Abstract
Context: Dyslipidemia is a major risk factor for the development of cardiovascular diseases. Many dyslipidemic patients do not achieve their target lipid levels with the currently available medications, and most of them may experience many side effects.
Objective: The present work aimed toward identifying a new class of novel nicotinic acid-carboxamide derivatives as promising antihyperlipidemic compounds.
Materials and methods: Six novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives were synthesized using acid chloride pathways. All structures were confirmed using 1H-NMR, 13C-NMR, IR, and HRMS. The evaluation of biological activity was conducted using Triton WR-1339-induced hyperlipidemic rats model.
Results: This study revealed that some of the newly synthesized novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives mainly C4 and C6 possessed significant antihyperlipidemic activities on lipid components TG and TC (p value <0.05).
Discussion and conclusion: This research opens the door for new potential antihyperlipidemic compounds derived from nicotinic acid that need further optimization of their biological activities.
Introduction
High serum low-density lipoprotein cholesterol (LDL-C) and elevated total cholesterol (TC) levels are the most prevalent markers for susceptibility to atherosclerotic heart diseasesCitation1,Citation2. World Health Organization has reported that high blood cholesterol contributes to approximately 56% of coronary heart diseases and 18% of global cerebrovascular diseases worldwide, which consequently cause about 4.4 million deaths each yearCitation3. Although LDL-C receives the main attention for clinical management, rising evidence indicates that high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), and very low-density lipoprotein (VLDL) play also a vital role in atherogenesisCitation4.
Although many antihyperlipidemic agents are available on the market, still large proportions of dyslipidemic patients do not achieve their target levels and all of the available agents are associated with many side effects. This may pose a challenge and limits their long-term use. So innovation of an alternative therapeutic agents is a hot area to work on. That’s why several research programs have been established to evaluate the potential antihyperlipidemic effect of different novel compounds, for the purpose of identifying promising hits for the treatment of hyperlipidemiaCitation5–12.
Triton WR 1339 (a nonionic detergent) has been widely utilized to induce acute hyperlipidemia in several experimental animalsCitation6,7,11,13–17. A single intraperitonial (i.p) dose of Triton WR-1339 administered to adult rats has been reported to increase TC, LDL-C, TGs and to reduce HDL-C levels to a maximum value in about 20 hCitation18.
Nicotinic acid is a well-studied agent for lowering TG and LDL-C, also for increasing HDL-C, which is used in a very high dose, up to 2000 mg dailyCitation19–21. Unfortunately, although nicotinic acid can be highly efficacious and positively alter the lipoprotein profile, particularly in individuals with atherogenic dyslipidemia, its usefulness has been restricted by side effects, mainly flushing that consequently limit its long-term useCitation22.
The aim of this study is to identify a new class of nicotinic acid carboxamide derivatives acting as a potential lipid lowering agents starting from nicotinic acid (niacin). The choice of carboxamide derivatives was based on results obtained by the same research group on indol carboxamide derivatives, where they found also that these compounds possessed lipid-lowering effectsCitation11,Citation12,Citation23,Citation24. So in this study we decided to prepare a hybrid chemical scaffold of two effective hypolipidemic agents using a well-established nucleus (nicotinic acid nucleus) as our starting point to synthesize a novel nicotinic carboxamide derivative bearing the benzophenone unit of fenofibrate drug (. We are proposing that this new combination may produce a promising candidate for the treatment of hyperlipidemia with potentially higher potency.
Figure 1. Schematic representation of the rational of the study.
Methods
Materials and equipments
All chemicals, reagents, and solvents were purchased from Aldrich Chemical Ltd (Dorset, UK) and Acros Organics (Geel, Belgium) and were used without further purification. Melting points were determined in open capillaries on a Stuart scientific electrothermal melting point apparatus (Staffordshire, UK). 1H-NMR and 13C-NMR spectra were recorded on Bruker-500 (500 MHz, Billerica, MA) at the University of Jordan. Deuteriated dimethyl sulfoxide (DMSO-d6) was used as solvents in sample preparation. Infra-red (IR) spectra were recorded using Shimadzu 8400F FT-IR (Kyoto, Japan) spectrophotometer at the University of Jordan. The samples were prepared by mixing 198 mg KBr with 2 mg of the target compound and then analyzed as thin solid films (KBr discs). High resolution mass spectra (HRMS) were measured in negative or positive ion mode using electrospray ionization (ESI) technique by collision-induced dissociation on a Bruker APEX-IV (7 Tesla) (Billerica, MA) instrument at the University of Jordan. The samples were dissolved in chloroform.
