Novel Thiadiazoline Spiro Quinoline Analogues Induced Cell death in MCF-7 cells via G2/M Phase Cell Cycle Arrest

A progression of novel thiadiazoline spiro quinoline derivatives were synthesized from potent thiadiazoline spiro quinoline derivatives . The synthesized compounds portrayed by different spectroscopic studies and single X-ray crystallographic studies. The compounds were assessed for in vitro anticancer properties towards MCF-7 and HeLa cells. The compounds showed superior inhibition action MCF-7 malignant growth cells. Amongst, the compound 4a showed signicant inhibition activity, the cell death mechanism was evaluated by uorescent staining, and ow cytometry, RT-PCR, and western blot analyses. The in vitro anticancer results revealed that the compound 4a induced apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest. The binding anity of the compounds with ERα and pharmacokinetic properties were conrmed by molecular docking studies. The compound structures are characterized by spectroscopic techniques and X-ray crystallographic studies. The cytotoxicity of compounds were examined towards MCF-7 cells and HeLa cells. The synthesized compounds showed good inhibitory activity towards the MCF-7 cells. Amongst, the parent unsbstituted thiadiazoline spiro quinoline compound 4a exhibited higher activity. The cell death mechanistic studies showeds that the compound 4a induced apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest. The binding anity and pharmacokinetic properties of the compounds were further evaluated by computational studies. Hence, the potent thiadiazoline spiro quinoline derivative 4a could serve for breast cancer therapy.


Introduction
Cancer is one of the rapidly growing diseases and causes millions of deaths worldwide. Cervical and breast cancers are the most prevalent reason of cancer death in women. 1 Presently, radiotherapy and chemotherapy are the most effective cancer treatments. However, due to the toxicity and side effects of the currently available drugs, there is an emerging need for new targeting drugs with a various mechanism of actions. In the clinical samples, the most breast carcinoma cells express the estrogen receptor alpha (ERα). 2 ERα is a nuclear hormone which promotes the proliferation of the breast carcinoma cells. In the therapeutic view, ERα targeting therapy is one of the essential treatments in breast cancer patients.
In this therapeutic context, the sulfur and nitrogen-containing thiadiazole pharmacophores were widely used in the medicinal chemistry eld for drug designing. 3,4 The recent reports have demonstrated that the thiadiazole incorporated heterocyclic scaffolds have signi cant inhibition effects against the proliferation of various cancer cell lines with different mechanisms of action. For example inverse agonist for estrogen receptor alpha ERα, 5 PPARγ peroxisome proliferators activated receptor co activator 1α signaling by ERα ligand, 6 inhibition of glutaminase 1, 7 inhibition of sphingosine kinase, 8 inhibition of c-Met, 9 inhibition of mitotic kinesin Eg5, 10 inhibition of Src hormology-2 domain-containing protein tyrosine phosphate-2, 11 suppression of IL-6/COX-2 intermediated JAK2/STAT3 signals, 12 etc.
The quinoline heterocycles are signi cant class compounds that are found in several natural products and synthetic scaffolds. The early reports discovered that the quinoline analogues inhibits the growth of cancer cells by targeted mechanism of action such as Estrogen receptor binding, 13 Estrogen receptor degrader, 14 topoisomerase 1 (Top1) inhibitors, 15 inhibition of histone deacetylase 6, 16 inhibition of c-KIT kinase, 17 ataxia telangiectasia mutated (ATM) kinase inhibition, 18 topoisomerase IIα (TOP2A) inhibition, 19 HSP90 inhibitors, 20 tubulin polymerization inhibitors, dihydroorotate dehydrogenase (hDHODH) inhibitors, 21 inhibition of AXL kinase, 22 Inhibition of mutant isocitrate dehydrogenase 1 (mIDH1), 23 inhibition of Rho-associated protein kinase (ROCK), 24 inhibition of aldehyde dehydrogense 1A1 (ALDH1A1), 25 etc. Because of these crucial activities of quinoline scaffolds, recently we have reported 8-nitroquinolone based compounds, that resulting good antitumor properties. [26][27][28][29] Prompted by the early reports of the thiadiazole and quinoline motifs, herein, we synthesized a novel thiadiazoline spiro quinoline from 8-nitro quinoline thiosemicarbazones. All the synthesized thiadiazoline spiro quinoline compounds were assessed in vitro anticancer properties against HeLa and MCF-7 cells. Amongst, compound 4a showed signi cant activity towards MCF-7 cells. The molecular cell death mechanisms were evaluated and the results con rmed that the compound 4a prompted apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest in MCF-7 cells.
Furthermore, pharmacokinetics properties and binding interaction between the compound and ERα protein were con rmed by docking studies.

