Heterocylcic Based Cucumin: Desin, Synthesis and Anticancer E cay Against Hela Cells

Rana Al-Kerm An-Najah National University Rola Al-kerm An-Najah National University Othman Hamed (  ohamed@najah.edu ) An-Najah National University https://orcid.org/0000-0003-1074-2887 Ashraf Sawafta An-Najah National University Mohammad Qneibi An-Najah National University Avni Berisha University of Prishtina: Universiteti i Prishtines Hasan Prishtina Omar Dagdage Sidi Mohamed Ben Abdellah University of Fes Graduate Normal School of Fes: Universite Sidi Mohamed Ben Abdellah de Fes Ecole Normale Superieure de Fes Ghaleb Adwan An-Najah National University


Introduction
Cancer is the second worldwide leading cause of death and one of the most common public health problems. World Health Organization (WHO) predicted a 70% increasing in cancer cases by the next few decades [1]. despite the advances in the standard available therapies including chemotherapy, radiotherapy, surgery, immunotherapy and hormone therapy, Chemotherapy continues to be the most effective therapeutic option and widely used treatment for different types of malignancies. However, the side-effects associated with the cancer chemotherapy such as hair loss, fatigue, vomiting, nausea, and even death in severe cases, limit the scope of chemotherapeutic drugs. Therefore, continuous research for more e cient and less toxic cancer drug that can selectively target cancer cells with minimal or no side effects on normal cells is going on [2]. Natural plants products have traditionally been a magni cent source of pharmaceutical agents for centuries. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; diferuloylmethane) ( Fig.1), is a phenolic, natural yellow to orange compound isolated from the plant Curcuma longa L.
Curcumin and its derivatives possess numerous interesting pharmacological properties and wide variety of biological activities including anti-inflammatory, antioxidant, anti-microbial, antiviral, anti-fungal, antimalarial, anti-angiogenesis, anti-carcinogenic, antitumor, as well as medicinal applications, such as; their use against Alzheimer's disease and in HIV therapies [4,5,[9][10][11][12][13]. Further, curcumin is non-toxic and safe even at high dosages. The non-toxicity and the natural origin of curcumin and its wide range biological activities ensure that curcumin and its derivatives are promising lead compounds in medicinal chemistry. Curcumin contains three important functional groups that mainly contribute to its biological activities, α,β-unsaturated β-diketo group; a seven carbon olefinic linker, and an ortho-methoxy phenolic hydroxy group. Structure activity-based researches have investigated that the α,β-unsaturated β-diketo group is an important functionality in anticancer activity, and the length of the olefinic linker is essential for the prevention of protein aggregation in Alzheimer's disease models. Moreover, the ortho-methoxy phenolic hydroxy group and methylenic hydrogen are responsible for the antioxidant activity. Curcumin is insoluble in both water and ether but it is soluble in ethanol, acetone, chloroform, methanol, dichloromethane, and dimethylsulfoxide. It exists in enolic and β-diketonic forms. therefore, it can undergo keto-enol tautomerism that are stabilized by intra-molecular hydrogen bonding between the enolic hydrogen atom and keto carbonyl oxygen. The enol is the predominant form of curcumin in solution due to the conjugation and intramolecular hydrogen bonding that contribute to its stability [6,7,9,[12][13][14].
To date, a series of novel curcumin derivatives that exhibit potential synergistic anticancer activity were synthesized. The results indicated that the prepared derivatives can inhibit breast cancer stem cells growth by hindering the mediated e ux mechanism of P-glycoprotein (P-gp). Glucoside of curcumin derivatives showed higher a nity to bind with P-glycoprotein than other derivatives of curcumin. Which have reduced the breast cancer stem cells growth [15]. The anticancer effect of the combination of 5uorouracil (5-FU) with curcumin against gastric cancer MKN45 and AGS cells was reported [16]. The results indicated that the combination of 5-FU and curcumin (2:1, mol/mol) enhanced cytotoxic effect compared to curcumin or 5-FU alone and showed synergistic effect. Moreover, the combination of 5-FU and curcumin also potentiated cytotoxicity in AGS cells compared to curcumin or 5-FU alone, but the effect was moderate. Further, synthesis of new heterocyclic derivatives based on curcumin was reported [17]. The in vitro inhibition capacity of the synthesized compounds was screened in two human cancer cell lines (breast cancer (MCF-7) and hepatocellular carcinoma (HEPG2) in addition to the normal cell line (human normal melanocyte, HFB4) in comparison to the known anticancer drugs: doxorubicin and 5urouracil. The anticancer activity results investigated that the synthesized products showed growth inhibition activity against HEPG2 cell line and MCF-7 cell line, but with varying intensities compared to the known doxorubicin and 5-urouracil anticancer drugs [17].
In the present work, we report synthesis of new curcumin-based benzodiazepines, diazepines and diazoles via an acid catalyzed condensation cyclization reaction of curcumin with different 1,2 diamines, pyridine, pyrazine, or pyrimidine. Moreover, synthesis of hydrogenated curcumin (H-curcumin) based amine using a two-step process was also reported. The in vitro anticancer activities of the synthesized heterocycles against HeLa cancer cells were screened using the MTT colorimetric assay.