Chemistry
General proceudre
A mixture of nicotinic acid (C1, 1 gm, Mwt 123.11, 8.1 mmol) and SOCl2 (1 mL, Mwt 118.97, 13.78 mmol) in 10 mL of dry benzene was placed in a round bottomed flask. The reaction mixture was refluxed for 48 h at 70 °C till the completion of the reaction. Solvents (dry benzene and SOCl2) were distilled out from the reaction mixture to afford nicotinoyl chloride (C2, 0.5 gm liquid, Mwt 141.56, 43.6%), as colorless liquid (Scheme 1).
Scheme 1. Preparation of N-(benzoylphenyl)pyridine-3-carboxamide derivatives (C4, C6, C8, C10, C12, and C14). (i) SOCl2, dry benzene, refluxed at 70 °C for 48 h, distillation to get rid from excess SOCl2 and dry benzene, (ii) refluxed at 70 °C for 18 h, followed by the addition of 1,4-dioxane and stirring for 24 h.
Next, a mixture of the corresponding amine (3.2 mmol) and nicotinoyl chloride (C2) (0.50 gm, Mwt 141.56, 3.5 mmol) was refluxed at 70 °C for 18 h in air condenser, then 15 mL of 1,4-dioxane was added. The reaction mixture was stirred for additional 24 h at room temperature. Next, 200 mL crushed ice was added to the reaction mixture, followed by suction filtration to afford the final product.
N-(3-benzoylphenyl)pyridine-3-carboxamide (C4)
A mixture of 3-amino-benzophenone (C3, 0.63 g, Mwt 197.24, 3.2 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce N-(3-benzoylphenyl)pyridine-3-carboxamide as pale yellow solid (C4, 0.50 gm, Mwt 302.3, 51.7%). Rf: 0.21 (CHCl3/MeOH, 96:4); Melting point: 154–156 °C. 1H-NMR (500 MHz, DMSO-d6): δ = 10.67 (s, 1H, NH amide), 9.12 (m, 1H, Ar-H), 8.78 (d, J = 3.4 Hz, 1H, Ar-H), 8.31 (d, J = 7.9 Hz, 1H, Ar-H), 8.21 (s, 1H, Ar-H), 8.12 (d, J = 8.1 Hz, 1H, Ar-H), 7.78 (d, J = 7.2, 2H, Ar-H), 7.71 (dd, J = 7.4, 7.4 Hz, 1H, Ar-H), 7.57–7.61 (m, 4H, Ar-H), 7.51 (d, J = 7.7, 1H, Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6): δ = 196.06 (ketone carbonyl), 164.80 (amide carbonyl), 152.75 (Ar-CH), 149.18 (Ar-CH), 139.50 (Ar-C Quaternary), 137.93 (Ar-C Quaternary), 137.47 (Ar-C Quaternary), 135.98 (Ar-CH), 133.21 (Ar-CH), 130.74 (Ar-C Quaternary), 130.10 (2 Ar-CH), 129.57 (Ar-CH), 129.08 (2 Ar-CH), 125.57 (Ar-CH), 124.65 (Ar-CH), 123.99 (Ar-CH), 121.75 (Ar-CH) ppm. IR (KBr disk): ν = 3302 (NH amide), 3117, 3063, 1959, 1813, 1681 (ketone carbonyl), 1658 (amide carbonyl), 1589, 1550, 1481, 1427, 709 cm−1. HRMS (ESI, negative mode): m/z [M-H+] 301.09560 (C19H13N2O2 requires 301.09715).
