Pharmacological Evaluation of Novel Isonicotinic Carboxamide Derivatives as Potential Anti-Hyperlipidemic and Antioxidant Agents

Article abstract of DOI:10.1002/ardp.201700024

Hyperlipidemia and oxidative stress have been implicated as contributing factors to the development of atherosclerosis and cardiovascular diseases (CVDs). Currently, a large number of antihyperlipidemic medications are conveniently available in the market

Full text of DOI:10.1002/ardp.201700024

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700024
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Full Paper
Pharmacological Evaluation of Novel Isonicotinic Carboxamide
Derivatives as Potential Anti-Hyperlipidemic and Antioxidant
Agents
Rana Abu Farha
1, Yasser Bustanji1, Yusuf Al-Hiari1, Sanaa Bardaweel1, Tariq Al-Qirim2,
Ghassan Abu Sheikha2, and Rabab Albashiti1
1
Faculty of Pharmacy, University of Jordan, Amman, Jordan
Faculty of Pharmacy, Alzaytoonah University of Jordan, Amman, Jordan
2
Hyperlipidemia and oxidative stress have been implicated as contributing factors to the development
of atherosclerosis and cardiovascular diseases (CVDs). Currently, a large number of antihyperlipidemic
medications are conveniently available in the market. Nonetheless, the majority of antihyperlipidem-
ics lack the desired safety and efficacy. Thus, the present study was undertaken to evaluate the
potential effect of novel N-(benzoylphenyl)pyridine-4-carboxamide and N-(9,10-dioxo-9,10-dihydroan-
thracenyl)pyridine-4-carboxamide derivatives in controlling hyperlipidemia and oxidative stress using
the Triton WR-1339-induced hyperlipidemic rat model for antihyperlipidemic activity and the DPPH
radical scavenging assay for antioxidant activity. This study revealed the antihyperlipidemic activities
of some of the newly synthesized, novel carboxamide derivatives, mainly C4 and C12 (p < 0.05). The
majority of the compounds displayed a relatively low or no DPPH radical scavenging effect, with C20
possessing the best radical scavenging effect (22%) among all. This research opens the door for new
potential antihyperlipidemic compounds derived from isonicotinic acid. N-(3-Benzoylphenyl)pyridine-
4-carboxamide (C4) was found to have promising lipid-lowering and antioxidant effects, which may
create a protective effect against CVDs, by reducing the LDL-C levels and diminishing the generation
of reactive oxygen species.
Keywords: Antihyperlipidemic / Antioxidant / Isonicotinic acid / Triton WR-1339
Received: January 23, 2017; Revised: July 20, 2017; Accepted: July 21, 2017
DOI 10.1002/ardp.201700024
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
World Health Organization (WHO) to contribute to approxi-
mately 56% of coronary heart diseases (CHDs) and 18% of
cerebrovascular diseases worldwide, which consequently lead
Introduction
Hyperlipidemias have been implicated as contributing factors
for the development of atherosclerosis and cardiovascular
diseases (CVDs) [1, 2]. Elevated blood total cholesterol (TC),
elevated low-density lipoprotein cholesterol (LDL-C), and
elevated triglyceride (TG) levels have been reported by the
to 4.4 million deaths each year [3].
The oxidative modification hypothesis of atherosclerosis
predicts that LDL-C particles must be oxidized before they
become a contributing factor for atherosclerosis [4–6]. LDL-C
particles oxidation and retention in the subendothelial space
of blood vessel is considered to be the initiating factor of
atherosclerotic lesion development [4–6]. Thus, in the
development of a new novel antihyperlipidmic agent, an
important area of interest is to search for an agent with
antioxidant activity that will protect endothelial and myocar-
dial functions and may serve as a better anti-atherosclerotic
agent.
Correspondence: Dr. Rana Abu Farha, Faculty of Pharmacy,
University of Jordan, Amman 11942, Jordan.
E-mail: abufarharana@yahoo.com
Fax: þ962-6-5300250
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Currently, a large number of antihyperlipidemic medications
are conveniently available in the market. Nonetheless, the
majority of antihyperlipidemics lack the desired safety and
efficacy, which limits their long-term use. Although nicotinic
acid has been historically used for its lipid-lowering and
antioxidant effect to prevent cardiovascular diseases and
atherosclerosis, the associated multiple side effects have
marred its popularity [7–11]. Therefore, tremendous efforts
were employed to synthesize and evaluate different nicotinic
acid derivatives for the treatment of hyperlipidemia with
improved anti-atherogenic effect and with less adverse
events [12–16].
Previously, our laboratory has reported the synthesis and
evaluation for antihyperlipidemic activity of nicotinic acid
derivatives, N-(benzoylphenyl)pyridine-3-carboxamide ana-
logs [16]. In this study, isonicotinic acid (which is an isomer
of nicotinic acid) was used as our starting point to synthesize
novel isonicotinic carboxamide derivatives bearing the
benzophenone unit of the fenofibrate drug (Fig. 1), as well
as to synthesize other carboxamide derivatives bearing the
anthraquinone unit to increase the rigidity of the compounds.
The potential lipid-lowering and antioxidant effects of these
compounds were evaluated using Triton WR-1339 hyper-
lipidemic rat model and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay, respectively. We are proposing that
this combination may produce a promising candidate for the
treatment of hyperlipidemia with potentially higher antioxi-
dant effect.
dry benzene, and the mixture was refluxed at 70°C for 48 h till
the formation of the corresponding isonicotinyl chloride (C2).
