Exploration of imidazole and imidazopyridine dimers as anticancer agents: Design, synthesis, and structure–activity relationship study

Dimerization of proteins/receptors plays a critical role in various cellular processes, including cell proliferation and differentiation. Therefore, targeting such dimeric proteins/receptors by dimeric small molecules could be a potential therapeutic approach to treating various diseases, including inflammation‐associated diseases like cancer. A novel series of bis‐imidazoles (13–18) and bis‐imidazo[1,2‐a]pyridines (19–28) were designed and synthesized from Schiff base dimers (1–12) for their anticancer activities. All the synthesized compounds were screened for anticancer activities against three cancer cell lines, including cervical (HeLa), breast (MDA‐MB‐231), and renal cancer (ACHN). From structure–activity relationship studies, imidazo[1,2‐a]pyridines (19–28) showed remarkable cytotoxic activities, with compounds 19 and 24 showing the best inhibitory activities against all three cell lines. Especially, both 19 and 24 were very effective against the breast cancer cell line (19, GI50 = 0.43 µM; 24, GI50 = 0.3 µM), exceeding the activity of the control adriamycin (GI50 = 0.51 µM). The in vivo anticancer activity results of compounds 19 and 24 were comparable with those of the animals treated with the standard drug tamoxifen. Therefore, the dimeric imidazo[1,2‐a]pyridine scaffold could serve as a potential lead for the development of novel anticancer agents.


| INTRODUCTION
Dimerization of proteins/receptors plays critical roles in various cellular processes, including cell proliferation and differentiation.
Dimers exist as homodimers, physical association of two identical proteins or heterodimers, or interaction of two different proteins. In some cases, dimerization is thought to be important for receptor function. For example, the cellular growth factors vascular endothelial growth factor and platelet-derived growth factor are monomers in the cell membrane, but ligand binding induces dimerization of these receptors. [1,2] Structural studies have shown that certain cytokines, such as human growth hormone and erythropoietin are bivalent, and one ligand binds simultaneously to two receptors and creates a 1:2 ligand-receptor complex. [3,4] Upon ligand binding, the estrogen receptor (ER) isoforms ERα and ERβ form homo-or heterodimers. [5][6][7] Heat shock protein 90, a chaperone protein which is responsible for the maturation of numerous signaling proteins including the proteins required for tumor growth, exists as a homodimer. [8] The presence of such dimeric proteins/receptors as therapeutic targets suggests that an interaction/disruption of these dimers by dimeric small molecules could be a potential therapeutic approach for treating various diseases. There are many natural and synthetic dimers with potential biological activities such as anticancer, [9] anti-HIV, [10] antimalarial, [11] antibacterial, [12] as well as opioid antagonist. [13,14] For example, indol-3-carbinol (I3C, Figure 1), which is found in cruciferous vegetables, [15] is a naturally occurring modulator of carcinogenesis that is dependent on the conversion of its dimer, diindolylmethane (DIM) [16,17] in the stomach during digestion. The structure-activity relationship (SAR) of DIM has been extensively studied and many analogs have been developed with the immune stimulatory G protein-coupled receptor 84 (GPR84) agonistic activities, especially the fluoro substituted DIMs (e.g., PSB-15160 and PSB-16671, see Figure 1). [18,19] GPR84 has been proposed as a drug target for acute myeloid leukemia and might have potential for treating other cancers as well. Similarly, numerous synthetic bis-indoles possess cytotoxic activities compared with its monomer. [20] Aaptamine ( Figure 1), a naphthyridine alkaloid isolated from marine sponge Aaptos aaptos, was found to possess cancer cell growth inhibitory activity, [21] while its dimer bis-aaptamine (Figure 1), isolated from the marine sponge Aaptos suberitoides showed potent cytotoxicity against P388 cell lines. [22] Research has been focused on the replacement of chloroquine by its dimeric piperaquine for the treatment of malarial disease caused by Plasmodium falciparum. [23] F I G U R E 1 Representative examples of biologically important dimers and their monomers F I G U R E 2 Rational design of bisimidazoles and bis-imidazopyridines from Schiff base dimers Schiff bases have been shown to exhibit a broad range of biological activities [24,25] and serve as a building block for the synthesis of biologically important heterocycles, such as imidazoles [26] and imidazopyridines. [27] The clinically useful anticancer drug, dacarbazine [28] holds imidazole moiety. Similarly, imidazopyridine drugs such as zolpidem [29] (used in the treatment of insomnia) and alpidem (as an anxiolytic agent), [30] are available in the market. Although a large number of imidazole and imidazopyridine analogs have been studied for various biological activities, their dimeric analogs are not well explored either synthetically and biologically.
Given the biological importance of Schiff bases, an attempt has been made in the present work to synthesize their 1,4dimeric analogs (Step I, Figure 2). The utility of Van Leusen synthesis was explored to prepare imidazole dimers from the corresponding Schiff base dimers (Step II, Figure 2). In addition to that, the hydrogen bonding property in the imidazole scaffold encouraged us to design the bis-imidazopyridine analogs as a fusion between the phenyl ring and the imidazole ring (Step III, Figure 2). All the prepared dimers of Schiff base, imidazole, and imidazopyridine were biologically evaluated for their anticancer activities. For selected compounds, anticancer activities demonstrated an in vivo model.

