Design, Synthesis, and Anticancer of Some New Amide Derivatives from Coumarin-3-Carboxylic Acid Combined with Carbohydrate

----------------------


INTRODUCTION
Carbohydrate-based low molecular weight gelators (LMWGs) have received increasing attention over recent years due to their interesting physical-chemical properties and many applications (Du et al., 2014)(Zhou et al., 2017. The gelators typically self-assemble and form fibrous supramolecular architectures that can immobilize certain solvents and form reversible gels. The driving forces for the formation of supramolecular gel networks are noncovalent forces such as hydrogen bonding, hydrophobic interactions, p-p stacking, CH-p interactions, and van der Walls forces, etc. Low molecular weight organogelators (LMOGs) have shown numerous applications for the formation of new supramolecular materials (Okesola & Smith, 2016) (Amabilino et al., 2017). Coumarin and its derivatives display a wide variety of biological activities, such as antitumor, anticoagulant, antispasmodic, antidiabetic, antibiotic, anti-HIV-1, and antioxidant activities (Salar et al., 2018) (Emami & Dadashpour, 2015). Interestingly, coumarin-3-carboxylic acids (also known as 3-carboxycoumarins) have some specific applications in a wide range of possibilities over other coumarin derivatives (esters and amides) (Markad et al., 2020)..carboxamides, prevalent in both natural and synthetic molecules, find use as drugs, insecticides, and synthetic or biopolymeric materials. (Pitzer & Steiner, 2016)Their preparation from carboxylic acids and amines is a challenging, highpriority reaction, particularly regarding atom economy (Bryan et al., 2018).
The production of amides directly from carboxylic acids and amines is inhibited by the energy barrier for the dehydration of stable ammonium carboxylate (Ramachandran & Hamann, 2021). Cancer is one of the most serious and complicated health disorders all over the world (El-Sayed et al., 2022). Owing to the incidence of resistance, toxicity, and lack of selectivity of the currently existing anticancer drugs, targeted therapies have recently been approved for the treatment of definite cancers such as lung, breast, pancreatic and colorectal cancers as well as lymphoma and leukemia (Gerber, 2008).

MATERIALS AND METHODS
The melting points were recorded through a Reichert Thermovar apparatus and are uncorrected. The infrared spectra were recorded using a Perkin-Elmer (model 1720) FTIR spectrometer. Nuclear magnetic resonance investigations were performed on a Bruker AC-300 or DPX-300 spectrometer (400 MHz 1H) (100 MHz 13C). The values of ppm (chemical shifts) are reported relative to TMS as a standard reference. The J values (coupling constants) are provided in Hz. The progress of the reaction was checked and guided by TLC using aluminum silica gel 60 F245. IR, 1H NMR, 13C NMR and elemental analyses were measured at the Micro Analytical Center at the Faculty of Science, Basra University, Iraq. 1-Synthesis of ((2R,3S,4S,5R,6R)-3,4,5trihydroxy-6-methoxytetra hydro-2Hpyran-2-yl) methyl 4methylbenzenesulfonate (2A)(El-Abid et al., 2021): Adding 1.2 equiv. of TsCl in a solution of 1equiv.of methyl -Dglucopyranoside in dry pyridine at 0 °C. The mixture was then gradually warmed up to room temperature. The reactions were monitored by TLC, mass spectrometry and H 1 NMR, between 2 and 24 hours of stirring. It is evident that, despite the use of an excess of TsCl. the conversion remains limited and unreacted glucose was still present in a significant amount. Penta-O-acetyl-β-D-glucopyranose (b2) (62.45g, 0.16 mol) and 2-bromoethanol (13.9 mL, 0.19 mol) were dissolved in dry CH2Cl2 (250 mL) in the dark and cooled to 0°C. BF3·Et2O (100 mL, 0.81 mol) was then slowly added. The mixture was then stirred at 0°C for 3 h and for 20 h at room temperature. After completion of the reaction, CH2Cl2 (50 mL) was added to the mixture, which was then poured into cold water (250 mL) with vigorous stirring. The organic layer was separated and washed repeatedly with water and saturated sodium bicarbonate. The organic phase was dried over anhydrous Na2SO4 and concentrated on a rotary evaporator. The resulting residue was purified by flash chromatography (stationary phase: silica gel; mobile phase: 2% methanol in CH2Cl2). The product was a white crystalline solid, with a yield of 37.01 g (50%). The preparation of O-acetyl methyl -D-glucopyranoside to a cold mixture of 2A&2B (1.0 g) and pyridine (50 mL) was added acetic anhydride (15 mL). The mixture was stirred overnight and then transferred to a separation funnel with EtOAc (50 mL). The solution was washed with 2 N HCl, saturated NaHCO3 and brine, dried over Na2SO4 and concentrated to afford 2.263 g of O-acetyl methyl--D-glucopyranoside, which was further purified by crystallization in EtOAc/hexane, in almost quantitative yield.