Excess SOCl2 and benzene were then distilled out from the
reaction mixture. Next, aminobenzophenone or aminoan-
thraquinone amine was added to the acid chloride (C2) and
the reaction mixture was mixed 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, the final product was precipitated by
the addition of crushed ice water, which was then filtered to
afford the corresponding amide. Structure elucidation and
confirmation of the target compounds was performed using
1H-NMR, 13C-NMR, IR, and HRMS analysis.
Biology
Induction of hyperlipidemia in experimental rats
As shown in Fig. 2, induction of hyperlipidemia in experimen-
tal rats (hyperlipidemic group, HG) using a single dose of
300 mg/kg Triton WR-1339 showed significant increase in
serum levels of LDL-C, TG, and TC compared with control
group (CG) after 18 h of Triton WR-1339 administration
(p < 0.05). These results were consistent with previously
published literature on the suitability of using this model in
the evaluation of hypolipidemic effect of different chemical
and natural compounds [18–21].
Acute toxicity study and selection of the experimental
dose
Based on the previous acute toxicity study for a related
compounds (N-(benzoylphenyl)pyridine-3-carboxamide deriv-
atives), doses of 20–100mg/kg were considered to be safe for
the evaluation of antihyperlipidemic effect [16]. In this study, a
dose–response curve was established at five different doses for
C6 compound (20, 30, 50, and 100 mg/kg) to evaluate the
potentialantihypelipidemiceffectofeachdose.Resultsshowed
that LDL-C, TC, and TG were significantly suppressed compared
to Triton WR-1339 group at all doses except for the 20 mg/dL
Results
Chemistry
In this study, the target carboxamide derivatives were
prepared using the acid chloride pathway, which gave
relatively good yields (range 27.7–58.5%) (Schemes
and 2) [17]. Isonicotinic acid (C1) was treated with SOCl2 in
1
Figure 1. Schematic representation of the
rationale of the study.
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Scheme 1. Preparation of N-(benzoylphenyl)pyridine-4-carboxamide derivatives (C4, C6, C8, C10, C12, and C14): (i) SOCl2, dry
benzene, refluxed at 70°C for 48 h, distillation to get rid of excess SOCl2 and dry benzene; (ii) refluxed at 70°C for 18 h, followed by
the addition of 1,4-dioxan and stirring for 24 h.
(p < 0.05). So a dose of 30 mg/kg was considered to be
suitable for the assessment of antihyperlipidemic activity for
all compounds.
suppression in the elevated plasma level of both TG and TC
compared to the HG (p < 0.05), which is consistent with its
reported effects [26].
The effect of acute administration of Triton WR-1339 on the
elevation of serum LDL-C level was evident with the
coadministration of C8, C10, C14, C16, C18, and C20, while
pretreatment with C4, C6, and C12 was associated with lower
LDL-C values (38.7 ꢁ 2.8, 124.2 ꢁ 15.1, and 34.6 ꢁ 3.1 mg/dL
respectively) compared to the HG (202.6 ꢁ 8.7 mg/dL)
(p < 0.05). C4 and C12 were the most potent among them.
Evaluation of antihyperlipidemic effect of novel
isonicotinic carboxamide derivatives
Theeffectsofpretreatmentwith30 mg/kgofnewlysynthesized
novel N-(benzoylphenyl)pyridine-4-carboxamide derivatives
(C4, C6, C8, C10, C12, and C14), N-(9,10-dioxo-9,10-dihydroan-
thracenyl)pyridine-4-carboxamide derivatives (C16, C18, and
C20), 30mg/kg nicotinic acid, and 65mg/kg fenofibrate on
serum lipid components are presented in Table 1.
At the recommended doses of nicotinic acid (30 mg/kg)
and fenofibrate (65 mg/kg) [22–25], nicotinic acid did not
show any significant differences in TG or TC compared to the
TG levels were significantly suppressed in
a similar
pattern to that of LDL-C by C4, C6, and C12 (78.5 ꢁ 4.0,
1899.8 ꢁ 148.3, and 49.3 ꢁ 4.2 mg/dL, respectively) compared
to the HG (2317.8 ꢁ 13.7 mg/dL) (p < 0.05). While C8, C10, C14,
C16, C18, and C20 showed almost similar elevation in TG
serum level compared to the HG.
HG (p ꢀ 0.05), while fenofibrate showed
a significant
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Scheme 2. Preparation of N-(9,10-dioxo-9,10-dihydroanthracenyl)pyridine-4-carboxamide derivatives (C16, C18, and C20): (i) SOCl2,
dry benzene, refluxed at 70°C for 48 h, distillation to get rid of excess SOCl2 and dry benzene; (ii) refluxed at 100°C for 18 h, followed
by the addition of 1,4-dioxan and stirring for 24 h.
RegardingTClevels,C8,C10,C16,C18,andC20didnotshowany
significant changes from the HG, where C4 and C12 showed
pronouncedactivitiesinloweringserumTClevel(77.0 ꢁ 4.2 and
51.5 ꢁ 5.2 mg/dL, respectively) in comparison to the HG
(398.8 ꢁ 17.3 mg/dL) (p < 0.05). Even though C6 and C14
showed lower potency compared to C4 and C12, still they
showed a significant reduction in serum TG levels (226.0 ꢁ 24.9
and 318.5 ꢁ 13.2 mg/dL, respectively) compared with the Triton
WR-1339-induced HG (398.8 ꢁ 17.3 mg/dL) (p < 0.05).