N-(4-benzoylphenyl)pyridine-3-carboxamide (C6)
A mixture of 4-amino-benzophenone (C5, 0.63 g, Mwt 197.24, 3.2 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce N-(4-benzoylphenyl)pyridine-3-carboxamide as pale yellow solid (C6, 0.87 gm, Mwt 302.3, 89.9%). Rf: 0.10 (CHCl3/MeOH, 96:4); Melting point: 158–160 °C. 1H-NMR (500 MHz, DMSO-d6): δ = 10.80 (s, 1H, NH amide), 9.14 (m, 1H, Ar-H), 8.79 (d, J = 3.8, 1H, Ar-H), 8.33 (d, J = 7.9, 1H, Ar-H), 7.99 (d, J = 8.6, 2H, Ar-H), 7.81 (d, J = 8.5, 2H, Ar-H), 7.74 (d, J = 7.2, 2H, Ar-H), 7.68 (dd, J = 7.3, 7.4 Hz, 1H, Ar-H), 7.56–7.61 (m, 3H, Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6): δ = 195.11 (ketone carbonyl), 165.06 (amide carbonyl), 152.85 (Ar-CH), 149.27 (Ar-CH), 143.51 (Ar-C Quaternary), 137.95 (Ar-C Quaternary), 136.10 (Ar-CH), 132.79 (Ar-CH), 132.49 (Ar-C Quaternary), 131.46 (Ar-C Quaternary), 130.77 (2 Ar-CH), 129.89 (2 Ar-CH), 128.98 (2 Ar-CH), 124.01 (Ar-CH), 119.97 (2 Ar-CH) ppm. IR (KBr disk): ν = 3309 (NH amide), 3101, 3039, 1982, 1928, 1905, 1828, 1681 (ketone carbonyl), 1643 (amide carbonyl), 1589, 1519, 1450, 1411, 865, 740, 702 cm−1. HRMS (ESI, negative mode): m/z [M-H+] 301.09594 (C19H13N2O2 requires 301.09715).
N-(2-benzoylphenyl)pyridine-3-carboxamide (C8)
A mixture of 2-amino-benzophenone (C7, 0.63 g, Mwt 197.24, 3.2 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce N-(2-benzoylphenyl)pyridine-3-carboxamide as pale yellow solid (C8, 0.30 gm, Mwt 302.3, 31.0%). Rf: 0.62 (CHCl3/MeOH, 96:4); Melting point: 133–136 °C. 1H-NMR (500 MHz, DMSO-d6): δ = 10.74 (s, 1H, NH amide), 8.76 (m, 1H, Ar-H), 8.70 (m, 1H, Ar-H), 7.98 (d, J = 7.7, 1H, Ar-H), 7.65–7.71 (m, 4H, Ar-H), 7.56 (dd, J = 6.8, 7.2 Hz, 1H, Ar-H), 7.52 (d, J = 7.4 Hz, 1H, Ar-H); 7.47 (d, J = 6.6 Hz, 3H, Ar-H), 7.38 (m, J = 7, 7.2 Hz, 1H, Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6): δ = 195.80 (ketone carbonyl), 164.45 (amide carbonyl), 152.17 (Ar-CH), 148.73 (Ar-CH), 137.70 (Ar-C Quaternary), 136.62 (Ar-C Quaternary), 135.50 (Ar-CH), 133.05 (Ar-CH), 132.50 (Ar-CH), 132.02 (Ar-C Quaternary), 130.74 (Ar-CH), 130.20 (Ar-C Quaternary), 129.95 (2 Ar-CH), 128.66 (2 Ar-CH), 125.55 (Ar-CH), 125.10 (Ar-CH), 123.90 (Ar-CH) ppm. IR (KBr disk): ν = 3186 (NH amide), 3117, 3063, 1982, 1913, 1890, 1828, 1674 (carbonyl), 1620 (amide carbonyl), 1589, 1535, 1450, 1411, 933, 763, 709 cm−1. HRMS (ESI, negative mode): m/z [M-H+] 301.09748 (C19H13N2O2 requires 301.09715).
