Design, Synthesis and Biological Evaluation of Morpholinated Isatin-Quinoline Hybrids as Potential Anti-Breast Cancer Agents.

Keeping in view the emerging need of potent and safer anti-breast cancer agents as well as pharmacological attributes of isatin, quinoline and morpholine derivatives, novel hydrazone linked morpholinated isatin-quinoline hybrids has been designed, synthesized and evaluated as anti-breast cancer agents. Synthesized hybrid compounds were preliminary screened against two breast cancer cell lines (MCF-7 and MDA-MB-231). Almost all synthetics showed potent inhibitory potential against hormone positive MCF-7 cells while inactive against hormone negative MDA-MB-231 cells. Potent compounds were further evaluated against L929 (noncancerous skin broblast) cell line and found highly selective for MCF-7 cells over L929 cells. Cell cycle analysis conrmed that most potent compound AS-4 (MCF-7: GI 50 = 4.36 µM) cause mitotic arrest at G 2 /M-phase. Due to higher selectivity toward estrogen receptor alpha (ERα) dependent MCF-7 cells various binding interactions of AS-4 with ERα are also streamlined, suggesting the capability of AS-4 in completely blocking ERα. Overall study suggest that, AS-4 can act as a potential lead for further development of potent and safer anti-breast cancer agents. NMR 19.58. Anal.