Evaluation of antioxidant effect of novel N-(benzoylphenyl)-
pyridine-4-carboxamide derivatives
Under experimental conditions, reference ascorbic acid
was tested at six different concentrations and showed a
potent scavenging effects on DPPH radicals with IC50
7.2 mg/mL (Fig. 3).
¼
The scavenging effects of the synthesized N-(benzoyl-
phenyl)pyridine-4-carboxamide and N-(9,10-dioxo-9,10-dihy-
droanthracenyl)pyridine-4-carboxamide derivatives on DPPH
radicals were tested and are presented in Table 2. These data
represent a preliminary screening on a single concentration
(100 mg/mL). Results showed that the majority of the tested
compounds displayed a relatively low or no DPPH radical
scavenging effect, with most of the compounds achieving less
than 22% DPPH radical scavenging effect, with C20 and C4
being the most potent among them.
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. ꢂp< 0.01 using Mann–Whitney
U-test.
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novel N-(benzoylphenyl)pyridine-4-carboxamide and N-(9,
10-dioxo-9,10-dihydroanthracenyl)pyridine-4-carboxamide
derivatives in controlling hyperlipidemia and oxidative
stress using hyperlipidemic rat model and DPPH radical
scavenging assay, respectively.
Triton WR-1339-induced hyperlipidemia in rats is a widely
accepted animal model [20, 21, 27–29], successfully used for
screening lipid-lowering activities of natural products and
chemical entities, since it is simple to handle and requires
shorter time to induce hyperlipidemia [30]. The administra-
tion of single intraperitoneal dose of Triton WR-1339 to adult
rats has been reported to increase TC, LDL-C, and TGs to reach
a maximum value in about 20 h and return to normal
thereafter [30].
Figure 3. DPPH radical inhibition by ascorbic acid. Results
The antihyperlipidemic activities of different isonicotinic
acid carboxamide derivatives, mainly C4, C6, C8, C10, C12, C14,
C16, C18, and C20, were evaluated in Triton WR-1339-induced
hyperlipidemic rats. In the current study, Triton WR-1339
(300 mg/kg, i.p.) administration caused alterations in the lipid
metabolism in about 18 h, which resulted in severe hyperlip-
idemia. The maximum effects on lipid components in our
model revealed similar pattern to previous report, where LDL-
C, TG, and TC peaked after 18 h from Triton WR-1339
administration [30].
Treatment with benzophenone amide derivatives C4 and
C12 (at a dose of 30 mg/kg) offered significant protection
against Triton WR-1339-induced hyperlipidemia by maintain-
ing serum LDL-C, TG, and TC nearly to normal levels compared
to Triton WR-1339 only treated group (p < 0.05). The activity
represent the average of three replicates.
Discussion
Hyperlipidemia and oxidative stress have been implicated as
essential contributing factors for the development of
atherosclerosis and CVDs such as hypertension, myocardial
infarction, peripheral vascular diseases, and CHDs [1, 2]. The
risk of CVDs can be reduced by using drugs with both
hypolipidemic and antioxidant effects. Despite the availability
of many drugs for treating hyperlipidemia, unfortunately,
most of them lack the desired safety. Thus, the present
study was undertaken to evaluate the potential effect of
Table 1. Effect of the novel C4, C6, C8, C10, C12, C14, C16, C18, C20, fenofibrate (FF), and nicotinic acid (NA) on plasma
lipid levels in Triton WR-1339-induced hyperlipidemic rats after 18 h.
Lipid profile components
Groups
LDL-C (mg/dL)
TG (mg/dL)
TC (mg/dL)
CG
HG
C4
C6
C8
C10
C12
C14
C16
C18
C20
FF
44.0 ꢁ 3.2ꢂ
202.6 ꢁ 8.7
38.7 ꢁ 2.8ꢂ
131.0 ꢁ 18.1ꢂ
2317.8 ꢁ 13.7
78.5 ꢁ 4.0ꢂ
86.1 ꢁ 6.5ꢂ
398.8 ꢁ 17.3
77.0 ꢁ 4.2ꢂ
124.2 ꢁ 15.1ꢂ
227.0 ꢁ 7.8
191.4 ꢁ 16.8
34.6 ꢁ 3.1ꢂ
1899.8 ꢁ 148.3ꢂ
2369.0 ꢁ 7.2
2349.0 ꢁ 41.0
49.3 ꢁ 4.2ꢂ
226.0 ꢁ 24.9ꢂ
381.5 ꢁ 14.9
339.2 ꢁ 31.7
51.5 ꢁ 5.2ꢂ
182.8 ꢁ 9.6
240.4 ꢁ 8.0
200.2 ꢁ 16.5
200.4 ꢁ 7.5
213.8 ꢁ 9.1
2272.4 ꢁ 2.74
2266.3 ꢁ 39.6
2406.2 ꢁ 66.8
2384.1 ꢁ 7.4
2358.2 ꢁ 8.6
1540 ꢁ 85.2ꢂ
392.5 ꢁ 39.1
318.5 ꢁ 13.2ꢂ
418.9 ꢁ 25.2
342.8 ꢁ 25.1
360.6 ꢁ 19.8
271.2 ꢁ 8.3ꢂ
392.5 ꢁ 39.1
NA
Values are expressed as means ꢁ SEM from six rats in each group. All groups received 300 mg/kg Triton WR-1339 i.p. injection
except for CG (control group), which received water as i.p. injection. Regarding oral gavage, CG and HG (hyperlipidemic group)
received: 6% DMSO/corn oil; C4: C4 þ 6% DMSO/corn oil; C6: C6 þ 6% DMSO/corn oil; C8: C8 þ 6% DMSO/corn oil; C10: C10 þ 6%
DMSO/corn oil; C12: C12 þ 6% DMSO/corn oil; C14: C14 þ 6% DMSO/corn oil; C16: C16 þ 6% DMSO/corn oil; C18: C18 þ 6% DMSO/
corn oil; C20: C20 þ 6% DMSO/corn oil; FF: fenofibrate þ 6% DMSO/corn oil; NA: nicotinic acid þ 6% DMSO/corn oil. TG:
triglyceride; TC: total cholesterol. C4, C6, C8, C10, C12, C14, FF, and NA were compared with the HG. ꢂp-value < 0.05 using Mann–
Whitney U-test.