2-(2-[(pyridin-3-ylcarbonyl)amino]benzoyl)benzoic acid (C10)
A mixture of 2-amino-benzophenone-2′-carboxylic acid (C9, 0.77 gm, Mwt 241.24, 3.2 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce 2-(2-[(pyridin-3-ylcarbonyl)amino]benzoyl)benzoic acid as pale yellow solid (C10, 0.115 g, Mwt 346.3, 10.4%). Rf: 0.51 (CHCl3/MeOH, 96:4); Melting point: 238–239 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 13.30 (broad s, 1H, COOH), 12.20 (s, 1H, NH amide), 9.15 (m, 1H, Ar-H), 8.84 (d, J = 4.4 Hz, 1H, Ar-H), 8.65 (d, J = 8.2 Hz, 1H, Ar-H), 8.32 (m, J = 6.4, 7.8 Hz, 1H, Ar-H), 7.99 (d, J = 7.5 Hz, 1H, Ar-H), 7.54–7.77 (m, 5H, Ar-H), 7.27 (d, J = 6.8 Hz, 1H, Ar-H), 7.17 (m, J = 7.3, 7.5 Hz, 1H, Ar-H) ppm. 13C-NMR (75 MHz, DMSO-d6): δ = 202.00 (ketone carbonyl), 166.87 (COOH), 163.62 (amide carbonyl), 152.79 (Ar-CH), 148.13 (Ar-CH), 141.17 (Ar-C Quaternary), 139.61 (Ar-C Quaternary), 134.90 (Ar-CH), 134.64 (Ar-CH), 133.13 (Ar-CH), 132.42 (Ar-CH), 130.03 (Ar-CH), 129.94 (Ar-C Quaternary), 129.86 (Ar-CH), 129.54 (Ar-C Quaternary), 127.40 (Ar-CH), 124.27 (Ar-C Quaternary), 124.09 (Ar-CH), 123.37 (Ar-CH), 120.52 (Ar-CH) ppm. IR (KBr disk): ν = 3325 (NH amide), 2507–3240 (OH), 3039, 1959, 1928, 1836, 1820, 1643 (ketone carbonyl), 1627 (acid carbonyl), 1566 (amide carbonyl), 1481, 1435, 1396, 1242 (C-O), 763, 709 cm−1. HRMS (ESI, positive mode): m/z [M + H+] 347.16389 (C20H15N2O4 requires 347.10263).
N-(2-benzoyl-5-methylphenyl)pyridine-3-carboxamide (C12)
A mixture of 2-amino-4-methylbenzophenone (C11, 1.56 gm, Mwt 211.26, 7.4 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce N-(2-benzoyl-5-methylphenyl)pyridine-3-carboxamide was obtained as pale yellow solid (C12, 0.819 gm, Mwt 316.6, 35.0%). Rf: 0.70 (CHCl3/MeOH, 96:4); Melting point: 123–125 °C; 1H-NMR (500 MHz, DMSO-d6): δ = 10.74 (s, 1H, NH amide), 8.77 (d, J = 1.7 Hz, 1H, Ar-H), 8.70 (d, J = 3.7 Hz, 1H, Ar-H), 7.98 (d, J = 7.9 Hz, 1H, Ar-H), 7.71 (d, J = 7.9 Hz, 1H, Ar-H), 7.65 (dd, J = 7.3, 7.5 Hz, 1H, Ar-H), 7.59 (d, J = 8.1 Hz, 2H, Ar-H); 7.46–7.50 (m, 2H, Ar-H), 7.36 (dd, J = 7.5, 7.4 Hz, 1H, Ar-H), 7.26 (d, J = 7.9 Hz, 2H, Ar-H), 2.32 (s, 3H, CH3) ppm. 13C-NMR (125 MHz, DMSO-d6): δ = 195.52 (ketone carbonyl), 164.41 (amide carbonyl), 152.68 (Ar-CH), 148.74 (Ar-CH), 143.48 (Ar-C Quaternary), 136.57 (Ar-C Quaternary), 135.53 (Ar-CH), 135.05 (Ar-C Quaternary), 132.32 (Ar-CH), 132.20 (Ar-C Quaternary), 130.65 (Ar-CH), 130.23 (Ar-C Quaternary), 130.18 (2 Ar-CH), 129.22 (2 Ar-CH), 125.49 (Ar-CH), 125.15 (Ar-CH), 123.90 (Ar-CH), 21.56 (CH3) ppm. IR (KBr disk): ν = 3294 (NH amide), 3039, 2924, 2862, 1990, 1921, 1874, 1844, 1681 (ketone carbonyl), 1620 (amide carbonyl), 1597, 1527, 1442, 933, 763, 709 cm−1. HRMS (ESI, positive mode): m/z [M + Na+] 339.11038 (C20H16N2NaO2 requires 339.11040).
