 µM for compounds 6j and 6n, respectively).
Maximum electrophilic reactivity index for atom O is also a semi-empirical descriptor with a negative sign coefficient value (− 120.804). This explains the weak anti-SARS-CoV-2 properties of compound 6a relative to 6b (descriptor value = 0.0117, 0.009 corresponding to estimated IC 50 = 27.990, 9.632 µM, respectively). Equation (1) can calculate the descriptor value.
Minimum atomic state energy for atom N is a topological descriptor with a negative coefficient value (-7.98889). This explains the weak anti-SARS-CoV-2 activity of compound 6o relative to 6m (descriptor value = 184.7028, 184.5203 corresponding to estimated IC 50 = 8.612, 108.928 µM, respectively).
The most appropriate QSAR validation technique is internal validation due to the short data set utilized. Statistical validation including, the standard division and Fisher criteria support the accuracy of the QSAR models. The comparative values of the model's coefficient (R 2 ) to their leave-one-out (R 2 cvOO) and leave-many-out (R 2 cvMO) coefficient values also validate the optimized QSAR models. The comparative predicted properties due to the QSAR models relative to the experimentally observed values, especially for the high potent analogs, also support the molecular models which can be considered in a future study for assigning higher potent hits/leads.

Experimental
Chemistry. 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 shifts were reported in parts per million (ppm) using the deuterated solvent peak or tetramethylsilane as an internal standard.

Synthesis of spiro-3-indolin-2-ones 6a-o (general procedure). A mixture of equimolar amounts
of the appropriate 1-alkylsulfonyl-3,5-bis(ylidene)-piperidin-4-ones 3a-h 66 (2.5 mmol) and the corresponding isatin 4a,b with sarcosine 5 in ethanol (20 ml) was boiled under reflux for the appropriate time. The separated solid upon refluxing was collected and crystallized from a suitable solvent affording the corresponding 6a-c,e-g,i,m-o. For the remaining synthesized agents, the clear reaction mixture was stored at room temperature (20-25 °C) overnight. The separated solid was collected and crystallized from a suitable solvent affording 6d,h,j-l.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information Files. The X-ray data have been deposited in the CSD with reference numbers CCDC 2087291, 2087292, 2087297, 2087299 and the Check-CIF files are also attached as supplementary files to this article.