Results And Discussion
Chemistry New novel thiadiazoline spiro quinoline analogues were synthesized by two step reactions (Scheme 1). The initial reaction involves the synthesis of quinoline-thiosemicarbazones from the corresponding reaction between thiosemicarbazides 1a-d and C-6 functionalized 8-nitro-2,3dihydroquinolines 2a-c. In the next step, the cyclization was carried out by the treatment of quinolinethiosemicarbazones with acetic anhydride at 90 °C for 4 hours. The heterocyclization promotes the Nacetylation of the quinoline-thiosemicarbazones and afforded 1,3,4-thiadiazoline core of the spiro compounds in good yield.

Crystal Packing and Molecular Geometry of 4d
The two molecules of compound 4d were crystallized in centrosymmetric triclinic unit cell. The combination of the structure was con rmed by R-factor of 6.34%. As indicated by Cremer and Pope analysis the thiadiazole ring adopted envelope conformation with the puckering coordinates of q 2 = 0.199(2) Å and f 2 = 30.76(1)° and the six-membered nitro-substituted heterocyclic ring adopts twisted boat conformation with the puckering coordinates of q 2 = 0.433(2) Å, f 2 = 219.96(1)° and q 3 = 0.279(2) Å.
The phenyl and nitrophenyl rings are oriented with an angle of 13.2(2)° to each other. Further, the nitro group was bent out from phenyl ring plane with dihedral angle of 9.9(2)°. Two acetyl groups (-COCH 3 ) are twisted out from each with a dihedral angle of 21.9(5)° to one another. The hydrogen bonds geometry of the compound was showed in table 1. Symmetry transformation used to produce equivalent atoms: #1 x, y+1, z; #2 x, y+1, z-1; #3 -x+2, -y+1, -z+1; #4x, y, z-1; #5 -x+2, -y+1, -z+2; The crystal packing was conquered by non-classical C-H···O and classical N-H···O interactions. The intraand intermolecular interaction can be destroyed by the graph-set motif for better understanding of any supramolecular architecture ( Figure 2). In this context, the intermolecular interactions which connect the molecules in crystalline lattices are explained with graph set notation. 30 The classical intramolecular hydrogen bond prompts to self-related S(6) motif through N2-H2N…O2 interactions. Further, two centro-symmetrically related intermolecular interactions are formed through C-H…O interactions leading to ring R 2 2 (20) and R 2 2 (24) motifs in the reversal centers of unit cell (Figure 3a & 3b). Further, these cento-symmetrically connected dimeric rings linked along b-axis and bc-diagonal of the unit cell leading to chain C(9) and C(10) motifs (Figure 3c & d). Further, the crystal packing features C-H…p and p…p interactions.