Experimental
General experimental

Materials and Methods
All chemicals used in this study were purchased from Aldrich Chemical Company and used as they were received. All prepared compounds were characterized by 1 H NMR, 13 C NMR, MS/MS and IR spectroscopy.
Nuclear Magnetic Resonance spectra were recorded on Varian Gemini 2000, 300 MHz instruments. Infrared spectra were recorded on a Shimadzu 820 PC FT-IR spectrometer. The solvent used in the NMR was DMSO-d 6 , 1 H NMR experiments were reported in δ units, parts per million (ppm) down eld from tetramethyl silane (TMS). All 13 C NMR spectra were reported in ppm relative to DMSO-d 6 (39.52 ppm).
The reactions progress was monitored using TLC analysis performed on silica gel plates, pre-coated with Merck Kieselgel 60 F254, and visualization was done using a UV lamp. The mobile phase used was hexane: ethyl acetate (6: 4) mixture. All melting points were uncorrected and were determined in an open capillary tube. Each melting point runs were carried out at least in duplicate. The sample puri cations were carried out by either crystallization or ash chromatography with silica gel (100-200) mesh.

General Procedure
Preparation of curcumin-based heterocyclic compounds In a round-bottomed ask equipped with a condenser and a magnetic stirring bar, curcumin (1.357 mmoles, 0.5 g) was dissolved in (30.0 mL) ethanol. The desired amine, pyridine, pyrazine, or pyrimidine (1.357 mmoles) was added to the curcumin solution followed by adding a few drops of the catalyst concentrated sulfuric acid. The reaction mixture was then re uxed in a para n oil bath until completion of the reaction (12 to 24 hr), which was monitored by TLC. The excess solvent was removed under reduced pressure using a rotary evaporator; the residue was washed with sodium bicarbonate solution (5%), ltered, washed again with water and dried. Residual starting materials were washed out from the product by suspending the product in either diethyl ether or ethyl acetate. The products were further puri ed by ash chromatography or crystallization. In the preparation of compound 2 glacial acetic acid A sample of 2, 3-diamino-5-bromopyrizine (0.1276 g, 0.678 mmole) was added to a solution of curcumin (0.678 mmole, 0.25 g) in ethanol (30.0 mL), followed by adding catalytic amount of H 2 SO 4 (1 drop). Then the solution was re uxed until reaction completion. The product was recrystallized from hexane/EtOAc    (7) A 2-hydrazinopyrimidine hydrate (0.15 g, 1.357 mmol) was added to a solution of 1,7-Bis(4-hydroxy-3methoxyphenyl)hepta-1,6-diene-3,5-dione (curcumin) (1.357 mmole, 0.  Preparation of H-Curcumin Based Amine 1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (8) A low-pressure reaction bottle was charged with a solution of curcuminoids 10.0 g in absolute ethanol (100 ml) and in the presence of Pd/C (0.35 g) which was used as a heterogeneous catalyst. The bottle was attached to the low-pressure hydrogenation apparatus and evacuated, and then hydrogen was admitted to a pressure slightly above 5 atm. The contents of the ask were shaken until absorption of hydrogen stopped (about 4 hrs). The catalyst was removed by ltration and ethanol was removed under vacuum to afford 4.6 g (91.8%) of pale-yellow gummy material. The gummy material was puri ed by ash chromatography using ethyl acetate as eluent. The prepared compound 8, was analyzed by 1

Preparation of stock solutions
Solutions of curcumin-based heterocyclics were prepared at a concentration of 400 μg per 1 mL of dimethyl sulfoxide (DMSO) solvent and then incubated at 4 °C.

