Synthesis and In Vivo Hypolipidemic Effect of Some N -(Benzoylphenyl)-Carboxamide Derivatives in Triton WR-1339-Induced Hyperlipidemic Rats

A series of N -(benzoylphenyl)-carboxamide derivatives ( 2a , 2b , 3a , 3b , 4a , 4b , 5a , 5b , 6a and 6b ) was prepared with good yields by reacting the corresponding carbonyl chlorides with aminobenzophenones at room temperature. This was followed by evaluating the hypotriglyceridemic and hypocholesterolemic effects of 3b , 5a and 5b . Triton WR-1339 (300 mg/kg) was intraperitoneally administered to overnight-fasted rats to induce hyperlipidemia. Rats were divided into six groups: control, hyperlipidemic, hyperlipidemic plus compounds 3b , 5a and 5b and hyperlipidemic plus bezafibrate. Results showed that after 18 h of treatment at a dose of 15 mg/kg body weight of each of the test compounds, the elevated plasma levels of triglycerides (TG) and total cholesterol (TC) were significantly lowered by compounds 5b and 3b ( p < 0.001) and by 5a ( p < 0.0001), compared to the hyperlipidemic control group. Compounds 3b and 5a significantly increased levels of high-density lipoprotein cholesterol (HDL-C) by 58 and 71%, respectively. In addition, compounds 3b and 5a caused significant reduction ( p < 0.0001) of low-density lipoprotein cholesterol (LDL-C) levels compared to the control group. These results suggest a promising potential for compounds 3b , 5a and 5b as lipid-lowering agents, which may contribute to reducing the risk of atherosclerosis and cardiovascular disease.


INTRODUCTION
There is a great deal of experimental and clinical evidence suggesting that hyperlipidemia is currently one of the main risk factors for atherosclerosis and cardiovascular diseases (CVDs) (Saleem et al., 2019). Consequently, decreasing any elevated plasma lipid levels could be a rational approach to the treatment and prevention of CVDs (Austin, Hokanson, Edwards, 1998;Ginghina, Iliescu, 2011;Harb, Bustanji, Abdallah, 2018;Rohilla et al., 2012;Stapleton et al., 2010).
Hyperlipidemia is normally treated with different groups of drugs including fibrates, among which is the well-known commercially available drug bezafibrate. In this context, treatment with fibrates results in a significant decrease in the level of plasma triglycerides (TG) in addition to a modest decrease in low-density lipoprotein (LDL)-cholesterol and an increase in highdensity lipoprotein (HDL)-cholesterol (Bkhaitan, 2017;Staels et al., 1998;Stancu, Sima, 2001). Fibrates, Triton WR-1339 is a globally accepted model for inducing acute hyperlipidemia in animal models to evaluate potential hypolipidemic drugs (Hayashi et al., 1981;Otway, Robinson, 1967). Published research showed that a single intraperitoneal administration of Triton WR-1339 to adult rats produced hyperlipidemia, where levels of cholesterol, triglycerides and phospholipids increased over 20 h and decreased thereafter (Al-Najdawi et al., 2014;Friedman, Byers, 1957;Schurr, Schultz, Parkinson, 1972;Sheikha et al., 2018). Triton WR-1339 causes an elevated plasma lipid profile by inhibiting the uptake of circulating lipoproteins by extrahepatic tissues, resulting in increased blood lipoprotein levels (Da Rocha et al., 2009;Schotz et al., 1957).
Accordingly, in continuation of our work in exploring the effects of different heterocyclic rings on improving the antihyperlipidemic kinetics and efficacy of new N-(benzoylphenyl)-carboxamide including bezafibrate, exert their effect through the activation of peroxisome proliferator-activated receptors (PPARs). PPAR-alpha induces apoA-I and apoA-II and lowers hepatic apoC-III production, which reduces the amounts of circulating triglyceride-rich lipoproteins (Hamadneh et al., 2017;Rubins et al., 1999;Shattat, 2014;Staels et al., 1998). Based on the correlation between hyperlipidemia and heart disease, development of new lipid-lowering agents is considered extremely important to overcome the adverse effects of current medications (Razali et al., 2018). In this respect, attention has been given to research focusing on the synthesis of N-(benzoylphenyl)-carboxamide derivatives with substantial antihyperlipidemic activity. In this class of compounds, studies have demonstrated the importance of a very large lipophilic aromatic ring represented by the benzoylphenyl moiety linked through a carboxamide to various heterocyclic rings such as furan, benzofuran, pyridine and others (Abu Farha et al., 2016;Al-Qirim et al., 2012;Hikmat et al., 2017;Jasim et al., 2018;Shattat et al., 2010). Figure 1 illustrates the common structural features between most of the proposed compounds (2-5, a, b), our previous imidazole derivatives (compounds I, (Jasim et al., 2018), and bezafibrate. All these compounds have an aryl carboxamide linkage in their structure; however, compounds I and the proposed ones in this work vary in aromatic heterocyclic rings.
derivatives, we report herein on the synthesis of such derivatives containing 1,5-dimethyl-1Hpyrazole, 2,5-dimethyloxazole, imidazole, thiazole and 1-methylindole rings. We also evaluated the pharmacological properties of the synthesized compounds om Triton WR-1339-induced hyperlipidemic rats used as a model. In addition, we only analyzed 3 compounds, specifically 3b and 5a and 5b, as potential antihyperlipidemic agents. The selection of the three compounds to test was based on the study of the effect of the size of the heteroatom in the ring and the steric hindrance effect on the biological activity.
NMR spectra were obtained using Bruker-Avance III 500 MHz spectrometers with DMSO-d6 as solvent and TMS as the internal standard. Chemical shifts (δ) are expressed in ppm units. High-resolution mass spectra (HRMS) were acquired (in positive or negative ion mode) using electrospray ion trap (ESI) technique by collision-induced dissociation on a BrukerAPEX-4 (7 Tesla) instrument. Thin layer chromatography (TLC) was performed using Merck aluminum plates pre-coated with silica gel PF254 (20 cm x 20 cm x 0.25 mm), and the plate was examined under a UV lamp (ƛ = 254 nm). Melting points were measured with SMP 10 Stuart apparatus and are uncorrected.

