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Design of 1,4-Dihydropyridine Hybrid Benzamide Derivatives: Synthesis and Evaluation of Analgesic Activity and Their Molecular Docking Studies

Purpose This study aims to determine the analgesic activity of 1,4-dihydropyridine hybrid benzamide derivatives. These hybrid derivatives were synthesized, and their analgesic activity was studied. The synthesis method applied was a one-step reaction involving a green chemistry approach. Methods The compounds were prepared via the amination method with a yield ranging between 82% and 93%. The title compounds were confirmed by means of IR, 1H and 13C NMR, and mass spectral analyses. The pharmacological activity of all the synthesized compounds was evaluated, and the analgesic activities were monitored in vivo (by tail immersion methods), with a digital analgesiometer. The drug response and damage of tail ata concentration of 10 mg/kg were measured by tail-flicking latency. Results The activity of compound 2c (81.35% activity at 5mg/kg) can be correlated with its salicylamidemoiety (13.99% activity at 5mg/kg), and diclofenac showed comparable activity (79.21% activity at 5mg/kg reference drugs). Compound 2c has a higher potential to inhibit COX proteins compared to diclofenac. The drug-like nature of the molecule 2c corresponds to its ADME properties. Conclusion In this study, all the synthesized compounds were found to possess significant analgesic activities; particularly, the performance of compound 2c is excellent. Thus, the preparative method described is an apt route for developing novel therapeutic formulations.


Introduction
A number of calcium channel blockers have been investigated as model molecules in the drug discovery sector, but few novel analgesics have been introduced. 1,2 It is well recognized that T-type calcium channels play an important role in pain transmission among different calcium channel subfamilies. [3][4][5][6] A literature search on selective T-blockers identified some chemically diverse molecules, such as R(−) efonidipine 7 (Figure 1), as a dihydropyridine (DHPs) derivative belonging to this class. Many studies have shown that DHPs may also inhibit T-type calcium channels as well, although DHPs are the most commonly assumed L-type calcium channel inhibitors. 8,9 Subsequently, T-type calcium channels have emerged as suitable pharmacological targets for the therapeutic intervention into neurophysiological disorders such as pain and epilepsy and can also be targeted by DHPs. 10 1,4-DHPs via a modified Hantzsch reaction under MW irradiation are reported. The pharmacological study showed good relaxant effects on the smooth muscles of the isolated rabbit gastric fundus, due to the blockade of Ca 2+ channels, with a mechanism similar to that of NIF.

Materials and Methods
All chemicals of analytical grade were procured from Sigma-Aldrich. The IR spectra of all compounds were recorded (Thermo scientific Nicolet iS5 F) in the range of 4000-400 cm −1 . The 1 H NMR and 13 C NMR (Bruker DRX-300) spectra were taken on a spectrometer at 300MHz; GCMS model Clarus SQ8 (PerkinElmer) was used to record the mass spectra. Elemental analysis (C, H, N, and S) was performed using an elemental analyzer (VarioEL III).

Pharmacological Activity Analgesic Activity
Compounds 2a-g, and 3a-g were screened for their analgesic activity. In total, five albino mice weighing 25-30g were selected and included in each of the four groups. A 12-hour-long light-dark cycle was followed and the laboratory temperature was maintained at ambient conditions (each animal was allowed a 1 week adaption before being experimented). They were administered (5 or 10mg/kg) with the compound under study, dissolved in DMSO, volume being 0.1 mL and the control animals were orally administered volume of 0.

Tail Flick Method
The tail flick method, previously reported was employed in this study, 43,44 on mice to determine the analgesia, to find the radiant heat. During this test, a high intensity beam of light of was absorbed on the tail. As the final outcome, a tail flick retort was taken in seconds. An analgesiometer with 5.4 amp conversion (tail flick method 1Nco) is used; 2-4 hours were set as the pre-drug response time, the damage occurred on tail corresponds to a time of 10-15 sec.
It is possible to attribute the analgesic effects produced by the synthesized test molecules to a central mechanism involving receptor systems or to a peripheral mechanism correlated to the inhibition of prostaglandins, leukotrienes, and other endogenous substances. Analgesia pre-emptively prevents central sensitization with anti-nociceptive treatment. Diclofenac has well-known anti-nociceptive effects. Diclofenac was administered intraperitoneally to rats before and after acute and inflammatory-induced pain to investigate the analgesic effects. 45 In general, NSAIDs are considered to be analgesic by inhibiting cyclo-oxygenase via a peripheral site of action. NSAIDs have been demonstrated to have central anti-nociceptive mechanisms. 46,47

Statistical Analysis
The mean and standard deviations (SEM) are provided; ANOVA was used to analyze the statistics of all variables after which Tukey's post hoc comparison test was done.