N-(2-benzoyl-4-chlorophenyl) pyridine-3-carboxamide (C14)
A mixture of 2-amino-5-chlorobenzophenone (C13, 0.68 gm, Mwt 213.68, 3.2 mmol) and nicotinoyl chloride (C2, 0.50 gm, Mwt 141.56, 3.5 mmol) was used to produce N-(2-benzoyl-4-chlorophenyl) pyridine-3-carboxamide as pale yellow solid (C14, 0.674 gm, Mwt 336.8, 62.5%). Rf: 0.73 (CHCl3/MeOH, 96:4); Melting point: 143–145 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 10.71 (s, 1H, NH amide), 8.67 (m, 2H, Ar-H), 7.43–7.92 (m, 10 H, Ar-H) ppm. 13C-NMR (300 MHz, DMSO-d6): δ = 193.38 (ketone carbonyl), 164.06 (amide carbonyl), 152.30 (Ar-CH), 148.20 (Ar-CH), 136.44 (Ar-C Quaternary), 135.05 (Ar-CH), 134.62 (Ar-C Quaternary), 133.75 (Ar-C Quaternary), 132.87 (Ar-CH), 131.54 (Ar-CH), 129.43 (2 Ar-CH), 129.28 (Ar-CH), 128.26 (2 Ar-CH), 126.79 (Ar-CH), 125.41 (Ar-C Quaternary), 123.38 (Ar-CH), 122.85 (Ar-C Quaternary) ppm. IR (KBr disk): ν = 3232 (NH amide), 3055, 1950, 1913, 1790, 1735, 1681 (ketone carbonyl), 1627 (amide carbonyl), 1597, 1527, 1404, 949, 825, 709 cm−1. HRMS (ESI, negative mode): m/z [M-H+] 335.05595 (C19H13ClN2O2 requires 335.05818).
Experimental animals
Seventy-two healthy male Wistar albino rats weighing around 200 gm were purchased from the Applied Science University animal houses. Prior to any experiment, animals were acclimatized for one week at a temperature of 25 °C with a 12 h light–dark cycleCitation25. All animals received ad libitum water and a standard rodent diet except when fasting was needed during the course of the study (18 h after treatment application).The ethical approval for conducting animal studies was obtained from the ethical and graduates studies council at the faculty of pharmacy/the University of Jordan (Ref No. 80/2014/165).
Triton WR-1339 model of dyslipidemia
Twelve healthy Wistar albino rats were divided into two groups (six in each): Triton WR-1339 group and control group (CG). Doses ranging from 200 to 400 mg/kg Triton WR 1339 have been reported to be used intraperitonialy for hyperlipidemia inductionCitation5,6,10,11,14,16,17,26,27. In this study, Triton WR-1339 was given at a dose of 300 mg/kg dissolved in water as intraperitoneal (i.p.) injection to induce dyslipidemia in rats, while the CG received i.p. water injection.
Acute toxicity study and selection of the experimental dose
Due to similarity in structure between synthesized compounds, acute toxicity experiment was conducted only on one representative compound (C8).
The experiment was conducted by selecting five different doses of C8 (200, 400, 600, 800, and 1000 mg/kg), which was orally administered to each group of six BALB/c mice. After oral administration, clinical observations were performed at 1, 2, 4, and 8 h after dose administration (day 1). After 24 h, the number of survived mice was counted in each group and survival rate was calculated based on Reed–Muench method as (number of live mice/total number of mice tested) × 100%Citation28. Survived mice were also observed for activity, behavior, and indications of toxicity or ill-health including: tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma for at least 14 daysCitation29.
A dose-response curve was then established at four different doses (20, 30, 50, and 100 mg/kg) for the aim of selecting the safest possible dose with significant efficacy to be used in the subsequent experiments.
Experimental design
Sixty male Wistar albino rats were randomly divided into 10 groups of 6 animals each. The first group, serving as a negative CG was received an i.p. injection of water and was gavaged with 6% DMSO/corn oil; the second group, serving as hyperlipidemic group (HG) was received 300 mg/kg Triton WR-1339 in water as i.p. injection and was gavaged with 6% DMSO/corn oil. In the remaining six groups, rats were injected with 300 mg/kg Triton WR-1339 in water i.p., followed by an intragastric administration of 30 mg/kg of the target compounds dissolved in 6% DMSO/corn oil. The last two group, fenofibrate group (FF) was injected intraperitoneally with Triton WR-1339 and intragastrically was treated with 65 mg/kg fenofibrate dissolved in 6% DMSO/corn oil, and nicotinic acid (NA) group was injected intraperitoneally with Triton WR-1339 and intragastrically was treated with 30 mg/kg nicotinic acid dissolved in 6% DMSO/corn oil.