Cell lines
The human cervical cancer cell lines (HeLa cells) were obtained from the American Type Culture Collection [ATCC], Manassas, VA, USA. Were grown in RPMI medium supplemented with 10% fetal calf serum, 1% non-essential amino acid, 1% l-glutamine, 1% penicillin streptomycin and 1% amphotericin B. All cells were grown in a humidi ed atmosphere of 95% air, 5% CO 2 at 37ºC, the culture medium was changed at least twice a week as needed. All chemicals used were purchased from Biological Industries except for the amphotericin B and MTT reagent from SIGMA Aldrich.
For screening experiment, the cells were grown into 12-well plates in 950 μl of RPMI medium (Biological Industries, USA) containing 5% FBS, 2×10 4 cells/well plating density. After that 50μl of diverse concentrations (400, 200, 100, 50, 25, and 12.5 μg /ml) of curcumin-based heterocyclics was added in duplicates to the prepared 12-well plates and incubated for 24 h at 37ºC, 5% CO 2 , 95% air and 100% relative humidity. An inverted microscope (Labomed, USA) was used to observe the morphological changes of the cells.

Cytotoxicity Assay
Cells at 70-80% con uence were detached from culture ask by removing the culture medium then adding 0.05% trypsin-EDTA and a suspension of 100 µl (2.0 × 10 4 cells/well) of viable cells were seeded in a 96well plate and incubated for 24 h at 37ºC. After the removal of media cells were treated with 50 µl stock solution (400 μg /ml) serially diluted to reach concentrations of (200, 100, 50, 25, and 12.5 μg /ml) of curcumin-based heterocyclics, then incubated for 24h at 37ºC to perform the MTT assay.

Results And Discussion
In preceding studies, a non-toxic and a naturally occurring curcumin was used as a skeleton for synthesizing various heterocyclic compounds including pyrazole, diazepine, benzodiazepine, diazoles, and isoxazole [19,20]. The antibacterial activity and cytotoxicity of the prepared curcumin-based heterocycles were tested against gram-positive and gram-negative bacteria. Some showed excellent antibacterial activities against the tested strains. Moreover, some of the prepared diazepines showed a synergistic effect with ampicillin antibiotic. The results indicated that the curcumin-based heterocycles are promising for designing a potentially active anticancer agent. The compound selected for this purpose are curcumin with heterocyclic moiety, since as shown in Figure 1, they have the functionalities that make ideal for this purpose.
Based on these results and to extend the work for development of curcumin-based reagents with better bioactivity. The anticancer activities of the prepared heterocycles against HeLa cancer cells were evaluated. Unsophisticated and convenient synthetic methods were employed in this work in preparation of benzodiazepines, diazepines, diazoles, and amines via condensation cyclization of curcumin with various 1,2 diamino and hydrazine compounds. Schemes I, II and III show a summary of the prepared compounds structures and reaction conditions.
In the preparation of curcumin-based benzodiazepines, diazepines and diazole, ethanol was used as a solvent and H 2 SO 4 as a catalyst. However, in the condensation reaction for making diazepine 2, curcumin was re uxed with diaminomaleonitrile in a glacial acetic acid, which was performed as a catalyst and a solvent. The reaction progress was monitored by TLC. Curcumin-based benzodiazepines, diazepines 2, 3, 4, 5, and 6 required more re ux time than diazole 7. Two methods were employed in puri cation of the prepared heterocycles, either column chromatography or crystallization. The puri ed products were analyzed by FT-IR, 1 H NMR, and 13 C NMR; The detailed descriptions of all the procedures and of the nal analytical characterization are summarized in the experimental part. The yield was quantitative for derivatives, it ranged between 21 % and 99 %.
Hydrogenated curcumin (H-Curcumin-based amine, 9) was prepared in a two-step process, as shown in (scheme III). The rst step involved the hydrogenation of curcumin 1 using a hydrogenation apparatus charged, absolute ethanol was used as a solvent and Pd/C was the catalyst. The pale-yellow gummy product 8 was puri ed by ash chromatography using ethyl acetate as eluent and analyzed by FT-IR, 1 H NMR, and 13 C NMR as described in the experimental part.
The second step involves condensation reaction of amination of H-Curcumin 8 with Ammonium acetate as shown in (scheme III).The progress of reaction was monitored using TLC. The synthesized product was puri ed as previously described in the experimental part. The structure of compound 9 was identi ed by FT-IR, 1 H NMR, and 13 C NMR.