Synthesis of the targeted compounds
General procedure for the synthesis of 4-imidazole carbonyl chloride (9) and N-methylindole-2-carbonyl chloride (11) Oxalyl chloride (20 ml) and 0.05 mL DMF were added to a solution of 4-imidazole carboxylic acid (10 mmol) or N-methylindole-2-carboxylic acid (10 mmol) at 0 °C. The mixture was stirred for 45 min and then refluxed for 90 min. After cooling, the excess oxalyl chloride was evaporated under reduced pressure, and the pale brownish residue was washed twice with dry toluene (15 ml) and then used for the next step without further purification.

General procedure for the synthesis of carboxamide derivatives (2-6, a-b)
To a cold stirred solution of a specified quantity of carbonyl chloride in 15 ml of chloroform, a suspended solution of aminobenzophenone and triethylamine (2 ml, 14 mmol), in 5 ml of chloroform was added, and the mixture was stirred for 48 h at room temperature. Next, organic liquids were evaporated under vacuum to dryness. Thereafter, 15 ml of dichloromethane were added, followed by extraction twice with a saturated solution of sodium bicarbonate (10 ml) and water (15 ml), sequentially. The organic layer was dried using anhydrous sodium sulfate and then evaporated under reduced pressure to obtain a crude material, which was purified by silica gel plate chromatography.

Animals and treatment
For the in vivo study, forty-eight 2-month-old male Wistar rats weighing 180-200 g, bred in the animal care center of the Faculty of Pharmacy, Al-Zaytoonah Private University, Amman, Jordan were used throughout this investigation. They were given free access to tap water ad libitum throughout the experimental duration (18 h). Rats were housed under standard conditions of 12 h light-dark cycle and under constant relative humidity (55 ± 15%) and temperature (22 ± 2 °C). All experiments were performed in accordance with the Guidelines for Animal Welfare Committee of Al-Zaytoonah University.

Pharmacological experimental design
Rats were fasted overnight and were randomly divided into six groups of eight animals each. The first group, serving as the normal control group (NC), received an intraperitoneal administration of normal saline; the second, hyperlipidemic control group (HC), received an intraperitoneal injection of Triton WR-1339 (300 mg/kg) dissolved in distilled water. Rats in the third, fourth and fifth groups were intraperitoneally injected with Triton, followed by an intragastric administration (15 mg/kg) of compounds 3a, 5a and 5b, respectively. In the last group (BF), rats were also intraperitoneally injected with Triton and intragastrically treated with bezafibrate (100 mg/kg) (Mori et al., 2004;Nakajima et al., 2009). After 18 h of Triton administration, animals were anesthetized, and blood was collected from the renal artery. The blood samples were immediately centrifuged (3000 rpm for 10 min) and the plasma was used for lipid analysis using an enzymatic method with an automatic analyzer (Model Erba XL-300, Mannheim, Germany).