Plausible Reaction Mechanism for Formation of Desired Compounds
A plausible mechanism of compound 4a-l with acetic anhydride is shown in Scheme 2. Initially, nucleophilic substitution with acetic anhydride on the NH group of 3a-l followed by the cyclization forms the mono acetyl intermediates II. And then the intermediates II gradually converted into diacetyl thiadiazoline compounds 4a-l.
In Vitro Anticancer Properties of Thiadiazoline Spiro Quinoline Analogues The recent reports revealed that the thiadiazole and quinolines are promising compounds which showed good anticancer activity towards tumor cells. Recently we have reported a new thiosemicarbazones, spiro pyrrolo oxindoles, indenoquinoxalines and pyarn derivatives from 8nitroquinolones that exhibited potential activity towards the cervical and breast cancer cells. [26][27][28][29] Hence, we selected speci ed cancer cell lines HeLa and MCF-7 to evaluate the cytotoxicity properties of the thiadiazoline spiro quinoline derivatives 4a-l. The toxicity of the compounds was evaluated by using MCF10A non-tumorigenic epithelial breast cells.
Initially, to evaluate the toxicity, various concentrations of the thiadiazoline spiro quinolines were treated with MCF10A normal breast cells. The compounds did not showed any signi cant toxicity up to 100 µM concentrations towards normal cells. Hence, to examine the cytotoxicity of the compounds, we used less than 100 µM of concentrations of compounds towards the cancer cells. The anticancer medication doxorubicin was utilized as positive control to analyze anticancer impacts of the compounds.
The MTT assay was performed to examine the inhibitory impacts 4a-l towards proliferation of HeLa and MCF-7 cells. The outcomes were summarized by IC 50 values that show the required concentration to inhibit the growth of 50% cells (Table 2). showed more potential inhibition activity than other ethyl and methyl and phenyl substitutions. On the observation of R 1 of the compounds, it was clear that the methyl and hydrogen substituted compounds showed better activity than chlorine substituted compounds. From the above perceptions, the order of inhibition activity of R substitution is H>CH 3 >CH 3 CH 2 >C 6 H 6 and R1 substitution is H CH 3 Cl. The compounds are more active against MCF-7 cells when compared with HeLa cells. Hence, we selected the potent compound 4a to examine mechanistic action towards MCF-7 cells.

Cell Morphology Analysis
The potent compound 4a was treated with breast cancer MCF-7 cells in two different concentrations (25µM & 50µM). The phase-contrast microscopy was used record the cell morphology changes. The compound 4a treated cells showed morphological changes like rounded, contracted and divided cell morphology whereas, the control cells exhibited usual morphology ( Figure 4). The morphology change of the cells showed that the compound 4a was prompted cell death in tested MCF-7 cells.

Apoptotic Cell Death Detection by Acridine Orange/ Ethidium Bromide (AO/EB)
The apoptosis provoked in the tested cancer cell was examined by acridine orange/ ethidium bromide (AO/EB) dual staining method. The MCF-7 cells were treated with 25μM and 50μM of the compound 4a for 24 hours and stained with AO/EB for 1 hour. The apoptotic morphology of the cells was examined in uorescence microscopy. The uorescence images showed orange stained cells with apoptotic fragmented, condensed chromatin nuclear morphology that showed late and early apoptotic dead cells. However, the non-treated control MCF-7 cells showed green uorescence with usual morphology ( Figure 5). The results revealed that the 4a prompted apoptotic cell death in treated MCF-7 cells.

DAPI Staining Analysis for Discovery of Nuclear Damaged Apoptotic Cells by DAPI
DAPI is the dye that ties strongly with damaged nuclear DNA and emits blue uorescence. 31 The MCF-7 cells were treated with 25μM and 50μM of the compound 4a for 24 hours and stained with DAPI for 1 hour. The nuclear damage morphology in the treated cells was examined by uorescence microscopy. In the uorescence microscopy images the compound treated cells were appeared with condensed chromatin and fragmented nuclear morphology and emit bright blue uorescence ( Figure 6). However, the non-treated control MCF-7 cells showed normal morphology with less blue uorescence. The results exposed that the compound 4a damaged nuclear DNA and prompted cell death in MCF-7 cells.