Anticancer activity
Curcumin and its derivatives have been known to have a wide variety of therapeutic effects, ranging from anti-in ammatory, chemo-preventive, anti-proliferative, and anti-metastatic. In this work a novel set curcumin-based heterocyclics were screened for their anti-tumor effect against HeLa cells. The in vitro cytotoxic and cytostatic effect of the prepared heterocyclics were evaluated using MTT test.
MTT assay was used to determine the cell viability by adding MTT solution to the plate and incubated for four hours, after that the isopropyl alcohol was added and incubated in dark for 15 minutes. The microplate reader (Labtech, UK) was used to measure the absorbance at 570 nm.

Cytotoxic effect of the curcumin-based heterocyclics on HeLa cells
The in vitro anticancer activities of a novel set of curcumin-based benzodiazepine, diazepines, diazoles, and H-curcumin based amine against HeLa cancer cell were evaluated. The results indicate that the prepared curcumin-based heterocyclics have varying cytotoxic effect on the HeLa cells at different concentrations. The viability of HeLa cells has reduced in the range of 4.48-14.57% within the studied concentration range as shown in (Table 1). In general, cells growth was decreased as the concentration of the prepared curcumin-based heterocyclics increased (Fig. 2).
Series 1: compound 2, series 2: compound 3, series 3: compound 4, series 4: compound 5 series 5: compound 6, series 6: compound 7, series 7: compound 9. Curcumin-based benzodiazepine and diazepines 2, 3, 4, 5, and 6 showed higher potency on HeLa cancer cells than the other tested compounds 7 and 9. The viability of HeLa cells that were treated with benzodiazepine and diazepines 2, 3, 4, 5, and 6 was reduced in the range of 4.48-6.73%. Curcumin-based diazepines 6 showed the highest cytotoxic effect on the HeLa cells at all concentrations. it reduced the viability of the tested HeLa cells in range of 4.48 % for the 400 μg /ml concentration to 4.95% for the 12.5 μg /ml concentration. Among the prepared curcumin-based benzodiazepine 3 and 5 are more effective against HeLa cancer cells than 4. As they reduced the viability of the tested HeLa cells in range of 4.78 % and 4.63% for the 400 μg /ml concentration to 5.95% and 5.9% for the 12.5 μg /ml concentration for 3 and 5, respectively. IC 50 of the prepared heterocyclestested on HeLa cancer cells were; compound 2 0.2490, compound 3 0.6811, compound 4 4.454, compound 5 2.120, compound 6 0.4572, and compound 7 1.245. (Fig. 3, Fig.4, Table 2) The lines represent the mean of three independent experiments carried out in triplicates.  5, Table 2). The lines represent the mean of three independent experiments carried out in triplicates.
Results of L6 cells showed that all the tested curcumin-based heterocyclics exhibited cytotoxic and cytostatic effects in a dose-dependent manner as presented in Figure 5, Figure 6, and Table 2 The reason for the low bioactivity of compound 9 could be due to the loss of the ole nic part of curcumin due to hydrogenation. As mentioned in the introduction this functionality is important functionality in anticancer activity. For comparison purpose we tried converting curcumin to curcumin amine as shown in  In vitro effects of curcumin-based heterocyclics on HeLa cells at different concentrations (μg/ml).

Figure 3
Cytotoxic effect of the prepared heterocycles on HeLa cancer cells.

Figure 4
Cytostatic effect of the prepared heterocycles on HeLa cancer cells.

Figure 5
Cytotoxic effect of the prepared heterocycles on L6 Cells (normal cells).

Figure 6
Cytostatic effect of the prepared heterocycles on L6 Cells (normal cells).

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