Statistical analysis
All antihyperlipidemic activity data are presented as the mean ± standard error of the mean (SEM) (n = 8). The data were analyzed using Student's t-test, and p < 0.05 was considered statistically significant (SPSS version 2015, IBM SPSS Statistics).

Chemistry
New carboxamide derivatives of heterocyclic compounds (2-6 a, b) were prepared in good yield (Scheme 1). 4-Imidazole carbonyl chlorides (9) and 1-methylindole-2-carbonyl chloride (11) were synthesized by reaction of their carboxylic acids with oxalyl chloride under reflux, whereas other carbonyl chloride derivatives were obtained from commercial sources. A large amount of oxalyl chloride was used since it acts as a solvent and chlorinating agent in the presence of one drop of DMF as catalyst. The synthesis of products 2-6 a, b was conducted by coupling of the corresponding carbonyl compound with aminobenzophenones, which are weak nucleophiles due to the delocalization of a lone pair of electrons (due to the resonance) at the nitrogen atom. To overcome this problem, triethylamine (weak organic base) was added to substitute the chlorine atom in carbonyl chlorides with trimethylamine, which would carry a positive charge via the nitrogen atom and consequently enhance the electrophilicity of the carbon atom of the carbonyl group. In addition, it enhances the nucleophilicity of the aromatic amine by abstracting a proton to attack the carbonyl group to give the products and triethylammonium chloride. It is worth mentioning that the present experimental procedure has advantages over a previously published one in terms of yield and reaction conditions for preparing compounds 4a and 4b (Jasim et al., 2018). In addition, compound 2a was originally prepared by converting 1,5-dimethyl-1H-pyrazole-3-carboxylic acid to the corresponding anhydride using polymer-bound 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide, followed by reaction with 3-aminobenzophenone (Parlow, Mischke, Woodard 1997). Progress of the reaction was monitored by TLC, which is based on the disappearance of the heterocyclic acyl chloride spot (limiting reactant). All target products were well-characterized by means of HRMS and NMR ( 1 H, 13 C, DEPT) techniques.

Effect of compounds 3b, 5a, 5b and bezafibrate on rats' plasma lipid profile
The effect of compounds 3b, 5a, 5b and BF on plasma lipid levels (TC, LDL-C, HDL-C and TG) of Triton WR-1339-treated rats after 18 h are shown in Table 1. The results revealed that the elevated plasma TG levels produced by Triton WR-1339 administration were significantly (p < 0.0001) reduced by 77, 88, 68 and 23% (p < 0.001) in rats treated with compound 3b, 5a, BF and 5b, respectively, as compared to the HC group. Values are presentd as the mean ± SEM ( n= 8) in each group. NC, normal control group; HC, hyperlipidemic control group; TC, total cholesterol; LDL-C, low-density lipoproteincholesterol; HDL-C, high-density lipoprotein-cholesterol; TG, triglycerides; HC is compared to NC.*p < 0.001, **p < 0.0001.

