Chemical Modification of Curcumin into Its Semi-Synthetic Analogs Bearing Pyrimidinone Moiety as Anticancer Agents

Natural products (NPs) continue to provide a structural template for the design of novel therapeutic agents and expedite the drug discovery process. The majority of FDA-approved pharmaceuticals used in medical practice can be traced back to natural sources, and NPs play a significant role in drug development. Curcumin, one of the most well-studied chemicals among the NPs, is currently the subject of intense investigation for its biological effects, including the prevention and treatment of cancer. Cancer has overtaken all other causes of death in the world today, with 19.3 million new cases and nearly 10 million deaths predicted in 2020. In the present investigation, we reported the synthesis of three semi-synthetic analogues of curcumin-bearing pyrimidinone moiety by the chemical modification of the diketone function of curcumin followed by their characterization by analytical techniques including infrared (IR), nuclear magnetic resonance (NMR), and mass spectral data. According to the National Cancer Institute (NCI US) methodology, the curcumin analogues (C1-C3) were tested for their anticancer efficacy against 59 cancer cell lines in a single dose assay. 1-(2,6-Dichlorophenyl)-4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-one (C2) demonstrated the most promising anticancer activity with mean percent growth inhibition (%GIs) of 68.22 in single dose assay at 10 µM. The compound exhibited >68 %GIs against 31 out of 59 cancer cell lines and was found to be highly active against all leukemia and breast cancer cell lines. The compound C2 showed a lethal effect on HT29 (colon cancer) with %GI of 130.44, while 99.44 %GI was observed against RPMI-8226 (Leukemia). The compound C2 displayed better anticancer activity against the panels of CNS, melanoma, ovarian, prostate, and breast cancer cell lines than curcumin and other anti-EGFR agents gefitinib and imatinib in single dose assay. The compound C2 also demonstrated potent anticancer activity in a 5-dose assay (0.001 to 100 µM) with GI50 values ranging from 1.31 to 4.68 µM; however, it was found to be non-selective with SR values ranging from 0.73 to 1.35. The GI50 values of compound C2 were found to be better than that of the curcumin against all nine panels of cancer cell lines. All of the curcumin analogues were subsequently investigated for molecular docking simulation against EGFR, one of the most attractive targets for antiproliferative action. In molecular docking studies, all the ligands were found to accommodate the active site of EGFR and the binding affinity of ligand C2 was found to be −5.086 kcal/mol. The ligand C2 exhibited three different types of interactions: H-bond (Thr790 and Thr854), π-cationic (Arg841), and aromatic H-bond (Asn842). The curcumin analogues reported in the current investigation may provide valuable therapeutic intervention for the prevention and treatment of cancer and accelerate anticancer drug discovery programs in the future.