Introduction
Cancer remains the most di cult disease to treat with second leading cause of deaths around the globe, responsible for approximately 9.6 million deaths in 2018. In fact 1 out of 6 deaths is due to cancer and low as well as middle income nations have 70 % share in it [1]. Rapidly dividing cells of the organs like breast, skin and uterine are more fond to mutations as compared to the cells of other organs of human body thus at high risk to develop cancer. Breast cancer is most commonly occurring cancer type in women and cause signi cant morbidity and mortality [2]. In 2019, it was responsible for 41760 deaths among women and men in US alone [3]. In India situation is more alarming where BC has 25-32% share in all cancer cases. A survey report issued by Indian Council of Medical Research (ICMR) in 2016 estimated 14.5 million new cancer patients at that time which were estimated to be lifted up to 17.3 million in current 2020 [4].
Estrogen is a primary female sex hormone that play an essential role in the growth and development of mammary glands. Interaction of estrogen with its estrogen receptors (ERα and ERβ) are reported to play an important role in proliferation of mammary cells. All these facts resulted in the emergence of selective estrogen receptor modulators (SERMs) in drug development eld. MCF-7 cells which are ERα dependent in nature and are sensitive to SERMs while MDA-MB-231 cells are ERβ dependent in nature in which the expression of estrogen, progesterone and HER2 receptors is absent thus also known as triple negative breast cancer cells [5][6][7][8]. US Food and Drug Administration (US-FDA) has already approved triarylethylene derivatives like raloxifene, tamoxifen and toremifene as SERMs with anti-breast cancer pro le [9]. Among them, tamoxifen is a rst line drug and widely prescribed for the treatment of breast cancer. It responds to approximately 70% of ERα cases while other need adjuvant therapy that generally relapse [10][11][12]. Additionally, tamoxifen adversely affect the endometrium leading to endometrial cancer while another SERM raloxifene has hot ashes, insomnia, dizziness, and melancholy like side effects [13]. Thus there is a global need to develop novel potent and safer anti-breast cancer agents.
Quinoline (benzopyridine) is a well know biologically active nucleus that has been widely distributed in natural products and represents a family of compounds called quinoline alkaloids. Some of the quinoline alkaloids are well known for their anticancer potential including berberine, camptothecin, chelidonine, chelerythrine, dictamine etc. Inspired from the anticancer potential of quinoline nucleus, synthetic anticancer analogues has been successfully developed by the researchers and are under clinical trial such as bosutinib, levatinib, cbazantinib and tipifarnib [25]. Halogen substituted quinoline compounds especially chloro substituted ones are currently gaining interest particularly due to their anticancer potential [26]. Some chloroquinoline based molecular hybrids ( Fig. 1) has been reported as potential antibreast cancer agents (5-8) [27][28][29][30].
Morpholine is a unique nucleus with nitrogen and oxygen embedded in it and highly popular in medicinal chemists due to its unique biochemical features. Oxygen atom present in the outer end signi cantly increase the a nity of morpholine ring with donor-acceptor type interactions with enzymatic receptors.
Oxygen atom also decrease the basicity of nitrogen present in the ring via electronegative effect. Due to its unique biochemical properties, researchers are widely exploiting this nucleus for developing anticancer drugs with minimal or no side-effects. Morpholine containing drug named ge tinib has been approved by US-FDA for the treatment of metastatic non-small cell lung cancer while various other morpholine containing compound like GDC-0941, WAY-600, Foretinib, Copanlisib etc are under clinical trials [31].
Molecular hybridization is a well-established stratagem in drug development in which two bioactive moieties are combined together with or without any linker to get a single hybrid molecule having properties of both parent moieties with higher potency, reduced toxicity and minimized resistance [18]. CUDC-907 is a well-known example for molecular hybridization which is a hybrid molecule of vorinostat (a potent histone deacetylase inhibitor approved by FDA in 2006 for treatment of T-cell lymphoma) and GDC-0941 (a phosphoinositide 3-kinase inhibitor) which was more effective in both in vitro and in vivo models with no systemic toxicity and resistance. This hybrid molecule has recently passed the phase I clinical trial for advanced solid tumor and lymphoma treatment and entered in phase II clinical trial. Thus molecular hybridization would be an e cient approach for the development of potent and safer drug candidates with minimum resistance [33].
Hydrazone is a versatile moeity in medicinal chemistry that has been widely employed in architecting broad range of pharmacologically active compounds including antibacterial, antifungal, anticonvulsant, analgesic and anticancer etc. Various isatin based hybrids linked to other biological moieties through hydrazone has been reported with admirable anticancer activity [24]. Due to dominant and favorable pro le of hydrazone in anticancer area, it has been selected to be utilized into target hybrid molecules (10) to connect Quinoline with morpholinated isatin (Fig. 1).
Considering the alarming heath issue of breast cancer and lack of potent and safer anti-breast cancer agents, present study targets the synthesis of hydrazone linked morpholinated isatin-quinoline hybrids and evaluation against MCF-7 and MDA-MB-231 cell lines along with effect on cell cycle. Compounds with promising activity were evaluated for cytotoxicity against L929 (noncancerous skin broblast) cell line. Furthermore, the binding interactions of most potent compound with target ERα were explored via molecular docking studies.

Chemistry
Synthesis of targeted hybrids was conducted via a series of chemical reactions (Scheme 1), initiated from various substituted isatins (11). Isatins were reacted with various alkylated morpholines (12) with halogen on alkyl end in the presence of K 2 CO 3 using DMF as solvent. Reaction was carried out at room temperature and monitored by TLC. After completion, reaction mixture was poured on crushed ice and precipitates so obtained were subjected to column chromatography to get pure 1-(2morpholinoalkyl)indoline-2,3-diones (13). Simultaneously, 4,7-dichloroquinoline (14) was treated with hydrazine hydrate in ethanol under re ux, yielded 7-chloro-4-hydarzinylquinoline after washing and crystallization with ethanol itself (15) [34]. 1-(2-morpholinoalkyl)indoline-2,3-diones and 7-chloro-4hydarzinylquinoline were further re uxed together in ethanol with few drops of glacial acetic acid and monitored for progress by TLC. After completion, reaction kept overnight at room temperature for precipitation. Precipitates obtained were further washed and recrystallized with ethanol to get desired morpholinated isatin-quinoline hybrids (AS-1 to AS-18). All chemical reactions were proceeded very smoothly and hybrids were obtained in decent yields. Chemical structures of targeted hybrids were characterized through 1 H and 13 C NMR along with elemental analysis and were accordance with assumed structures.