Cell Cycle Analysis
Flow cytometry cell cycle method was used to determine the cell cycle in tested MCF-7 cells. The MCF-7 cells were treated with the compound 4a in IC 50 concentration and then the cell cycle was investigated. The control untreated cells were exhibited 62.11% of cells in G0-G1 phase, 18.70% of cell in G1/S phase, and 15.18% of cells in G2/M phase. But the treated cells exhibited 21.44% of cell in G0-G1 phase, 9.47% of cells in G1/S phase, and 67.67% of cell in G2/M phase (Figure 7). From the ow cytometry results it was observed that the G2/M phase cells were extensively increased in tested cells. However, the G0-G1 and G1/S phase cells were reduced in treated cells. The cell cycle results exposed that the compound 4a prompted cell cycle arrest in G2/M phase.

Annexin V-FITC and PI Staining Assay
The ow cytometry annexin V-FITC and PI staining method were employed to quantify died cells. The IC 50 concentration of the compound 4a was ERα positive MCF-7 cells and stained with annexin V-FITC and PI. Afterward the stained cells and untreated control cells were analyzed by owcytometer. The results showed 62.99 percentage of necrotic cells, 0.30 percentage of late apoptotic cells and 0.33 percentage of early apoptotic cells (Figure 8). From the results, it was con rmed that the compound 4a induced cell death MCF-7 cells.

Inhibition of Estrogen Receptor ERα by compound 4a
Estrogen Receptor ERα is one of the hormone that promotes the proliferation of breast carcinoma cells. 32 ERα targeting therapy is one of the essential therapies for breast cancer treatment. Commonly, the compound binds with the ERα results in the decrease of mRNA levels of ERα. Hence, we have investigated levels of ERα mRNA in the 4a treated cells. The well-known ERα positive breast cancer drug tamoxifen was utilized as positive control to evaluate the inhibiting ability of 4a. The ERα positive MCF-7 cells were treated with the compound 4a in IC 50 concentration. Afterward the the ERα levels were determined by RT-PCR. The results showed signi cant decrease of ERα mRNA in treated cells (Figure 9). The inhibition of ERα by 4a was further con rmed by western blot analysis. After treatment, the proteins were taken out from the cells and the ERα levels were examined. The western blot results revealed that the compound 4a was signi cantly inhibiting the ERα levels in the treated cells which are similar to the standard Tamoxifen results (Figure 9c). The results revealed that that the compound 4a induced cell death by inhibition of ERα. From the above in vitro cytotoxic results, we conclude that the compound 4a induced apoptosis by G2/M phase cell cycle arrest and inhibition of estrogen receptor alpha (ERα) in MCF-7 cells.

Molecular Interaction of ERα with Compounds
Further, the interactions of the compounds with breast cancer ERα protein was examined by computational docking studies. The ERα protein was downloaded from Protein data bank PDB with the ID of 3ERT. The Schrodinger maestro module protein making wizard was used to set up the PDB structure, 33 and Schordinger ligPrep module was employed to prepare the compound structures. 34 The prepared compounds and the protein structures were exposed to molecular docking by using Glide module. 35 The results of the thiadiazoline spiro quinolines 4a-l showed better binding a nities with estrogen receptor alpha protein (Table 3). Amongest the compound 4a showed the most elevated binding a nity with a glide score of -5.925. The compound 4a showed hydrogen bonding interaction with ERα residues of CYS530, LEU 546 through acetyl group and a nitro group of the compound (Figure 10). The compounds 4i, 4d, and 4f, also showed good binding a nities by forming hydrogen bonding interaction with ERα. The drug-like ADME (adsorption, distribution, metabolism, and excretion) properties, Lipinski and Jorgensen rules were examined by pharmacokinetic studies using computational Qikprop module of Schrodinger. 36 The Lipinski's rules states that the molecular weight of the drug should be less than 500, the hydrogen bond donor of the drug should be ≤5, hydrogen bond acceptor of the drug should be ≤10, and the octanol / water partition coe cient (QPlogP o/w ) should be less than <5. The computational results of the compounds were satis ed the above rules (Table 4). Particularly, the potent compound 4a showed the molecular weight is 349.364 a.m.u., hydrogen bond donor is 2, hydrogen bond acceptor is 8 and the octanol/water partition coe cient is 0.8. These results revealed that the compound 4a ful lled the Lipinski's rules.
The Jorgensen's drug-like rules are the solubility in water QPlogS should be greater than -5.7, the cell permeability of Caco-2 QPP Caco value should be greater than 22nm/s, and the primary metabolic reaction value #metab 7 values should be less than 7. The computational results of the compounds were satis ed the above rules (Table 4). The selected active compound 4a ful lled the rules with the QPlogS value of -3,669, QPP Caco value of 66.525, and #metab value of 4. The above results indicated that the compound 4a is orally available. Further, the synthesized compounds ful lled the additional properties like QP log HERG values, partition coe cient of brain/blood QP log BB, the permeability of MDCK cell QPP MDCK, permeability of skin Qp log Kp, blood serum albumin binding capacity QP log Ksha, percentage of oral absorption, and Vander wall's polar surface area of oxygen and nitrogen atoms PSA ( Table 5). The results demonstrated that the compounds having drug like properties with strong with strong binding sites.