Introduction
Currently, a major challenge facing the modern scientific community is the development of novel anticancer drugs with fewer side effects. Research on natural products (NPs) has been boosted recently because NPs are thought to be relatively safer than synthetic ones [1,2]. The use of NPs for medicinal purposes has gained popularity during the past few decades. Many of the FDA-approved medications used in clinical practices came from natural sources [3,4]. Many anticancer agents have been obtained by the chemical modification of NPs. A few examples of such modifications are given in Figure 1 [5][6][7][8][9][10][11][12][13][14]. Similarly, curcumin, one of the key chemical components found in turmeric (Curcuma longa Linn.), has been utilized to prepare a variety of semi-synthetic analogues [15][16][17][18][19][20][21][22]. Medicinal chemists have identified four main sites to bring about chemical modification in curcumin to form semi-synthetic congeners, including active methylene (-CH 2 -), aryl side chain, diketone group, and carbon-carbon double bonds (-CH=CH-) with improved bioactivity [23,24]. In the current study, we described the chemical modification of the diketone function of curcumin into their pyrimidine analogues as well as their antiproliferative activity. The chemical modification is outlined in Figure 2. The structural alteration was found to enhance biological activities by enhancing stability, reducing rotational freedom, and diminishing metal-chelation characteristics [25]. Our research team found that curcumin analogues have been demonstrated to have anticancer, antimalarial, and anti-HIV effects in the past [26][27][28]. Numerous biological activities, such as those that are antibacterial, anticancer, antioxidant, antimalarial, anti-inflammatory, anti-Alzheimer's, and anti-HIV, have been reported [15][16][17][18][19][20].
Cancer has overtaken all other causes of death in the world today, with 19.3 million new cases and nearly 10 million deaths predicted in 2020 [29]. Nowadays, systemic chemotherapy is used in conjunction with large-mass surgical excision of the tumor and radiation therapy. Chemotherapy often associated with numerous toxic effects and scientists from all over the world are working to find safer cancer treatments. As active ingredients derived from natural sources were presumed to be safe, our reliance on nature increased. The majority of anticancer drugs today in clinical practices are from natural sources [4,30]. The epidermal growth factor receptor (EGFR) is found on the surface of some normal cells and is involved in cell growth. The EGFR protein participates in cell signaling pathways that regulate cell survival and division. Sometimes, mutations in the EGFR gene cause EGFR proteins to be made in higher than normal amounts in a number of cancer cell lines, including those from breast, colon, non-small cell lung, renal, melanoma, ovarian, and prostate cancers. This causes cancer cells to divide more rapidly [31][32][33][34][35]. Curcumin analogues have also been identified as EGFR inhibitors, hence we selected EGFR as a potential target for molecular docking studies [2,22,26]. We examine the binding insight of curcumin analogues against the active site since it is a rational target for several anticancer treatments (such as Gefitinib, Erlotinib, Cetuximab, Panitumumab, and others) as well as the most widely researched receptor in the tyrosine kinase superfamily [36][37][38][39].   (7), acronycine (9), combretastatin A-4 (11), 3 -O-methylnordihydroguaiaretic acid (15), and daidzein (17)] by chemical modification.  Cancer has overtaken all other causes of death in the world today, with 19.3 million new cases and nearly 10 million deaths predicted in 2020 [29]. Nowadays, systemic chemotherapy is used in conjunction with large-mass surgical excision of the tumor and radiation therapy. Chemotherapy often associated with numerous toxic effects and scientists from all over the world are working to find safer cancer treatments. As active ingredients derived from natural sources were presumed to be safe, our reliance on nature increased. The majority of anticancer drugs today in clinical practices are from natural sources [4,30]. The epidermal growth factor receptor (EGFR) is found on the surface of some normal cells and is involved in cell growth. The EGFR protein participates in cell signaling pathways that regulate cell survival and division. Sometimes, mutations in the EGFR gene cause EGFR proteins to be made in higher than normal amounts in a number of cancer cell lines, including those from breast, colon, non-small cell lung, renal, melanoma, ovarian, and prostate cancers. This causes cancer cells to divide more rapidly [31][32][33][34][35]. Curcumin analogues have also been identified as EGFR inhibitors, hence we selected EGFR as a potential target for molecular docking studies [2,22,26]. We examine the binding insight of curcumin analogues against the active site since it is a rational target for several anticancer treatments (such as Gefitinib, Erlotinib, Cetuximab, Panitumumab, and others) as well as the most widely researched receptor in the tyrosine kinase superfamily [36][37][38][39].

Isolation of Curcumin
Curcumin was isolated by a conventional method as per the reported procedure and nearly 250 mg of curcumin was isolated with 80 g of the ground tumeric powder [2,40].

Isolation of Curcumin
Curcumin was isolated by a conventional method as per the reported procedure and nearly 250 mg of curcumin was isolated with 80 g of the ground tumeric powder [2,40].

Antiproliferative Activity
The anticancer activity of the curcumin analogues (C1-C3) was carried out against 60 NCI cancer cell lines derived from nine different panels (breast, colon, CNS, leukemia, melanoma, non-small cell lung, ovarian, renal, and prostate cancer cell lines) at a single dose (10 µM) and five dose assay as per the National Cancer Institute US [43][44][45][46]. The results of anticancer activity of compounds C1-C3 in single dose assay at 10 µM are given in the Table