Biological evaluation
Synthesized compounds were evaluated for their cytotoxic activities on two breast cancer cell lines using MTT assay. One was MCF-7, which is ERα dependent and hormone positive cell line while another one was MDA-MB-231, which is ERβ dependent and hormone negative cell line. Preliminary screening of test compounds was performed by using initial concentration of 100 µM each. Compounds showing percentage growth inhibition (Table 1) greater than 60 % were only considered as active and further evaluated for GI 50 values ( Table 2) using different concentrations of test compounds against sensitive breast cancer cell line along with L929 (noncancerous skin broblast) cell line. In preliminary screening all synthesized compounds showed potent growth inhibition potential against MCF-7 cell line while found inactive against MDA-MB-231 at 100 µM concentration that makes the hybrid molecules, selective inhibitors of hormone positive breast cancer. Among the screened hybrids compounds, compounds with two carbon alkyl chain between morpholine and isatin (AS-1 to AS-6) showed more sensitivity toward MCF-7 cells as compare to those with three (AS-7 to AS-12) and four carbon chain (AS-13 to AS-18). Suggesting that two carbon chain length between morpholine and isatin is most suitable for anticancer activity. Furthermore, only hybrids with two carbon chain length between morpholine and isatin (AS-1 to AS-6) were able to show growth inhibition more than 60 % thus explored for GI 50 values. Unsubstituted isatin in hybrid molecule (AS-1) give GI 50 values of 28.27 µM which is even lower than AS-5 and AS-6. Thus, the overall preference order for R becomes F > Cl > Br > OCH 3 > CH 3 > H and for carbon chain length between isatin and morpholine, it is n = 1 > 2 > 3 that generates a beautiful structure activity relationship. Active compounds when further evaluated against L929 cell line, most of them (AS-1, AS-2, AS-5 and AS-6) were found inactive with GI 50 values above 100 µM while AS-3 and AS-4 showed GI 50 values of 78.63 and 52.42 µM respectively. Most potent compound showed selectivity index of 12.03 between MCF-7 and L929 cell line that makes it highly selective for breast cancer cells over normal broblast cells.   . Accuracy of docking protocol was validated by docking co-crystallized ligand 4-hydroxytamoxifen into its binding site. The program was capable to reproduce best t con rmation of 4-hydroxytamoxifen in chain A with root mean square deviation (RMSD) value of 0.7756, indicating the reliability of docking protocol. After that AS-4 was docked into 4-hydroxytamoxifen binding site, and best pose with − 10.1584 score was selected for discussion (Fig. 3).
Overall binding pattern of AS-4 with its binding site disclose that compound is well settled in the cavity which is stabilized through various electrostatic interactions. Major interactions of AS-4 with ERα include π-σ, π-π stacked, π-alkyl, salt bridge attractive charge, halogen interaction, C-H bond, π-donor hydrogen bond and conventional hydrogen bond interaction. Chloroquinoline moiety of AS-4 is well stabilized in the cavity formed by Ala350, Leu387, Met357, Trp383, Leu536 and Leu354 (hydrophobic residues). Chloroquinoline moiety is seemed to be sandwiched between Trp383, Ala350, Leu387 and Leu536 stabilized through π-σ, π-π stacked and π-alkyl type interactions. Long distanced (π-orbital; d = 2.843 Å) π-alkyl interaction of chloro group on quinoline with aromatic residue of Trp383 is also observed. Short distanced conventional hydrogen bond interaction (H-bond acceptor; d = 1.876 Å) is observed between the Asp351 and hydrazone linkage that proves importance of this linkage in strong binding of AS-4 with active site. Halogen type interaction was observed between ouro group at isatin and Met528. Additional π-donor hydrogen bond interaction was also observed between isatin and Cys530. Alkyl chain between isatin and morpholine seems to interact with Asp351 through C-H bond interaction. A salt bridge attractive charge interaction is also observed between nitrogen group of morpholine and carboxylic oxygen of Asp351. Additional C-H bond interactions are observed between Asp351 and morpholine moiety. Another conventional hydrogen bond (H-bond acceptor; d = 2.692 Å) is observed between the oxygen of morpholine and Met528. Overall study seems to propose that AS-4 has been adequately decorated with small, rigid and planer groups showcasing ideal scaffold that is able to complete the pharmacophoric need for ERα inhibition.

