Synthesis, Molecular Docking and Pharmacological Investigation of Heterocyclic Amine Derivatives as Potential Anticancer Agents

Cancer is life threatening disease that causes great damage to health worldwide. Studies shown that hypoxia is the major contributor to tumor and cancer development due to overexpression of carbonic anhydrase. To encounter such cells abnormalities, demanding new drugs or novel analogs of currently in use. Therefore, the search for new pharmacoactive moieties with considerable effective activity against such tumors and cancers is needed. The implication of heterocyclic amine and acetamide derivatives well known as chemotherapeutic agents. Heterocyclic amine morpholine was taken as principal products and its new derivatives were synthesized after being designed computationally via molecular docking. A series of some of its new synthetic analogs i.e heterocyclic amine derivatives 1(a-o) were successfully synthesized and screened for their anticancer and carbonic anhydrase inhibitory potential. Most of the compounds showed good results possessing reasonable carbonic anhydrase inhibitory activity particularly compounds 1c, 1d, 1h and 1i showed very reasonable carbonic anhydrase inhibitory activity whereas compound 1h showed maximum inhibition comparable to acetazolamide. Similarly, four of the synthesized compounds showed good anticancer activity particularly compound 1b, 1c, 1h, and 1i showed reasonable, whereas compound 1h have better IC 50 value comparable to cisplatin when evaluated via in vitro MTT assay.


Introduction
Cancer has been an endless combat worldwide with a lot of advancement in remedies and defensive therapies. The disease is illustrated by cells frequently growing with the failure to be stopped, creating tumors with the potential to be metastatic 1 . Latest studies in cancer therapy have shown that hypoxia is the major contributor to tumor and cancer development which is due to overexpression of carbonic anhydrase [2][3][4][5] . Nevertheless, the instantaneous intensifying diversi cation of life threating diseases like tumors and related cancers demanding new drugs or novel analogs of the drugs which are already in use. Therefore, the present time requires the search for new pharmacoactive moieties with considerable effective activity against such tumors and cancers [6][7][8] . Carbonic anhydrase has 16 distinct isozymes that include membrane-bound isozymes (CA IV, CA IX, CA XIII), cytosolic isozymes (CA I, CA II, CA III, CA VII, and CA XIII), and mitochondrial isozymes (CA VA and CA VB); salivary isozymes (CA VI) are secreted isozymes. 9 . CA overexpression is a feature of certain physiological condition [10][11][12][13] , Due to their high therapeutic potential, CAs have emerged as promising pharmacological candidates. Inhibitors of the CA enzyme are commonly used for diseases like mountain sickness, acidic ulcers, glaucoma, and epilepsy 14 .
Carbonic anhydrase is another signi cant therapeutic target in the treatment of obesity, and as antiobesity medicines 15,16 . Transmembrane enzymes have shown to be overexpressed in hypoxic tumors notwithstanding their poor distribution. Because of this, new compounds that can effectively inhibit tumor development and cancer proliferation are urgently needed 17,18 . Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible and Epacadostat (EPA) is inhibitor of indoleamine 2,3dioxygenase 1 19,20 .
Numerous unique strategies have been used in this respect, including the identi cation of novel targets, structural changes to existing compounds, the combination of two pharmacophores in a single molecule, and the use of structural variations such as spacers or linkers 21 . The heterocyclic amine and its acetamide derivatives have long been recognised for their potency as chemotherapeutic agents, as evidenced by their numerous patents 22,23 . Several activities are related with heterocyclic amine and its acetamide derivatives [24][25][26][27] . Keeping in view the importance of heterocyclic amines, a series of some of its new synthetic analogs were synthesized and screened for anticancer and carbonic anhydrase activity.
The heterocyclic amine morpholine was taken as principal products and its new derivatives were synthesized after being designed computationally via molecular docking.

Materials
A digital Gallen-hamp (MPD-BM-3.5) equipment was used to record melting points (M.P) of synthesized compounds. Thin layer chromatography(TLC) was used to monitor the progress of the reactions. Proton NMR, 13 C-NMRspectra were done on a Bruker AM300 in DMSO at 300 and 75 MHz respectively. FT-IR spectra were measured by using NICOLET IS10 spectrophotometer (ν max in cm -1 ).

Docking studies Ligand selection and optimization
The synthesized chemical structures (1a) to (1o) were used as ligand molecules and their structures were drawn in Discovery Studio Visualizer 2017, saved in PDB format which were further adapted to pdbqt format using Autodock Tools 1.5.6. The AutoDock Tools gave gasteiger charges to all atoms, added polar hydrogens and removed non-polar hydrogens from all the ligands.