Blood collection and lipid profile analysis
Based on literature recommendations, blood samples (2 mL each) were collected after 18 h of drug administrationCitation18. Rats were anesthetized using inhaled diethyl ether, blood was collected from retro-orbital plexus in plain tubes and allowed to clot for 10 min at room temperature. The blood samples were then centrifuged at 4000 rpm for 10 min and stored at 4 °C until used.
Lipid components (TC and TG) were measured directly using commercially available enzymatic colorimetric assay kits by the automatic analyzer (Model Erba XL-300, Mannheim, Germany) at Alzaytoonah University of Jordan.
Statistical analysis
All data were entered and analyzed using SPSS© 19 (SPSS Inc., Chicago, IL). Results were presented as means ± standard error of the mean (SEM). To investigate any differences between groups, non-parametric Mann–Whitney U test was used. For all statistical analysis, a p value of less than 0.05 was considered statistically significant. All tests were two-tailed.
Results
Chemistry
The acid chloride pathway was utilized to prepare the target compounds with a relatively good yield (Scheme 1)Citation30. Nicotinic acid (C1) was treated with SOCl2 in dry benzene, and the mixture was refluxed at 70 °C for 48 h till the formation of the corresponding nicotinyl chloride (C2). Then distillation was carried out to get rid from excess SOCl2 and benzene. Next, the acid chloride (C2) was mixed with aminobenzophenone amines and refluxed over 18 h at 70 °C, followed by the addition of 1,4-dioxane as a solvent and the reaction mixture was stirred for additional 24 h at room temperature. Finally, crushed ice water was added to precipitate the product, which was then filtered to afford the corresponding amide with relatively acceptable yields (10–90%).1H-NMR, 13C-NMR, IR, and HRMS analysis were used in structural elucidation and confirmed the proposed structures of the target compounds.
Triton WR-1339 model development
By comparing Triton WR-1339-treated group (HG) with negative CG, it was evident that Triton WR-1339 resulted in severe hyperlipidemia (high TG and high TC) in Wistar rats 18 h after the administration of a single dose of 300 mg/kg (p value < 0.05), which was consistent with previously published literatureCitation18. This indicates the suitability of the model and the selected dose which was chosen based on literature data concerning hyperlipidemia inductionCitation5,Citation10,Citation14,Citation16 ().
Figure 2. Effect of Triton WR-1339 on lipid profile after 18 h. Values are means ± SEM from six rats in each group. TG: triglyceride; TC: total cholesterol. *p Value < 0.01 using Mann–Whitney U test.
Acute toxicity study and selection of the experimental dose
Acute toxicity study revealed the nontoxic nature of C8 at the doses of 200–1000 mg/kg (). No lethality was observed for all selected doses after 1 day till day 14. Also mice did not suffer from any clinical signs of toxicity during this time period.
Table 1. Determination of compound (C8) acute toxicity using 5 different doses (200, 400, 600, 800, and 1000 mg/kg). Experiment was conducted on 5 groups of BALB/c mice (6 mice per each group).
One-tenth of the dose, in which no behavioral alterations were observed, was considered safe for further assaysCitation31. Hence, doses of 20–100 mg/kg were considered safe for subsequent experiments. The selection of the dose to be administered was based on dose-response curve experiment which was established at the following doses (20, 30, 50, and 100 mg/kg) to evaluate the compound effect on lipid components. It was found that starting from a dose of 30 mg/kg, lipid components (TG and TC) were significantly suppressed (p value <0.05) compared to Triton WR-1339 group. So due to similarity in structure between all synthesized compounds, a fixed dose of 30 mg/kg was considered a suitable dose for the assessment of antihyperlipidemic activity for all compound.
Effect of novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives, nicotinic acid, and fenofibrate on rat plasma lipid profile
The effect of novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives, nicotinic acid, and fenofibrate on plasma lipid components (TG and TC) was investigated 18 h after treatments application ().
Table 2. Effect of the novel C4, C6, C8, C10, C12, C14, FF, and NA on plasma lipid levels in Triton WR-1339-induced hyperlipidemic rats after 18 h.
In our study, fenofibrate dose (65 mg/kg) and nicotinic acid (30 mg/kg) were selected based on literature recommendationsCitation9,32–34. At the recommended doses, fenofibrate showed a significant suppression in the elevated plasma level of both TG and TC compared to HG (p value <0.05) which is consistent with its reported effectCitation35. While nicotinic acid did not show any significant differences in TG or TC compared to HG (p value ≥0.05).