Antiproliferative Activity
The anticancer activity of the curcumin analogues (C1-C3) was carried out against 60 NCI cancer cell lines derived from nine different panels (breast, colon, CNS, leukemia, melanoma, non-small cell lung, ovarian, renal, and prostate cancer cell lines) at a single dose (10 µM) and five dose assay as per the National Cancer Institute US [43][44][45][46]. The results of anticancer activity of compounds C1-C3 in single dose assay at 10 µM are given in the Table 1 Figure 3. The anticancer activity of curcumin analogues C1-C3 were compared with curcumin and other EGFR inhibitors, Gefitinib and Imatinib, and their comparative anticancer activity at 10 µM is given in Table 2. The mean %GIs of individual panels was calculated from the single dose assay data. The compound C2 displayed better anticancer than curcumin and standard drug imatinib and gefitinib activity against CNS, melanoma, ovarian, prostate, and breast cancer cell lines. The curcumin analogues displayed mean growth percent inhibition of 68.22 (%GI = >68), and were further selected for screening in a 5-dose assay [47].  The 5-dose assay was carried out as per the reported method [48][49][50]. The compound C2 showed strong antiproliferative activity in a 5-dose assay against 58 NCI cell lines, with GI50 values ranging from 1.31 to 4.68 µM, TGI values ranging from 1.05 to >100 µM, and LC50 values between 6.41 and >100 µM. The compound C2 displayed superior anticancer activity than curcumin in 5-dose assay (  (Table 1) [51]. The anticancer activity of compounds C2 against nine panels of 58 NCI cancer cell lines in terms of GP and Log10 molar concentration are shown in Figure 5.    The 5-dose assay was carried out as per the reported method [48][49][50]. The compound C2 showed strong antiproliferative activity in a 5-dose assay against 58 NCI cell lines, with GI 50 values ranging from 1.31 to 4.68 µM, TGI values ranging from 1.05 to >100 µM, and LC 50 values between 6.41 and >100 µM. The compound C2 displayed superior anticancer activity than curcumin in 5-dose assay (Figure 4). The compound C2 displayed the most promising antiproliferative activity against HL-60(TB) (GI 50  as the value of SR was found to be less than three (Table 1) [51]. The anticancer activity of compounds C2 against nine panels of 58 NCI cancer cell lines in terms of GP and Log 10 molar concentration are shown in Figure 5.

Molecular Docking Studies
Curcumin and its analogues' anti-EGFR action were well-documented in the literature [2,22,26]. The molecular docking against EGFR (PDB ID: 2J5F) was carried out at the binding site of 34-JAB in the current work as per the reported protocol [52]. The molecular docking score and types of interaction of curcumin analogues are summarized in Table 3. Three types of interaction were observed for the ligands (C1-C3) including H-bond, πcationic, and halogen bonds with a binding affinity of −4.936 to −5.117 kcal/mol. The ligand C1 showed an aromatic H-bond with the residue Asp855 ( Figure S4). The ligand C3 showed two types of interactions including H-Bond (with residue Thr790, Thr854, Lys875 via water molecule); halogen bond (with the residue Lys875); aromatic H-bond (with the residue Asp855) (Figure 6). The ligand C2 was found to be the most promising compound and displayed the most significant anticancer activity. The ligand C2 showed three types of interactions including H-bond of methoxy function of phenyl ring with the residue Thr854 via water molecule, π-cationic interaction of the 4-hydroxy-3-methoxyphenyl with the residue Arg841, and halogen bond interaction of one of the o-chloro function with the water molecule. Furthermore, the ligand C2 displayed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723. The 3D interactions of ligand C2 against the active site of EGFR are shown in Figure 7. Thr854 via water molecule, π-cationic interaction of the 4-hydroxy-3-methoxyphenyl with the residue Arg841, and halogen bond interaction of one of the o-chloro function with the water molecule. Furthermore, the ligand C2 displayed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723. The 3D interactions of ligand C2 against the active site of EGFR are shown in Figure 7. Thr854 via water molecule, π-cationic interaction of the 4-hydroxy-3-methoxyphenyl with the residue Arg841, and halogen bond interaction of one of the o-chloro function with the water molecule. Furthermore, the ligand C2 displayed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723. The 3D interactions of ligand C2 against the active site of EGFR are shown in Figure 7.

Discussion
Three new curcumin analogues (C1-C3) were reported in the current work. Curcumin and substituted phenyl urea in glacial acetic acid were stirred at 80 °C for 8 h to obtain 1-aryl-4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-ones (C1-C3) in good yields (75-80%). All the title compounds were characterized by analytical techniques, followed by their anticancer evaluation and molecular docking studies. One of the curcumin analogues (C2) displayed the most promising anticancer activity with a mean GP of 31.78 (% GI = 68.22). Nearly 31 cancer cell lines were found to be highly sensitive against the compound C2 with %GIs of >68. Two of the curcumin analogues (C1 and C2) displayed lethal effects on HT29 colon cancer cell lines with %GIs of 105.09 and 130.44. The anticancer activity of compound C2 was found to be better than that of curcumin and anti-