Target protein optimization
For protein optimization the combined crystallized ligands and molecules of water and were removed from protein structures. The Zinc ion present in 6VKG was not removed as it can result information of ionic interactions between Ligand and target protein.

Analysis of target active binding sites
The binding site was de ned and analyzed on the bases of co-crystallized ligands present in protein structures and search space was con ned to get poses of ligands as much similar in conformation as possible to co-crystallized ligands. The docked poses along with score were obtained by using Auto Dock Vina. The poses with best score were rescored by help of x-score. The co-crystallized ligands were also docked as reference to compare and verify results.

Pharmacological studies
In Vitro CA Inhibition Assay Results of CA assay were presented in as the mean triplicate experiments (± SEM) and expressed as % inhibitions: ( Table 1). Compound 1h has exhibited maximum percent enzyme inhibition (88.3%) in comparison to control acetazolamide. The increased inhibitory activity of compound 1h might be due to hydrophobic groups that leads to better binding interaction with inner hydrophobic pockets of CA 29 .

MTT assay
The percentage of cell growth inhibition in triplicate experiments was expressed as [1-(A/B)] *100, and the IC50 values were calculated by using Graph-pad prism6 as presented in Table 2. Most of the synthesized compounds have shown better cell growth inhibition in MTT assay due to acetamide moiety present in all compounds 30 . Table 2 The effects of compounds (1a-1o) on the viability of Resistant Ovarian cancer cell lines A2780 cisR (n = 3).  Docking study The results of docking show that compounds have reasonably good binding with tubulin, carbonic anhydrase IX and very high a nity for telomerase reverse transcriptase enzyme. The compounds having bulkier heterocycle ring structure were more active as compared to compounds with simple phenyl and benzyl functional group 32 . Compounds 1h, 1i and 1l have the very promising binding a nity in case of binding with Tubulin (1JFF), Auto Dock vina has given highest score to 1h similarly in X-Score, 1h was scored to have highest binding a nity where as 1i and 1l also scored to have reasonable a nity. Binding a nity showed that the tubulin can be a good target for these compounds. The compounds were docked in taxol binding site which has a bulkier structure therefore the synthesized compounds with bulkier structure showed good binding a nity and most these compounds can be good candidates for tubulin inhibition, as whole or fragments 33 .
The compounds under study share a lot of characteristics with BIBR1532, a reported highly potent telomerase inhibitor. BIBR1532 have amide functional group along with naphthalene ring system 34 . The synthesized compounds have morpholine ring system which is more polar characteristics due to presence of oxygen and nitrogen atoms 35 . Similarly, most of the synthesized compounds have carbonyl functionality in the form of ester, amide and thioate esters, which having carbonyl functional group, can have same interactions in binding pocket as BIBR1532 36 . The compound 1h, and 1l showed good activities but compound 1i shown highest a nity for Telomerase Reverse Transcriptase enzyme although it is slightly lower than BIBR1532. It is due to the fact that the binding pocket is highly hydrophobic and compound 1i contain toluene with 4-isopropyl group, being more hydrophobic, is a better candidate for further development of telomerase inhibitors 37 . Using AutoDock vina the binding energy of the compound 1i was taken in the same docking pocket, resulted good binding energy value i.e. -9.8 Kcal/mol near to BIBR1532 i.e. -10.4 Kcal/mol as presented in Table 4. The results show that para-cymene functional group (4-isopropyltoluene) along with morphline ring can be a potent inhibitor of telomerase reverse trancriptase. The compound 1m has shown a high predicted binding a nity with carbonic Anyhdrase IX enzyme. The predicted value is even higher than that of epacadostat, an experimental inhibitor of same enzyme and was co-crystallized with protein structure used for docking 38 . The compound 1d, 1f, 1h, 1g, 1n, 1o and also showed reasonably good hydrophobic and hydrophilic interactions. Zinc is also playing role in binding of Ligand to protein (Fig. 1, Fig. 2, Fig. 3). The results are suggestive that compound 1m should be evaluated for its carbonic anhydrase IX inhibitory activity through in-vitro testing. The Docking results overall suggest that the compounds are pharmacologically active and have a very good potential for further testing as an inhibitor of Carbonic Anhydrase IX and they can also be considered as fragments or leads for further drug development.  Two-dimensional (2D) presentation of binding interactions of synthesized compounds with the amino acid residues of the binding site of Telomerase Reverse Transcriptase (PDB ID: 5CQG)