Results indicate that animals treatment with 30 mg/kg of newly synthesized novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives C4 and C6 significantly suppressed Triton WR-1339-induced elevation in TG level (76.0 ± 20.1 and 72.2 ± 9.4 mg/dL, respectively) compared to HG (2317.8 ± 13.7) (p value <0.05). Even though C8 showed lower potency compared to C4 and C6, but still it showed a significant reduction in serum TG level compared Triton WR-1339-induced HG (1717.7 ± 296.5 mg/dL versus 2317.8 ± 13.7 mg/d respectively, p value <0.05). No significant differences in serum TG were associated with the administration of C10, C12, and C14 (2370.2 ± 11.9, 2336.0 ± 5.3, and 2213.0 ± 70.2 mg/dL, respectively) compared to Triton WR-1339 untreated group (2317.8 ± 13.7 mg/dL) (p value ≥0.05).
Regarding TC, all the compounds except C10 showed an evident prevention in the elevation of serum TC levels 18 h after Triton WR-1339 administration (p value <0.05). In a similar pattern to their effects on TG, C4 and C6 were the most active agents against TC among all investigated compounds in the present work (49.7 ± 12.7 and 66.0 ± 7.6 mg/dL, respectively).
Discussion
The present study evaluated the significant antihyperlipidemic activities of different nicotinic acid carboxamide derivatives; mainly C4, C6, C8, C10, C12, and C14 in Triton WR-1339-induced hyperlipidemic rats. The choice of carboxamide derivatives was based on results obtained by the same research group on indol carboxamide derivatives, where they found also that these compounds possessed lipid-lowering effectsCitation11,Citation12,Citation23,Citation24.
Triton WR-1339 has been widely utilized to induce acute hyperlipidemia in several experimental animalsCitation6,11,14,16,17. This model is the most widely accepted model for screening the potential hypolipidemic effect of different natural products and chemical entities, since it is simple to handle and requires shorter time to induce hyperlipidemiaCitation18.
In this study, Triton WR-1339 at a dose of 300 mg/kg was found to be suitable to significantly induce hyperlipidemia compared to CG. The maximum effects on lipid components in our model revealed similar pattern to previous report, where TG and TC peaked after 18 h from Triton WR-1339 administrationCitation18. This proves the feasibility of using this model to evaluate hypolipidemic activity of different compounds.
Our results showed that benzophenone amide derivatives C4 and C6, at a dose of 30 mg/kg, significantly reduced serum TG and TC levels compared to Triton WR-1339 group (p value <0.05), which may represent promising leads for the treatment of hyperlipidemia. The activity was reduced for C8 and it was almost abolished for C10, C12, and C14.
It is also important to highlight that compounds which are derived from 2-aminobenzophenone were either less active (C8) or inactive compounds (C10, C12, and C14) compared to N-(3-or 4-benzoylphenyl)pyridine-3-carboxamide (C4 and C6) which found to be active hypolipidemic compounds. This might imply that active hits (C4 and C6) favor an extended structure. This finding was consistent with a previous work by Al-Hiari et al.,Citation11 where researchers found that N-(3-or 4-benzoylphenyl)-1H-indole-2-carboxamide derivatives were more active compared to those compounds derived from 2-aminobenzophenone, which may also suggest that activity is dependent on the three dimensional extended structure.
Finally, since our experiment was conducted only on a single dose of the compounds (30 mg/kg), it is recommended to conduct further studies at higher doses, and to perform an extensive investigation of the effect of different functional groups or substitution (either on pyridine or benzophenone by using different linker instead of amide linker) on the hypolipidimic activities.
Conclusion
Based on results obtained from the present study, this study revealed the antihyperlipidemic activities of several novel N-(benzoylphenyl)pyridine-3-carboxamide derivatives. It is recommended to perform extensive investigations of the effect of different functional groups or substitutions on the biological activities, as well as to investigate the potential mechanism of action for these compounds. This could provide a basis for the discovery and development of new promising leads as potential antihyperlipidemic agents.
Declaration of interest
The authors report no declarations of interest.
Acknowledgements
The authors wish to thank the Deanship of Scientific Research at the University of Jordan and the Scientific Research Support Fund for their generous funds.
References
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