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Table 2. In vitro antioxidant activity of synthesized N-
(benzoylphenyl)pyridine-4-carboxamide and N-(9,10-dioxo-
9,10-dihydroanthracenyl)pyridine-4-carboxamide derivatives
using DPPH radical scavenging assay.
Regarding the antioxidant activities, our results showed that
the majority of the tested compounds displayed a relatively
low or no DPPH radical scavenging effect, with most of the
compounds achieving less than 22% DPPH radical scavenging
effect. This finding was comparable to results obtained
previously by Ogata et al. [14], who have studied the
scavenging capacity of 23 kinds of nicotinic and isoncotinic
acid-related compounds using DPPH radical scavenging assay.
Ogata et al. [14] found that nicotinic acid and isonicotinic
acids as a nucleus have scavenging rates of 27.4% and 40.9%,
respectively. The structural modification of both kinds of
nucleus resulted in the reduction of their antioxidant capacity
to less than 20% in all cases, except for the hydrazide
derivative (nicotinic hydrazide and isonicotinic hydrazide),
which have achieved 100% DPPH radical inhibition ability.
Also mild antioxidant capacities of different nicotinic acid
derivatives, namely thionicotinic acid, thioamide, and thioni-
trile, were reported [31], with thionicotinic acid showing the
most potent antioxidant radical scavenging activity (33.20%)
while the other two compounds exerted almost no
activities [31].
DPPH radical inhibition
Compounds
(%)a)
C4
C6
C8
C10
C12
C14
C16
C18
C20
12.80 ꢁ 4.3
8.90 ꢁ 2.5
5.40 ꢁ 6.5
0.10 ꢁ 2.4
1.40 ꢁ 0.8
NA
1.20 ꢁ 2.3
NA
21.90 ꢁ 0.7
Concentration used for each compound ¼ (100 mg/mL). NA:
not active.
a)Results represent the average of three replicates, data are
expressed as mean ꢁ SD.
In spite that the scavenging rates for most of our
compounds were not significant, C4 demonstrated a mild
was reduced for C6 and it was almost abolished for C10, C12,
C14, C16, C18, and C20. Thus, C4 and C12 may represent
promising leads for the treatment of hyperlipidemia.
Since the active compounds C4, C6, and C12 showed a
significant reduction in TG, LDL-C, and TC levels, it is
suggested that those compounds may antagonize the activity
of Triton WR-1339 by stimulating lipoprotein lipase enzyme,
which is the main target in Triton WR-1339-induced hyper-
lipidemic rat model.
scavenging effect of 12.8%. This makes C4
a distinct
compound among all compounds, because of its wide range
of biological activities including antioxidant and antihyperli-
pidemic effects. These dual activities will create a protective
effect against CVDs, by reducing LDL-C levels and diminishing
the generation of reactive oxygen species. This finding
specifically makes C4 a promising lipid lowering and antioxi-
dant agent.
Activities were shown to be mainly associated with
benzophenone derivatives, while being concealed with
anthraquinone derivatives. Rigidity of the compounds seems
to play an important role in compounds activities, where the
more rigid anthraquinone derivatives were not active
compared to benzophenone derivatives.
Finally, since our experiments were conducted only on a
single dose of the compounds (30 mg/kg for hypolipidemic
evaluation and 100mg/mL for antioxidant evaluation), it is
recommended to conduct further studies at higher doses, and
to perform an extensive investigation of the effect of different
functional groups or substitutions (either on pyridine or
benzophenone by using different linkers instead of amide
linker) on the hypolipidemic and antioxidant activities.
Based on the results obtained from the present study, this
study revealed the antihyperlipidemic activities of some novel
isonicotinic 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.
Regarding benzophenone derivatives, it is important to
highlight that the three-dimensional energy-minimized
structures of these compounds may play a role in their
hypolipidemic activities, where an extended linear structure is
found to be active, while the non-extended form is less active/
inactive. This is related to the attachment point of the amide
linker where compounds with amide nitrogen near to the
carbonyl moiety (at position 2 in benzophenone) were less
active/inactive (with the exception of C12). This position may
create intramolecular hydrogen bonding between the car-
bonyl and the amide nitrogen, thus contributing to the non-
extended structure and also preventing those two moieties
from participating in H-bonding with the amino acid residue
of the receptor. C12 lacks such interaction due to steric 3-
methyl substituent. These suggestions are in concordance
with previously obtained results on N-(benzoylphenyl)pyri-
dine-3-carboxamide [16], where activities were decreased in
general by switching the amide linker from positions 3 or 4 to
position 2.
Experimental
Chemistry
General
All chemicals solvents, and reagents were purchased from
Acros Organics (United Kingdom) and Aldrich Chemicals
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(United Kingdom) and were utilized without any purification.