Conclusion
New thiadiazoline spiro quinoline derivatives were prepared from quinoline based thiosemicarbazones. The compound structures are characterized by spectroscopic techniques and X-ray crystallographic studies. The cytotoxicity of compounds were examined towards MCF-7 cells and HeLa cells. The synthesized compounds showed good inhibitory activity towards the MCF-7 cells. Amongst, the parent unsbstituted thiadiazoline spiro quinoline compound 4a exhibited higher activity. The cell death mechanistic studies showeds that the compound 4a induced apoptosis by inhibition of estrogen receptor alpha (ERα) and G2/M phase cell cycle arrest. The binding a nity and pharmacokinetic properties of the compounds were further evaluated by computational studies. Hence, the potent thiadiazoline spiro quinoline derivative 4a could serve for breast cancer therapy.

Experimental Section
Chemistry The analytical grade solvents and chemicals were bought from Loba Chime and Sigma Aldrich. The JASCO FT-IR 4100 spectrometer utilized for record the IR spectrum. The values of IR frequencies were presented in cm -1 . The Bruker advance 400 MHz NMR spectrometer employed analyse proton and carbon NMR spectrum. The chemical shift δ values presented in ppm. The Bruker AXS KAPPA APEX-2 diffractometer was used to analyze the single crystal structures. The CHNS elemental analyzer Perkin Elmer 2400 series II was used for elemental analysis.

Synthesis of Thaiadiazoline Spiro Quinolines 4a-b
The 8-nitro quinoline-thiosemicarbazones 3a-l were synthesized from the reaction of thiosemicarbazides 1a-d and 8-nitroquinolones 2a-c in methanol using few drops of acetic acid as catalyst. In the next step, the thiosemicarbazones 3a-l were re uxed in 20 ml of freshly distilled acetic anhydride at 90 °C for 3 to 5 hours. After completion of the reaction the whole mixture was poured into crushed ice. The obtained products 4a-b were ltered and recrystaled in methanol.

X-ray crystallographic analysis
All the crystals of the product were acquired by slow evaporation method. The good quality and transparent crystals were analyzed for X-ray crystallographic analysis using 0.71073 Å of X-ray wavelengths. The data of the solved crystals were given in table S1, S2, S3. The crystals ere re ned by SHELXL-2014 full-matrix least-squares calculations. Geometrically calculated bond distance of hydrogen atoms are -CH = 0.93 Å (for aromatic), -CH = 0.97 Å (for CH 2 ) and compelled to ride on the concerned parent atom with U iso (H) = 1.2 or 1.5 U eq (parent atom). The N-H hydrogen was isotropically re ned and placed from electro density map. The ORTEP diagram of the compounds was shown in Figure 1.

MTT Cytotoxicity Study
The MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was utilized to examine the cytotoxicity of synthesized compounds. The MCF-7 and HeLa cells were purchased from National Centre for Cell Science (NCCS) Pune. The HeLa and MCF-7 cells were placed in growth medium containing 96 well plates. The compounds were serially diluted and seeded to cells containing wells for 24 hours at 37°C and moisten with 5% CO 2 . Then the growth medium was suctioned and altered with MTT. After 4 hours incubation the MTT also removed and the acidi ed ethanol was added for the lysis step. The spectrometer was used to analyze the cell viability. The experiment was repeated three times as triplicate and then the IC 50 was determined from absorbance values.