4-General Procedure For The Synthesis of Azide Sugars 4A & 4B (Hao et al., 2020):
(0.01mol) from compound (3A&3B) was dissolved in (15 ml) from DMF and add of 0.01 mol from sodium azide reflex the reaction at 120 C O , the reaction course was followed by TLC ethyl acetate: hexane as eluent. The crude material was purified by column chromatography ethyl acetate: hexane and concentrated under a rotary evaporator to give the desired compound. Chemical Formula(4A): C13H19N3O8; 80% Yield, The IR spectrum showed the absence of azide absorption band starting in ν (2101cm −1 ). Chemical Formula(4B): C16H23N3O10; 85% Yield, The IR spectrum showed the absence of an azide absorption band starting in ν (2101cm −1 ). 5-General procedure for the synthesis of amino sugars 5A and 5B.(de Oliveira et al.,

2005):
A solution of azido sugar 4A & 4B (1 mmol) in methanol (5 mL) was stirred under H2 atmosphere (1 atm) for 16 h in the presence of 10% Pd-C. (10% w/w of azido sugar). The catalyst was then removed by filtration and the solvent was evaporated under reduced pressure. The obtained residue was purified by chromatography on Silica Florisil using 4% MeOH in CH2Cl2 as the eluent affording the corresponding amino sugar 5A and 5B, respectively. Chemical Formula (5A): C13H21NO8, the IR spectrum showed the disappearance of azide and the appearance absorption band of amine starting in ν (3339 cm −1 ). Chemical Formula (5B): C16H25NO10, the IR spectrum showed the disappearance of azide and the appearance absorption band of amine starting in ν (3386 cm −1 ). 6-General procedure for the synthesis of amide sugars 6A and 6B. (Fattahi et al., 2018): To a magnetically stirred solution of 3-carboxylic acid coumarin (1 mmol) in chloroform was added 1 mmol of N, N'diisopropylcarbodiimide and the reaction mixture was stirred at room temperature for 1 h. After this period, the amine (1mmol) was added and the reaction mixture was stirred at room temperature for the indicated time until the starting materials were totally consumed as checked by TLC. Then, the solvent was separated by filtration.  04, 153.76, 153.18, 125.68, 118.00, 77.38, 77.06, 76.74, 63.01, 61.91, 40.30, 39.69, 33.79, 32.25, 30.71, 29.69, 28.91, 26.23, 26.00, 25.56, 25.26, 25.19, 24.89, 24. Compounds (0.3 mmol), were dissolved in methanol (5 mL), and sodium ethoxide (2.04 mg,0.03 mmol) was added thereto. Then the reaction mixture was stirred at room temperature until the disappearance of starting material (6 hours) and concentrated. Then it was extracted with 40 mL of diethyl ether three times and then the organic phase was treated with 100 mL of water (three times). Subsequently, the diethyl ether phase was dried by adding anhydrous sodium sulfate (Na2SO4) and then filtered. The diethyl ether was evaporated and then the residue was dried Chemical Formula)7A): C17H19NO8, yield 75%, light pink, m.p =180C o , The IR spectrum showed ν 3557, 3322 cm −1 , 1576 cm −1 carbonyl amide. Chemical Formula)7B): C18H21NO9, yield 80%, pink, m.p =188C o , The IR spectrum showed ν 3409 cm −1 , 1613 cm −1 carbonyl amide.

Molecular Docking Studies:
On an Intel Xenon W 3565 CPU running Ubuntu business version 14.04, the Glide module of the Schrodinger program was installed, and docking experiments with highly active and moderately active compounds were conducted. The RCSB protein data repository was the chosen source, and it provided the protein structure. The Chemdraw 18.0 Perkinelmer program was used to design the specific ligands.

Ligand Preparation:
ChemDraw was used to clean up the structure for bond alignment and draft the ligands for the docking experiment. Then, to reduce energy as ligands were introduced to the workstation, the OPLS3e (Optimized Potentials for Liquid Simulations) force field was implemented in Ligprep (Version 2019-1, Schrodinger). Since it enables the assignment of bond order, the addition of hydrogens to ligands, and the conversion of the 2D structure to 3D, this minimization is helpful for docking investigations. The best ligand conformations output file was used in docking studies.

Protein Preparation:
The Schrodinger protein preparation wizard (2019-1 version) was the primary protein preparation and minimization tool. The protein had a hydrogen atom added to it, and charges were also assigned. Het states were generated with Epik at a pH of 7.0 2.0. The protein is preprocessed and then refined and modified by evaluating the water molecules and other factors in the working space. The most important water molecules were unaltered, whereas all other molecules save those containing heteroatoms were removed. Finally, the OPLS3 force field was used to achieve maximum protein minimization. Using the ligand that was cocrystallized with the protein of interest (PDB-4GQR), a grid was constructed that represents the active site of the chosen target. Root mean square deviation (RMSD) was used to verify the protein after the last docking step with the co-crystal ligand in XP mode, and the value was found to be in the region of 0.46.