| Chemistry
The first series of 1,4-isomers (1-6) of dimeric Schiff bases were prepared from terephthalaldehyde and a variety of amines that diverge the electronic effects of the aromatic ring. The substituents on the phenyl ring of the amine were electron-donating methyl groups (ortho-and para-substitution) as shown in compounds 2, 3 and para-methoxy group as indicated in 4, electron-withdrawing chloro group at para-position as demonstrated in compound 5 along with the parent compound 1 (unsubstituted one). To study the hydrophobic effect, the benzene ring was replaced by the cyclohexyl group as shown in compound 6. The next series of Schiff bases (7)(8)(9)(10)(11)(12) were derived from isophthalaldehyde to visualize the effect of biological activity on 1,3-positional isomers (Scheme 1). The substitution pattern was the same as that of the earlier series. Further, to explore the effect of 1,2-positional isomers of the corresponding Schiff bases, we intended to react various amines with phthalaldehyde. However, the reaction did not yield the desired product, probably due to the steric factor.
Compound 30 was synthesized from the reaction of aniline and benzaldehyde (equimolar ratio) using the reaction condition of Scheme 1. The chemical structures of the synthesized compounds are summarized in Table 1.
Next, imidazole analogs were synthesized from the corresponding dimeric Schiff base by Van Leusen imidazole synthesis [31] using ptoluenesulfonylmethyl isocyanide (TOSMIC) in the presence of K 2 CO 3 .
The structures of the synthesized compounds were characterized by 1 H, 13 Table 2.
At first, Schiff base dimers 1-12 were tested in all three cell lines (Table 2). Unfortunately, none of them showed any anticancer activity. Next, the corresponding imidazoles were screened.
Compound 13 without any substitution on phenyl rings showed Methyl substitution at the para-position on the phenyl ring (14) increased the cell growth inhibitory activity against all three cancer  among all three cell lines selected (see Figure 4).
ERs (ERα, ERβ) are prominent in breast cancer cells and therefore current breast cancer drugs like tamoxifene, rolaxifene, toremifene act as either agonist or antagonist by binding to this receptor. [32,33] As mentioned earlier, the ERs form homo-or heterodimers upon ligand binding. Based on these facts and to account for the mechanistic pathway of the imidazopyridines dimers, particularly toward breast cancer cell line, we believe that the two effective positional isomers 19 and 24 bind to the ERs and upon binding, the receptors could be dimerized. However, the mechanistic studies are under process and the results will be explored elsewhere.  The values outside the normal range of the hematological parameters are diagnostic for cancer. [34] In Table 3 recurrence. [35] In Table 4, CEA and CA are elevated in cancerinduced rats (Group II) whereas the antigens were reduced due to the effect of compound 19 (Group III) and compound 24 (Group IV), which was also comparable to the drug tamoxifen (Group V).
Liver marker enzymes act as significant indicators of malignant disorders. [36] The concentrations of liver marker enzymes in the serum, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and acid phosphatase (ACP) in the experimental groups are shown in Table 5. A marked upsurge in the levels of serum marker enzymes was found in cancerinduced rats (Group II) compared with the control group (Group I)
To study the SARs of each class, all the synthesized compounds were

| General procedure for the preparation of Schiff bases 1-12 and 30
To a solution of corresponding dialdehyde (

Collection of samples
In the experiment design, the animals were divided into five groups each consisting of six rats. After the experimental regimen (16 weeks), the animals were killed by cervical dislocation under mild chloroform anesthesia. Blood was collected in EDTA and centrifuge tubes by an incision made in the jugular veins and serum was separated by centrifugation at 2,000 rpm for 20 min and utilized for various biochemical assays. The liver and mammary tissue were excised immediately and thoroughly washed with ice-cold physiological saline and blotted dry. A part of the tissues such as liver and breast were removed and fixed in 10% formalin for histopathological study.

Hematological parameters
The hematological parameters such as hemoglobin, PCV, WBC, RBC, and platelets, were assayed. The whole blood sample was analyzed for the changes in the blood cells using SYSMEX Xs-800 i automatic hematology analyzer (Bio-Rad Laboratories, Inc.). [40] Principle

| Estimation of aspartate transaminase
The enzyme catalysis the following reaction  4.2.4 | Estimation of alanine transaminase [40] Principle The enzyme catalyses the following reaction -+α -→ + -L Alanine oxoglutarate ALT pyruvate L glutamate.
The oxaloacetate is measured by the reaction with 2,4-dinitrophenyl hydrazine giving a brown-colored hydrazone after the addition of NaOH. The color developed is read at 520 nm.  [41] Principle

| Estimation of alkaline phosphatase
The method used was that of King and Armstrong in which disodium phenyl phosphate is hydrolyzed with the liberation of phenol and inorganic phosphate. The liberated phenol is measured at 700 nm with Folin-Ciocalteu reagent. 4.2.6 | Estimation of ACP [42] Principle

Reagents
The method used was that of King  4.2.7 | ELISA of CEA and CA 15-3 [43] Quantitative estimation of tumor markers namely, CEA and CA 15-3 was carried out by solid phase ELISA using the UBI MAGIWELL (USA) enzyme immunoassay kit. The desired numbers of coated wells were secured in the holder. Ten microliters of standards, controls, or serum samples were then dispensed into appropriate wells. One well was saved for the blank, to which standards or enzyme conjugate should not be added. Fifty microliters of antibody solution was dispensed into each well expect the blank well. The wells were then incubated for 1 hr at room temperature. After incubation mixture the wells were rinsed with working washing buffer (50 ml) five times. Then, 100 µl of solution A (a phosphate buffer solution containing hydrogen peroxide) and 100 µl solution B (tetramethylbenzidine solution) was added into each well including the blank well. This was then incubated for 30 min at room temperature, after which, the enzyme reaction was stopped by the addition of 50 µl of stop reagent and the intensity of the color was measured with microreader at 450 nm.