AO/EB Dual staining assay
The apoptosis induced by the compounds was found by AO/EB dual staining process. The MCF-7 cancer cells were placed and developed in 24-well plate. Then 25μM and 50μM of the compound 4a was added to the cells and keep alive for one day. After that the treated cells were washed with PBS and stained with 5μg/mL of AO/EB for 1 hour. The stained cells were again washed with PBS and examined by Nikon ECLIPSE, TS 100 uorescence microscopy (excitation at 480/30 nm) to distinguish the apoptotic cells.

DAPI Staining Method
The compound induced nuclear damage in the treated cells was found by DAPI staining method. The MCF-7 cancer cells were placed and developed in 24-well plate. Then 25μM and 50μM of the compound 4a was added to the cells and keep alive for one day. After that the treated cells were washed with PBS and stained with 5μg/mL of DAPI for 1 hour. The stained cells were again washed with PBS and examined by Nikon ECLIPSE, TS 100 uorescence microscopy (excitation at 510 nm) to distinguish the apoptotic nuclear damage.

Cell Cycle Analysis
The ow cytometry method is used to examine the cell cycle. The MCF-7 cancer cells were placed and developed in 24-well plate. Then IC 50 concentration of the compound 4a was added to the cells and keeps alive for one day. After that the treated cells were washed with PBS and stained with 50 mg/mL of propidium iodide for 30 minutes. After staining the cells were examined by ow cytometry (BD bioscience, San Jose, CA, USA) for cell cycle distribution and DNA content.

Annexin V-FITC and PI Staining Assay
The apoptotic cells were quantitatively analyzed by ow cytometry annexin V-FITC and PI staining method. The MCF-7 cancer cells were placed and developed in 24-well plate. Then IC 50 concentration of the compound 4a was added to the cells and keeps alive for one day. After that the treated cells were suspended with binding buffer (200μL). Then the cells were stained with 5μL of propidium iodide and 10μL of annexin V-FITC for 30 minutes. After staining the cells were examined by ow cytometry (BD FACS caliber, USA) to quantify the apoptotic cells.

Gene expression analysis by RT-PCR
The reverse transcriptase-poly chain reaction RT-PCR method was used to examine the ERα levels. The MCF-7 cancer cells were placed and developed in 24-well plate. Then IC 50 concentration of the compound 4a was added to the cells and incubated for 24 hours. After that the levels of ERα in the treated cell was analysed by RT-PCR method and the β-actin was used as internal control. The conventional thiazole method was used to isolate the total RNA from the treated cancer cells. The cDNA conversion of separated RNA was performed by reverse transcription of M-MLV reverse transcriptase. From the RT product the level of β-actin and ERα was quanti ed using semi-quantitative PCR ananlysis.
The agarose gel was used to resolve the PCR product then then ethidium bromide was used to stain the resolved products to visualized in UV light. The experiment was repeated thrice as triplicate and the

Western blot analysis
The western blot method is used to analyze the level of ERα protein. The MCF-7 cancer cells were placed and developed in 24-well plate. Then IC 50 concentration of the compound 4a was added to the cells and incubated for 24 hours. The treated cells were washed with PBS and lysed with SDS (sodium dodecyl sulfate). The 10% SDS-PAGE gels was used to resolve the proteins from the lysates. The Gels were moved on nitrocellulose membranes and blocked with 5% of non-fat dry milk, incubated with primary antibodies of rabbit anti-ERα (1: 1000, CST) and, mouse antibody anti-β-actin. Then the membrane was washed with PBS. Then the goat anti-rabbit secondary antibody was added to the washed membrane and incubated for 1 hour. After incubation, the membranes were washed and examined by enhanced chemiluminescence ECL.