Molecular Docking
The compound showed an interaction during molecular docking studies using Autodock vina 1.1.2, 48 and there exist a binding mode between compounds 2C and the COX proteins. Coordination geometry of the COX-2 protein complexed with SC-558 (PDB ID: 1CX2) inhibitor 49

ADME and Molecular Property Predictions
By using Lipinski's "rule of five", 51 the molecular property of compound 2c was calculated and the approach was theoretical and related to ADME and toxicity. The parameters of Liminski were predicted by Swiss ADME, a web-based tool. 52 The measure of evaluation corresponds to the topological polar surface area (tPSA). 53 The bioavailability is driven by gastrointestinal absorption. 54 Calculation of percentages: % ABS = 109 -(0.345 × tPSA).

Results and Discussion Chemistry
A previously reported 55 method was used to synthesize 1,4-dihydropyridine 1a-h, and an amination method was used to prepare 2a-g, and 3a-g through reactions of compounds 1a-h with benzamide (Scheme 1). Compounds 2a-2g and 3a-3g of 1 H NMR and 13 C NMR spectra were given in the (Supplementary Material Figures 1-28

Analgesic Activity
At a dose of 10 mg/kg, compound 2c was more effective (60 min) than diclofenac or salicylamide. The variations in analgesic activities are shown in Figure 2 and the results are summarized in Table 1. These are excellent target compounds with sensible active properties, based on the results of in vivo tests. As a result of electron-donating groups present on the aryl ring, the activity development took place and also the presence of 4-chlorophenyl moeityin compound 2c is enhanced the activity compared to un-substituted derivative. Compound 2c has 2-hydroxybenzamide group and it shows the best activity compared to diclophenac.

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The relationship between the structure of a compound and its analgesic activity is illustrated by several rules. In Figure 3, we see that in the para position there is a phenyl group which acts as a lipophilic moiety, whereas the hydrogen bonding related to −C=O− and −NH groups act as hydrophilic centres. Hence, the 1,4-dihydropyridine derivatives with salicylamide are important pharmacophoric agents showing biological activity.
Compound 2c, which possesses a 1,4-dihydropyridine structure with 4-Cl-Ph, showed significant analgesic activity and it was more effective due to electron withdrawing groups (4'-Cl). Compound 3c which is having a 1,4-dihydropyridine (DHP) ring of 4'-substitution of hydroxybenzamide was less active.
However, compounds 3a-g were less active than other compounds due to two nearby halogen groups interfering with the amide functionality. Literature indicates that thiophenecarboxamide has the greatest pharmacological effects. 56 Similarly, compounds 2b and 2c showed moderate-to-good analgesic activities compared to the un-substituted 2-hydroxybenzamide derivative. Compounds 2a-g show an activity in the range 12.01-94.07% at 60 min duration, and these results are comparable with that of the reference drug.
A selective COX-2 inhibitor is a better choice for stopping the pain-stimulating action of cycloxygenase-2 enzyme, but COX-2 inhibitors are associated with a wider range of side effects. 57,58 The use of selective COX-2 inhibitors or coxib drugs can antagonize COX-2 enzyme selectively, which is essential. We found that compound 2c had good docking scores (−9.7 kcal/mol) against COX-2 enzyme, while our standard drug Diclofenac had docking scores of -7.5 kcal/mol. Therefore, compound 2c may be a promising candidate for selectively inhibiting COX-2.
The selective COX-2 inhibitor is more effective than the conventional NSAIDs and has low GI and high cardiovascular side effects compared to the latter. 1,4-dihydropyridine hybrid benzamide is a COX-2 inhibitor with a high degree of selectivity towards its target. It provides an alternative to other selective and traditional NSAIDs in treating patients with arthritis and other painful conditions.