1H-NMR and 13C-NMR spectra were recorded on a Bruker-500
(500 MHz) at the University of Jordan. Deuteriated dimethyl-
sulfoxide (DMSO-d6) was used as solvent in sample prepara-
tion. Infrared (IR) spectra were recorded using Shimadzu
8400F FT-IR 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). Melting points were determined in open
capillaries on a Stuart scientific electrothermal melting point
apparatus (United Kingdom). 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 T) instrument at the
University of Jordan. Thesampleswere dissolved in chloroform.
Plate Reader Biotek ELX800 with BioTek’s Gen5TM (USA) was
used for measuring absorbance for antioxidant experiment.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
129.08 (2 Ar-CH), 125.79 (Ar-CH), 124.75 (Ar-CH), 122.04 (2 Ar-
CH), 121.86 (Ar-CH) ppm. IR (KBr disk): n ¼ 3325 (NH amide),
3055, 1944, 1874, 1828, 1751, 1658 (ketone carbonyl), 1604
(amide carbonyl), 1550, 1481, 848, 717 cmꢃ1. HRMS (ESI,
negative mode): m/z [MꢃHþ] 301.10570 (C19H13N2O2 requires
301.09715).
N-(4-Benzoylphenyl)pyridine-4-carboxamide (C6): A mixture
of isonicotinoyl chloride (C2, 0.50 g, Mwt 141.56, 3.5 mmol)
and 4-amino-benzophenone (C5, 0.63 g, Mwt 197.24,
3.2 mmol) was used to produce N-(4-benzoylphenyl)pyri-
dine-4-carboxamide as pale yellow solid (C6, 0.566 g, Mwt
302.3, 58.5%). Rf: 0.39 (CHCl3/MeOH, 96:4); Melting point:
150–152°C. 1H-NMR (300 MHz, DMSO-d6): d ¼ 10.94 (s, 1H, NH
amide), 8.84 (m, 2H, Ar-H), 7.57–7.98 (m, 11H, Ar-H) ppm.
13C-NMR (75 MHz, DMSO-d6): d ¼ 194.64 (ketone carbonyl),
164.27 (amide carbonyl), 149.53 (2 Ar-CH), 142.67 (Ar-C
Quaternary), 142.36 (Ar-C Quaternary), 137.35 (Ar-C Quater-
nary), 132.37 (Ar-CH), 132.24 (Ar-C Quaternary), 130.98 (2
CH-Ar), 129.42 (2 Ar-CH), 128.51 (2 Ar-CH), 122.05 (2 Ar-CH),
119.64 (2 Ar-CH) ppm. IR (KBr disk): n ¼ 3363 (NH amide), 3039,
1936, 1851, 1820, 1782, 1681 (ketone carbonyl), 1643 (amide
carbonyl), 1597, 1527, 1404, 848, 740, 702 cmꢃ1. HRMS (ESI,
negative mode): m/z [MꢃHþ] 301.10357 (C19H13N2O2 requires
301.09715).
General synthesis procedure
A mixture of isonicotinic acid (C1, 1 g, Mwt 123.11, 8.1 mmol)
and SOCl2 (1 mL, Mwt 118.97, 13.78 mmol) was placed in a
round bottomed flask with 10 mL of dry benzene. The
reaction mixture was refluxed for 48 h at 70°C until the
reaction was completed. Dry benzene and SOCl2 solvents were
distilled out from the reaction mixture to afford isonicotinoyl
chloride (C2, 0.6 g liquid, Mwt 141.56, 51.8%), as colorless
liquid (Scheme 1).
Later, the corresponding amine (3.2 mmol) was added to
isonicotinoyl chloride (C2) (0.50 g, Mwt 141.56, 3.5 mmol) and
the reaction mixture was refluxed at 70°C for 18 h in air
condenser. Fifteen milliliters of 1,4-dioxane was then added
and 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-(2-Benzoylphenyl)pyridine-4-carboxamide (C8): A mixture
of isonicotinoyl chloride (C2, 0.50 g, Mwt 141.56, 3.5 mmol)
and 2-amino-benzophenone (C7, 0.63 g, Mwt 197.24,
3.2 mmol) was used to produce N-(2-benzoylphenyl)pyri-
dine-4-carboxamide as pale yellow solid (C8, 0.317 g, Mwt
302.3, 32.7%). Rf: 0.50 (CHCl3/MeOH, 96:4); Melting point:
115–117°C; 1H-NMR (300 MHz, DMSO-d6): d ¼ 10.83 (s, 1H, NH
amide), 8.69 (d, J ¼ 4.8 Hz, 2H, Ar-H), 7.36–7.73 (m, 11 H, Ar-H)
ppm. 13C-NMR (75 MHz, DMSO-d6): d ¼ 195.33 (ketone car-
bonyl), 163.82 (amide carbonyl), 150.18 (2 Ar-CH), 141.04
(Ar-C Quaternary), 137.15 (Ar-C Quaternary), 135.96 (Ar-C
Quaternary), 132.59 (Ar-CH), 132.59 (Ar-CH), 131.45 (Ar-C
Quaternary), 130.32 (Ar-CH), 129.47 (2 Ar-CH), 128.19 (2 Ar-
CH), 125.19 (Ar-CH), 124.61 (Ar-CH), 121.08 (2 Ar-CH) ppm. IR
(KBr disk): n ¼ 3271 (NH amide), 3063, 1982, 1944, 1836, 1805,
1681 (ketone carbonyl), 1620 (amide carbonyl), 1581, 1535,
1442, 925, 756, 702 cmꢃ1. HRMS (ESI, negative mode): m/z
[MꢃHþ] 301.09653 (C19H13N2O2 requires 301.09715).