Conclusion
In the present study, hydrazone linked morpholinated isatin-quinoline hybrids has been designed as antibreast cancer agents and synthesized in good yields that were characterized by using 1 H and 13

Materials and measurements
All chemicals and reagents were procured form Sigma Aldrich, Spectrochem and CDH, India and utilized without any further puri cation. All yield mentioned are refer to the isolated compounds after puri cation process. Characterization of synthesized compounds was done via spectroscopic techniques such as 1 H and 13 C NMR along with elemental analysis. NMR spectra were recorded on Avance III HD 500 MHz Bruker Biospin and JOEL 400 MHz using DMSO-d 6 . Chemical shifts in 1 H NMR were reported in δ values relative to TMS as internal standard (0.00 ppm). Splitting patter in obtained 1 H NMR spectra are reported as s: singlet, d: doublet, m: multiplet, br: broad peak and coupling constants (J) in hertz (Hz).

Cell cycle analysis
Most potent compound AS-4 against MCF-7 cell line was subjected to cell cycle phase distribution analysis [37] and was performed using BD Cycletest plus DNA Kit (BD Biosciences) according to manufacturer's instructions. MCF-7 cells (4×10 5 cells/well) were seeded in 6-well plates allowed for attachment. After 24 h, cells were treated with GI 50 concentration of AS-4. After treatment, oating as well as adhered cells were collected in falcon tubes (15 mL) and subjected to centrifugation for 5 minutes.
The cell pellet obtained after centrifugation was washed twice with PBS. After this, cell pellet was xed by using ethanol (70%, 1 mL) and kept at -20°C for 2 hr. After that, cells were washed with PBS again. Then 250 µL of solution A (trypsin buffer) was added to each tube and allowed to stand for 10 minutes at room temperature followed by the addition of 200 µL of solution B (trypsin inhibitor and RNase buffer). After 10 minutes, 200 µL of cold solution C (PI stain solution) was added and incubated for 1 hour in dark on ice. The stained cells were analyzed using BD Accuri software by ow cytometry (BD Accuri C6 Flow Cytometer, BD Biosciences).

Molecular docking study
Crystal structure of human estrogen receptor alpha (ERα) in complex with its selective antagonist 4hydroxytamoxifen (PDB entry: 3ERT; Resolution: 1.9 Å) was retrieved from Protein Data Bank [38].
Preparation of structures was done using the drug design platform LeadIT [39]. Co-crystalized ligand 4hydroxytamoxifen from chain A was used to de ne the binding site in ERα with the radius of 6.50 Å.
Structure of AS-4 was drawn on ChemDraw Ultra (2013), and its energy was minimized by using MM2 force eld in Chem3D Ultra software (Cambridge, USA) [40]. Prepared AS-4 structure was used as protonated in aqueous solution and docked into prepared binding site of ERα using the FlexX docking module in LeadIT. All FlexX solutions yielded were scored by using a consensus scoring function (CScore) and ranked accordingly. Top best pose with the highest score was selected for further investigation of the interactions [18]. 3D enzyme-hybrid and monomer-hybrid interactions were visualized using Discovery Studio Visualizer [41]. A.Various hybrid molecules containing isatin (1-4, 6, 7), chloroquinoline (5-8) and morpholine (3,(8)(9) nucleus having anti-breast cancer activity; B. Designed morpholinated isatin-quinoline hybrids (10).