Docking Studies
Compound 2c and the control diclofenac were studied for their docking performances with COX enzyme proteins 1CX2 and 2OYE using Autodock Vina program. The 2c displayed more binding affinity (−9.7 kcal/mol) to the 1CX2 protein than shown in the case of 2OYE protein (−8.7 kcal/mol). However, it has only a lower binding affinity towards proteins 1CX2 and 2OYE than the control, diclofenac. The binding energy values of these two proteins are −7.5 and −8.2 kcal/ mol, respectively. Because of its amide moiety, compound 2c showed remarkable binding affinity towards the COX proteins, 1CX2 and 2OYE, compared to diclofenac. Protein-ligand bonds are stabilized by hydrogen bonding, and the favorable bond distance between the donor and acceptor is less than 3.5 Å. 59 A comparison of hydrogen bond distances existing in diclofenac and compound 2c showed a value less than 3.5 Å. A hydrogen bond is formed between compound 2c and the receptor 1CX2 (bond length: 2.18), Tyr122 (bond length: 2.84) and the hydrogen bonding interactions of Lys546 (bond length: 1.88) were found to be complex. Residues Arg44, Arg61, Lys79 and Pro542 have shown complex hydrophobic interactions. Diclofenac, used as a control forms only one hydrogen bond with 1CX2. A hydrogen bond was formed in the residue Gln461 (bond length: 2.24). It was found that the hydrophobic interactions among Cys36, Pro40, Cys41, Cys47, Leu152 and Pro153 were complex. Figure 4 illustrates the hydrophobic interactions formed between amino acids in 1CX2 protein and compound 2c. In Figure 5, diclofenac was shown as a control. A hydrogen bond is formed between compound 2c and receptor 2OYE. The residue His207 was involved in hydrogen bonding (2.16 bond length) and in hydrophobic interactions, residues His386, Val447, and Glu454 took part. The Glu454 is involved in electrostatic interactions and hydrogen bonds were formed only once between diclofenac and receptor 2OYE in the case of the control diclofenac. The hydrogen bonding interaction with residue Val349 (bond length: 2.79) was complex in nature. As a result, Tyr348, Leu352, Tyr385, Phe518, Ile523, Ala527 and Leu531 formed complex hydrophobic interactions and amino acid residues in 2OYE form hydrogen bonds and hydrophobic interactions. Figure 6 shows  Figure 7 shows diclofenac as a control. The results display that compound 2c has a remarkable inhibition capability than the control, diclofenac and other compounds in both COX proteins, these results are shown in Table 2.

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ADME and Molecular Property Prediction
Bioactive compounds are increasingly used as therapeutic agents due to their bioavailability in the case of oral doses. 60 According to the study, hydrogen binding capacity, reduction of molecular flexibility, intestinal absorption, and low polar surface area were the main predictors. 61 With one violation of Lipinski's "rule of five" for MW >500 daltons, compound 2c obeys Lipinski's "rule of five" without violation; and the control diclofenac obeys Lipinski's "rule of five" without violation ( Table 3). The number of rotatable bonds and the binding of receptors or channels define the conformational changes which can be measured by counting the number of rotatable bonds in the molecules. In both compounds, 2c and diclofenac, there are less than 10 rotatable bonds and there are no chirality centers, hence have low conformational flexibility. In addition to passive molecular transport across membranes, the topological polar surface area (tPSA) is correlated to the blood-brain barrier. 53 Compound 2c and diclofenac have tPSA value of 144.83 and 49.33 Å 2 , respectively. In contrast, compound 2c does not meet the criteria for gastro-intestinal absorption and is to be given later via oral administration, while diclofenac does. The latter was expected to penetrate the blood-brain barrier more readily (tPSA > 90 Å 2 ) than compound 2c. The side effects of the central nervous system are compacted or abstracted in the case of compound 2c, whereas diclofenac is free of such harmful effects. The absorption percentages of compound 2c and diclofenac were 59.04% and 82.98%, respectively, which indicatethe diclofenac has high bioavailability than 2c. The oral administrative route creates an acceptable bioavailability (>50%). Compound 2c has only poor water solubility (-logS value of −6.29), whereas that of diclofenac was moderate (-logS value of −4.65). When 2c was administered, liver dysfunction was not anticipated as a side effect, because it has been projected as non-inhibitors of CYP2D6 and diclofenac has the property of inhibiting the enzyme CYP2D6 as expected. The P-glycoprotein (P-gp) belongs to the family ofATP-binding cassette transporters. It is involved in intestinal absorption, drug metabolism, and brain diffusion, and its inhibition may alter the bioavailability and protection. 62 As a result of the extra accumulation of phospholipids in tissues, drug-induced phospholipidosis occurs. 63 According to our study, diclofenac and compound 2c are not P-gp substrates nor they induce phospholipidosis. Compound 2c has a respectable ADME and toxicity profile, low gastrointestinal absorption, and no blood-brain barrier penetration. As a result, compound 2c was recognized as having druglike properties as perLipinski's "rule of five", with only one exception, ie, the violation of MW > 500; the projected parameters are acceptable within the allowed range.