Synthesis of the N-(benzoylphenyl)pyridine-
4-carboxamide derivatives (Scheme 1)
N-(3-Benzoylphenyl)pyridine-4-carboxamide (C4): A mixture of
isnicotinoyl chloride (C2, 0.50g, Mwt 141.56, 3.5mmol) and 3-
amino-benzophenone (C3, 0.63 g, Mwt 197.24, 3.2 mmol) was
used to produce N-(3-benzoylphenyl)pyridine-4-carboxamide as
pale yellow solid (C4, 0.50 g, Mwt 302.3, 51.7%); Rf: 0.11 (CHCl3/
MeOH, 96:4); Melting point: 134–136°C; 1H-NMR (500 MHz,
DMSO-d6): d¼ 10.72 (s, 1H, NH amide), 8.80 (d, J ¼ 5.7 Hz, 2H, Ar-
H), 8.21 (m, 1H, Ar-H), 8.13 (d, J ¼ 8.1 Hz, 1H, Ar-H), 7.88 (d,
J¼ 5.9 Hz, 2H, Ar-H), 7.78 (d, J¼ 7.2 Hz, 2H, Ar-H), 7.71 (dd,
J¼ 7.5, 7.4 Hz, 1H, Ar-H), 7.59 (dd, J¼ 7.9, 8.2 Hz, 3H, Ar-H), 7.52
(d, J ¼ 7.7 Hz, 1H, Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6):
d¼ 196.01 (ketone carbonyl), 164.73 (amide carbonyl), 150.78 (2
Ar-CH), 148.70 (Ar-C Quaternary), 142.06 (Ar-C Quaternary),
139.27 (Ar-C Quaternary), 137.94 (Ar-C Quaternary), 137.44 (Ar-
C Quaternary), 133.23 (Ar-CH), 130.10 (2 Ar-CH), 129.60 (Ar-CH),
2-(2-[(Pyridin-4-ylcarbonyl)amino]benzoyl)benzoicacid(C10):
A mixture of isonicotinoyl chloride (C2, 0.50 g, Mwt 141.56,
3.5 mmol) and 2-amino-benzophenone-20-carboxylic acid (C9,
0.77 g, Mwt 241.24, 3.2 mmol) was used to produce 2-(2-
[(pyridin-4-ylcarbonyl)amino]benzoyl)benzoic acid as pale
yellow solid (C10, 0.307g, Mwt 346.3, 27.7%). Rf: 0.58
(CHCl3/MeOH, 96:4); Melting point: 246–248°C; 1H-NMR
(300 MHz, DMSO-d6): d ¼ 12.28 (s, 1H, COOH), 11.12 (s, 1H,
NH amide), 8.13 (m, 1H, Ar-H), 7.50–7.83 (m, 4H, Ar-H), 7.31 (m,
2H, Ar-H), 7.21 (m, 1H, Ar-H) 7.17 (m, 1H, Ar-H), 7.14 (m, 2H,
Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6): d ¼ 192.93 (ketone
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carbonyl), 165.93 (COOH), 163.90 (amide carbonyl), 151.63 (2
Ar-CH), 138.68 (Ar-C Quaternary), 137.09 (Ar-C Quaternary),
134.30 (Ar-CH), 133.60 (Ar-CH), 133.43 (Ar-CH), 131.62 (Ar-CH),
130.61 (Ar-C Quaternary), 130.29 (Ar-C Quaternary), 130.04
(Ar-C Quaternary), 129.70 (Ar-CH), 128.61 (Ar-CH), 124.26 (Ar-
CH), 121.32 (Ar-CH), 120.90 (Ar-CH) ppm. IR (KBr disk): n ¼ 3302
(NH amide), 2607–3263 (OH), 3101, 3032, 1959, 1928, 1898,
1851, 1643 (ketone carbonyl), 1589 (acid carbonyl), 1543
Mwt 141.56, 3.5 mmol) and 1-amino-anthraquinone (C15,
0.71 g, Mwt 223.23, 3.2 mmol) was used to produce N-(9,10-
dioxo-9,10-dihydroanthracen-1-yl)pyridine-4-carboxamide as
orange solid (C16, 0.478 g, Mwt 328.3, 45.5%), Rf: 0.22 (CHCl3/
MeOH, 96:4); Melting point: 258–260°C. 1H-NMR (500 MHz,
DMSO-d6): d ¼ 13.10 (s, 1H, NH amide), 9.12 (d, J ¼ 6.4 Hz, 1H,
Ar-H), 8.94 (d, J ¼ 5.7 Hz, 2H, Ar-H), 8.30 (m, 1H, Ar-H), 8.21 (m,
1H, Ar-H), 8.06 (d, J ¼ 7.4 Hz, 2H, Ar-H), 7.97–8.02 (m, 4H, Ar-H)
ppm. 13C-NMR (125 MHz, DMSO-d6): d ¼ 187.53 (ketone
carbonyl), 182.54 (ketone carbonyl), 164.52 (amide carbonyl),
151.53 (2 Ar-CH), 141.68 (Ar-C Quaternary), 141.02 (Ar-C
Quaternary), 136.50 (Ar-CH), 135.42 (Ar-CH), 135.33 (Ar-CH),
134.40 (Ar-C Quaternary), 134.10 (Ar-C Quaternary), 132.78
(Ar-C Quaternary), 127.73 (Ar-CH), 127.05 (Ar-CH), 126.14 (Ar-
CH), 123.18 (Ar-CH), 121.42 (2 Ar-CH), 119.18 (Ar-C Quater-
nary) ppm. IR (KBr disk): n ¼ 3194 (NH amide), 3109, 3070,
1994, 1851, 1782, 1689 (ketone carbonyl), 1666 (ketone
(amide carbonyl), 1481, 1435, 1396, 1234 (C-O), 763, 702 cmꢃ1
.