Induction of hyperlipidemia with Triton WR-1339
Shown in Figure 2 are the results pertaining to the plasma levels of TC, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) of all groups treated for 18 h. Triton WR-1339 caused a significant elevation in plasma TC, TG and LDL-C (p < 0.0001) levels in the HC group in comparison with the NC group (n = 8). Our results showed that the increase in plasma TC concentration in HC was 122% as compared to NC, whereas the LDL-C level in HC was elevated by 73% as compared to NC. At the same time, the TG level in HC was increased by 791%, while a significant (p < 0.0001) decrease of 45% in the HDL-C level occurred after Triton WR-1339 injection.
After Triton administration, TC levels were significantly (p < 0.0001) decreased by compounds 3b and 5a by 54 and 56%, respectively, and reduced by 25% by 5b (p < 0.001). Compounds 3b and 5a significantly reduced the elevated LDL-C levels by 19 and 30% (p < 0.0001) compared to HC-treated rats (Table I). No significant differences in LDL-C levels were observed with compounds 5b and BF compared to HC-treated rats.
All tested compounds, except compound 5b, significantly increased HDL-C levels. In fact, compounds 3b and 5a and BF significantly elevated HDL-C levels by 58, 71 and 73%, respectively (p < 0.0001), as compared to HC. To be more specific, the order of the hypolipidemic activity was 5a > 3b > 5b.
In this Investigation, we determined the antihyperlipidemic activity of aminobenzophenone thiazole derivatives and aminobenzophenone oxazole derivative. Our findings demonstrated that the replacement of the oxygen atom in the nucleus ring with sulfur significantly affected the biological activity of the compounds. Previous works have shown the importance of the heteroaromatic ring for biological activity. We suggested that the heteroaromatic ring interacts with the proper target(s) through hydrogen bonding. Our results confirmed that the heterocyclic ring plays a critical role in activity. Our results also showed that compound 5a is more active than 5b even though both contain the aminothiazole ring, but they differ in the position of the benzophenone carboxamide linkage. Apparently, the para derivative, compound 5b, allows alignment extension of the molecule with the proper target in relation to the different orientation obtained from the meta isomer 5a. Therefore, the distance between the heteroatoms in the nucleus and the points of interaction in the proper target would be affected. In case of compound 5a, the meta derivative allows orientation of the nitrogen atom in the thiazole nucleus to accept an H-bond while the para derivative should not allow an exact orientation to fit with the target losing the feature of H-bond acceptance.
On the other hand, the activity of dimethyloxazole benzophenone carboxamide derivative (compound 3b) would not be affected by the influence of the position of the benzophenone carboxamide linkage, as both heteroatoms of the ring are capable of hydrogen bond interaction with the proper target(s). The effect of the dimethyl groups shows no significant variation in biological activity in comparison with substituted heteroaromatic rings in previous published works, which support our findings pertaining to the chemical nature of the heterocyclic ring. In addition, it is obvious that the presence of the two methyl groups in the oxazole ring did not provide the expected benefit to the compound, producing almost the same efficacy as compound 5a. Moreover, the results clearly showed that a small heterocyclic ring provides the same, if not better, pharmacological activity as bigger and more substituted heterocyclic derivatives. Additionally, our results confirmed that bioisosteric replacement is possible and potentially useful in the benzoylphenyl carboxamide class. As has been previously reported, benzoylphenyl carboxamides containing a substituted and unsubstituted imidazole ring reduce TG levels by 54-65% (Jasim et al., 2018), whereas benzoylphenyl carboxamides containing a furan ring reduce TG by almost by 70% (Hikmat et al., 2017). On the other hand, benzoylphenyl carboxamides containing an indole moiety reduced triglycerides by almost 70% and benzoylphenyl carboxamides containing 5-fluoro-1Hindol reduced triglycerides by almost 90%. (Al-Najdawi et al., 2014;Hamadneh et al., 2017). All these findings confirm that the nature of the heterocyclic ring attached to the benzoylphenyl carboxamide moiety is essential for antihyperlipidemic activity.
The observed increase in plasma TG in Tritontreated rats was mainly due to an increase in the secretion of very-low density lipoproteins (VLDL) by the liver, in addition to a reduction of VLDL and LDL catabolism. Furthermore, the significant decrease in plasma HDL-C levels in a Triton-treated animal results mostly from movement of the apo A-1 protein from the HDL surface (Friedman, Byers, 1957). Accordingly, while most of the lipids in VLDL are TG and while cholesterol represents a minor proportion of VLDL, it is not surprising that the anti-hypertriglyceridemic activity of compounds 3b, 5a and 5b was significantly higher than the antihypercholesterolemic activity. This observation suggests that our compounds are able to restore, at least partially, the catabolism of β-lipoproteins as hypothesized by many researchers with other antihyperlipidemic agents (Pérez et al., 2000(Pérez et al., , 1999 CONCLUSIONS In summary, new N-(benzoylphenyl)-carboxamide derivatives were successfully prepared and characterized using NMR and HRMS data. The lipid-lowering activities of compounds 3b, 5a and 5b were evaluated using Triton WR-1339-induced hyperlipidemic rats. Interestingly, compounds 3b, 5a and 5b caused improvement in the lipid profile such as reducing hypercholesterolemia and hypertriglyceridemia. At the same time, compounds 3b and 5a significantly elevated HDL levels in Triton-induced hyperlipidemic rats, suggesting that these compounds could be possible candidates in the management of patients with lipid abnormalities. These findings are consistent with our previous published data, which confirm that compounds possessing N-(benzoylphenyl)carboxamide nuclei linked to the heterocyclic core have antihyperlipidemic activity. The results of this study and previous ones are very encouraging and advance the need to determine the agents with the best pharmacokinetic profile to move ahead with their clinical application.