Discussion
Three new curcumin analogues (C1-C3) were reported in the current work. Curcumin and substituted phenyl urea in glacial acetic acid were stirred at 80 • C for 8 h to obtain 1-aryl-4,6-bis((E)-4-hydroxy-3-methoxystyryl)pyrimidin-2(1H)-ones (C1-C3) in good yields (75-80%). All the title compounds were characterized by analytical techniques, followed by their anticancer evaluation and molecular docking studies. One of the curcumin analogues (C2) displayed the most promising anticancer activity with a mean GP of 31.78 (% GI = 68.22). Nearly 31 cancer cell lines were found to be highly sensitive against the compound C2 with %GIs of >68. Two of the curcumin analogues (C1 and C2) displayed lethal effects on HT29 colon cancer cell lines with %GIs of 105.09 and 130.44. The anticancer activity of compound C2 was found to be better than that of curcumin and anti-EGFR drugs (gefitinib and imatinib) against CNS, melanoma, ovarian, prostate, and breast cancer cell lines, it was also found as active against all the cell lines of leukemia and breast cancer cell line panels ( Table 2). The compound C2 was further studied in 5-dose assay in which GI 50 values were found to be ranging from 1.31 to 4.68 µM, however, it was found to be non-selective with SR values ranging from 0.73 to 1.35. The GI 50 values of compound C2 were found to be better than that of the curcumin against all nine panels of cancer cell lines. The anticancer activity of compound C2 (mean % GI = 68.22) was found to be promising more than the previously reported work [2]. Chemically altering the diketone function to produce pyrazole and primidone analogues was shown to be more promising than doing so to produce bigenelli type curcumin analogues [2,26,53,54]. Curcumin analogues with 3-chloro-4-fluoro (C3) substitution in the phenyl ring at 1-pyrimidine showed mean %GI of 14.33, while 4-chloro (C1) substitution showed mean %GI of 20.37 and 2,6-dichloro (C2) substitution showed the most promising anticancer activity with %GI of 68.22. Three types of interaction were observed for the ligands (C1-C3) including H-bond, π-cationic, and halogen bonds with a binding affinity of -4.936 to -5.117 kcal/mol. The ligands C1 and C2 bind to the EGFR active site efficiently, whereas the ligand C3 binds less efficiently. The most active compound (C2) showed H-bond interaction with residue Thr854 through the water molecule, π-cationic interaction with residue Arg841, and halogen bond interaction with the water molecule. It also showed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723.

Molecular Docking Studies
The molecular docking against EGFR was performed for the ligands, C1-C3. The EGFR (PDB: 2J5F) X-ray crystal structure with a resolution of 3.00 Å; R-value 0.194 (observed) was obtained from the protein data bank (https://www.rcsb.org/structure/2j5f) [55]. The ligands C1-C3 were saved as mol file and the docking was done as per the protocol reported [52].

Conclusions
Curcumin was successfully isolated from the ground turmeric powder and chemically modified to prepare three semi-synthetic analogues bearing pyrimidinone nucleus. All the compounds (C1-C3) were synthesized in good yields and characterized by analytical data of IR, NMR, and mass spectroscopy. The anticancer activity of curcumin analogues was evaluated in a single-dose experiment at 10 µM. The anticancer activity of compound C2 was found to be promising with %GI of 68.22 percent, and superior to curcumin, gefitinib, and imatinib against CNS, melanoma, ovarian, prostate, and breast cancer cell lines. The compound C2 displayed promising anticancer activity and has been further evaluated in 5-dose assay displayed anticancer activity with GI 50 values ranging from 1.31 to 4.68 µM; however, found to be non-selective with SR values ranging from 0.73 to 1.35. The compound C2 was found to be more active than curcumin against all nine panels of cancer cell lines. Since EGFR was found to be over-expressed in a number of cancer cell lines, including those from breast, colon, non-small cell lung, renal, melanoma, ovarian, and prostate cancers we selected EGFR as a potential target and the mechanism behind the anticancer activity of the title compounds. We further studied the binding insight of our compounds (C1-C3) against the active binding site EGFR where 6-acrylamido-4-anilinoquinazoline usually bind [52]. In molecular docking studies all the ligands were found to accommodate in the active site of EGFR and the binding affinity of ligand C2 was found to be −5.086 kcal/mol. The ligand C2 exhibited three different types of interactions: H-bond (Thr790 and Thr854), π-cationic (Arg841), and aromatic H-bond (Asn842). Furthermore, the ligand C2 displayed good interaction with residues Ala743, Val726, Thr790, Lys745, Leu799, Asp800, Gly721, Leu781, Cys775, Leu844, Cys797, Ser720, and Phe723. The curcumin analogues reported in the current investigation may provide valuable therapeutic intervention for the prevention and treatment of cancer and accelerate anticancer drug discovery programs in the future.

Data Availability Statement:
The authors confirm that the data supporting the study's findings are included in the article and its supplementary information.