HRMS (ESI, negative mode): m/z [MꢃHþ] 345.08426
(C20H13N2O4 requires 345.08698).
N-(2-Benzoyl-5-methylphenyl)pyridine-4-carboxamide (C12):
A mixture of isonicotinoyl chloride (C2, 0.50g, Mwt 141.56,
3.5 mmol) and 2-amino-4-methylbenzophenoneC11, 1.56 g,
Mwt 211.26, 7.4 mmol) was used to produce N-(2-benzoyl-5-
methylphenyl)pyridine-4-carboxamide obtained as pale yellow
solid (C12, 0.541 g, Mwt 316.6, 53.4%). Rf: 0.34 (CHCl3/MeOH,
96:4); Melting point: 130–132°C; 1H-NMR (300 MHz, DMSO-d6):
d ¼ 10.80 (s, 1H, NH amide), 8.69 (m, 2H, Ar-H), 7.48–7.70 (m, 7H,
Ar-H), 7.38 (d, J ¼ 6.3 Hz, 1H, Ar-H), 7.27 (d, J ¼ 7.2 Hz, 2H, Ar-H),
2.33 (s, 3H, CH3) ppm. 13C-NMR (75 MHz, DMSO-d6): d ¼ 195.07
(ketone carbonyl), 163.88 (amide carbonyl), 150.17 (2 Ar-CH),
143.01 (Ar-C Quaternary), 140.73 (Ar-CQuaternary), 135.99(Ar-
CQuaternary),134.57(Ar-CQuaternary),131.88(Ar-CH),131.55
(Ar-CH), 130.28 (2 Ar-CH), 129.70 (2 Ar-CH), 128.74 (Ar-C
Quaternary), 125.08 (Ar-CH), 124.62 (Ar-CH), 121.09 (2 Ar-CH),
21.06 (CH3) ppm. IR (KBr disk): n ¼ 3232 (NH amide), 3070, 2924,
2862, 1990, 1944, 1836, 1797, 1681 (ketone carbonyl), 1620
(amide carbonyl), 1573, 1535, 1442, 933, 840, 763cmꢃ1. HRMS
(ESI, negative mode): m/z [MꢃHþ] 315.11281 (C20H15N2O2
requires 315.11280).
carbonyl), 1635 (amide carbonyl), 1581, 1519, 1404, 709 cmꢃ1
.
HRMS (ESI, positive mode): m/z [MþHþ] 329.09195
(C20H13N2O3 requires 329.09207).
N-(4-Hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)pyri-
dine-4-carboxamide (C18): A mixture of isonicotinoyl chlo-
ride (C2, 0.50 g, Mwt 141.56, 3.5 mmol) and 1-amino-4-
hydroxyanthraquinone (C17, 0.77 g, Mwt 239.23, 3.2 mmol)
was used to produce N-(4-hydroxy-9,10-dioxo-9,10-dihy-
droanthracen-1-yl)pyridine-4-carboxamide as orange-red
solid (C18, 0.238 g, Mwt 344.3, 21.6%), Rf: 0.14 (CHCl3/MeOH,
96:4); melting point: 254–256°C. 1H-NMR (500 MHz, DMSO-
d6): d ¼ 13.03 (s, 1H, NH amide), 9.05 (d, J ¼ 9.4 Hz, 1H, Ar-H),
8.93 (d, J ¼ 3.2 Hz, 2H, Ar-H), 8.30 (dd, J ¼ 4.1, 6.7 Hz, 2H, Ar-H),
8.27 (dd, J ¼ 3.5, 5.1 Hz, 1H, Ar-H), 7.99 (d, J ¼ 4.1 Hz, 2H, Ar-H),
7.95 (d, J ¼ 4.2 Hz, 2H, Ar-H), 7.58 (d, J ¼ 9.4 Hz, 1H, Ar-H) ppm.