Receptor Grid Generation:
A receptor grid was created around the protein by choosing the inhibitory ligand (X-ray posture of the ligand in the protein; PDB:4GQR). With a partial atomic charge of 0.25, the Vander Waal radii of the receptor atoms were scaled to 1.00, and a box was created around the centroid of the ligand. Docking and Analysis: The above-described ligand and protein were used as input in a molecular docking experiment. XP Visualiser (Version 2019-1, Schrodinger) was used to assess the docking study's findings. The compounds' SMILES format was created with OSIRIS Datawarrior. The Glide module in Schrodinger was used for docking experiments of the planned and produced compounds. We used XP mode (extra precision) for all docking calculations. The protein's atomic weight was reduced by a factor of 0.8, and the atoms had a partial atomic charge of less than 0.15. The bestdocked confirmation was selected based on the Glide docking score. XP visualizer was used to delving deeper into the interactions between these docked conformations. Under local anesthetic and guided by ultrasound imaging, oncologists took true-cut biopsies of patients' liver cancers from the tu mor core and sent them on to the cell transpl ant team unit.
• Primary Cell Exposure To 7A & 7B: Cell suspension with 1×10 5 per ml was transferred to microplate 96-plate wells with each 120 μl well with cell suspension exposed to various concentrations of 7A & 7B (12.5, 25, 50, 100, 200 μg/ ml Using B-D-glucopyranose as a starting material, the synthesis technique involved treating the reaction for 3 hours with TSCl in dry pyridine to generate a first derivative 2A, also in high yield. In order to preserve the hydroxyl group, the product reacts with acetic anhydride in the same solvent, yielding 3A. treated with 2-bromoethanol in dry CH2Cl2 at 0 degrees Celsius in the dark. the second derivatives 1,2,3,4,6-penta-O-acetyl--D-glucopyranose as starting material. For example, BF3+Et2O3B. Both 3A and 3B are introduced into the Due to the potency of the nucleophile (N3), the SN2 reaction with sodium azide in DMF resulted in a high yield of azide carbohydrate from 4A&4B. Amino sugars 5A and 5B are synthesized via the reduction of azide carbohydrate derivatives in a hydrogen peroxide environment (1 atm) in methanol for 16 hours. This section outlines the general synthesis pathway for sugar derivatives. Scheme1.

Synthesis of Amide Derivatives:
Amide bond formation from coumarin 3-carboxylic acid with aminosugar in CCl4 present DCC to product 6A&6B respectively. Scheme 2.
Scheme 2: synthesis of amide sugar derivatives All the synthesized compounds were characterized by IR, 1 H, 13 C NMR and Mass spectroscopy. The appearance of the singlet at 8.5-8.6 ppm in 1 H NMR confirms the amide NH unit. Figure (1).
One peak appeared around 170 ppm in 13 C NMR confirming the presence of carbonyl carbon in all the titled derivatives & Three peaks at 14.47, 11.55 and 10.08 in 13 C NMR confirm the isopropyl unit (Fig. 2).
The FT-IR of the Removal protection group of compounds 7A & 7B shows, Peak appearance when 3557 ,3409 cm -1 due to OH groups.

Molecular Docking Study:
The Molecular Operating Environment (MOE-Dock) software version 2014.0901 was used to do docking simulations for targets 6A, 7A, 6B, and 7B in order to evaluate their promising in vitro inhibitory effects and comprehend their binding mechanisms within the EGFRWT, EGFRT790M, and HER-2 active sites. The Protein Data Bank files for the relevant EGFRWT, EGFRT790M, and HER-2 enzymes (PDB codes: 1M17, 3UG2, and 3RCD, respectively) were selected and downloaded.

Selection Of Protein Crystal Structures: Crystallographic Structures of Protein:
Mitogen-activated protein kinase (JNK1) are provided in the Protein Data Bank pdb code (A1 chain with) (4AWI). This section evaluates and chooses the 4AWI crystal structure for docking. The MOE structure organization technique corrected the faults in the protein by identifying the biggest and ligand pockets, inserting the tested compounds in these pockets for docking, ligand interaction, computing the total binding energy, and calculating the H-bond and Van der Waals (VDW) energies. The cytotoxicity potentials of 7A were evaluated by MTT assay against liver cancer primary tissue culture after 48 hr., which appears that 7A exhibited selective cytotoxicity against liver cancer cells isolated from Iraqi patients with inhibitory concentration (IC50) 49.23 μg/ml as shown in Figure (3) and Table (3).  Determine the Anticancer Activity of 7B Against Liver Carcinoma: The cytotoxicity potentials of 7B were evaluated by MTT assay against liver cancer primary tissue culture after 48 hr., which appears that 7B exhibited selective cytotoxicity against liver cancer cells isolated from Iraqi patients with inhibitory concentration (IC50) 86.49 μg/ml as showed in Figure (5) and Table (2).  The Determination of The Effect of 7A &7B on Apoptosis (DNA Fragmentation) by Using the Acridine-Orange / Ethidium Bromide (AO/EB) Technique: The fluorescence microscope image of the primary cell culture of human liver cancer cells stained with AO / EtBr and representing the apoptosis method.as show in Figure (

CONCLUSION
A series of new coumarin derivatives containing amide bonds with monosaccharide derivatives. The synthesized compound 7A & 7B showed excellent anticancer activity.