13C-NMR (125 MHz, DMSO-d6): d ¼ 188.42 (ketone carbonyl),
186.42 (ketone carbonyl), 164.42 (amide carbonyl), 159.41 (Ar-
C Quaternary C-OH), 151.49 (2 Ar-CH), 141.70 (Ar-C Quater-
nary), 135.85 (Ar-CH), 135.49 (Ar-CH), 135.27 (Ar-C Quater-
nary), 134.03 (Ar-C Quaternary), 132.60 (Ar-C Quaternary),
131.06 (Ar-CH), 127.83 (Ar-CH), 127.57 (Ar-CH), 126.93 (Ar-
CH), 121.43 (2 Ar-CH), 117.77 (Ar-C Quaternary), 115.11 (Ar-C
Quaternary) ppm. IR (KBr disk): n ¼ 3433 (NH amide),
3171–3294 (OH), 3101, 3032, 1952, 1689 (ketone carbonyl),
1627 (ketone carbonyl), 1581 (amide carbonyl), 1489, 1465,
N-(2-Benzoyl-4-chlorophenyl)pyridine-4-carboxamide
(C14): A mixture of isonicotinoyl chloride (C2, 0.50 g, Mwt
141.56, 3.5 mmol) and 2-amino-5-chlorobenzophenone (C13,
0.68 g, Mwt 213.68, 3.2 mmol) was used to produce N-(2-
benzoyl-4-chlorophenyl)pyridine-4-carboxamide as pale yel-
low solid (C14, 0.437 g, Mwt 336.8, 40.5%). Rf: 0.73 (CHCl3/
MeOH, 96:4); Melting point: 154–156°C; 1H-NMR (300 MHz,
DMSO-d6): d ¼ 10.79 (s, 1H, NH amide), 8.66 (m, 2H, Ar-H),
7.47–7.67 (m, 10H, Ar-H) ppm. 13C-NMR (300 MHz, DMSO-d6):
d ¼ 193.38 (ketone carbonyl), 163.95 (amide carbonyl), 150.15
(2 CH-Ar), 140.68 (Ar-C Quaternary), 136.39 (Ar-C Quaternary),
134.43 (2 Ar-C Quaternary), 133.78 (Ar-C Quaternary), 132.93
(Ar-CH), 131.58 (Ar-CH), 129.46 (2 Ar-CH), 129.33 (Ar-CH),
128.29 (2 Ar-CH), 126.86 (Ar-CH), 121.09 (2 Ar-CH) ppm. IR (KBr
disk): n ¼ 3236 (NH amide), 3124, 3063, 1936, 1851, 1805, 1797,
1689 (ketone carbonyl), 1620 (amide carbonyl), 1581, 1519,
1396, 949, 833, 748, 686 cmꢃ1. HRMS (ESI, negative mode): m/z
[MꢃHþ] 335.05652 (C19H12ClN2O2 requires 335.05818).
786, 748, 725 cmꢃ1
. HRMS (ESI, negative mode): m/z
[MꢃHþ] 343.07151 (C20H11N2O4 requires 343.07133).
N-(9,10-Dioxo-9,10-dihydroanthracen-2-yl)pyridine-4-carbox-
amide (C20): A mixture of isonicotinoyl chloride (C2, 0.50 g,
Mwt 141.56, 3.5 mmol) and 2-amino-anthraquinone (C19,
0.71 g, Mwt 223.23, 3.2 mmol) was used to produce N-(9,10-
dioxo-9,10-dihydroanthracen-2-yl)pyridine-4-carboxamide as
green solid (C20, 0.835 g, Mwt 328.3, 79.5%), Rf: 0.15 (CHCl3/
MeOH, 96:4); Melting point: 300–302°C. 1H-NMR (500 MHz,
DMSO-d6): d ¼ 11.41 (s, 1H, NH amide), 9.14 (m, 1H, Ar-H), 8.68
(m, 1H, Ar-H), 8.18–8.41 (m, 6H, Ar-H), 7.90 (d, J ¼ 3.7 Hz, 2H,
Ar-H) ppm. 13C-NMR (125 MHz, DMSO-d6): d ¼ 182.18 (ketone
Synthesis of the N-(9,10-dioxo-9,10-dihydroanthracenyl)-
pyridine-4-carboxamide derivatives (Scheme 2)
N-(9,10-Dioxo-9,10-dihydroanthracen-1-yl)pyridine-4-carbox-
amide (C16): A mixture of isonicotinoyl chloride (C2, 0.50 g,
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carbonyl), 181.39 (ketone carbonyl), 163.30 (amide carbonyl),
145.32 (Ar-C Quaternary), 143.83 (Ar-C Quaternary), 134.58
(Ar-CH), 134.29 (2 Ar-CH), 133.96 (Ar-C Quaternary), 133.04 (2
Ar-C Quaternary), 128.82 (Ar-C Quaternary), 128.30 (2 Ar-CH),
126.73 (2 Ar-CH), 126.65 (2 Ar-CH), 125.06 (Ar-CH), 117.30
(Ar-CH) ppm. IR (KBr disk): n ¼ 3433 (NH amide), 3063, 1898,
1674 (ketone carbonyl), 1635 (ketone carbonyl), 1589 (amide
carbonyl), 1534, 1496, 1419, 717 cmꢃ1. HRMS (ESI, negative
mode):m/z[MꢃHþ] 327.08375 (C20H11N2O3 requires 327.07642).
of different target compounds (30 mg/kg of C4, C6, C8, C10,
C12, C14, C16, C18, C20, nicotinic acid [NA] and 65 mg/kg
fenofibrate [FF]) dissolved in 6% DMSO/corn oil.
Blood sample collection and biochemical evaluation
Eighteen hours following drug administration, 2-mL blood
sample was collected by retro-orbital sinus puncture from
each rat under mild anesthesia using inhaled diethyl
ether [30]. Blood samples were collected in plain tubes and
serum was separated by centrifugation at 4000 rpm for 10 min
and stored at 4°C for biochemical evaluation.
Since TG level exceeded 400 mg/dL in this model, we cannot
rely on Friedewald formula in calculating LDL-C level [34, 35].
So serum TC, LDL-C, and TG were measured directly using
commercially available enzymatic colorimetric assay kits by
the automatic analyzer (Model Erba XL-300, Germany,
Mannheim, Germany) at Alzaytoonah University of Jordan.
Biological assays
Animals
Adult healthy male Wistar albino rats weighing 180–200 g
were procured from the Applied Science University animal
house. Rats were housed in polypropylene cages in room at a
temperature of 25°C with a 12-h light-dark cycle. Animals
were allowed to acclimatize for
7 days prior to any
experimentation, and they received water and a standard
rodent diet ad libitum. The study protocol was approved by
the ethical and graduates studies council at the Faculty of
Pharmacy/the University of Jordan (Ref. No. 80/2014/165).
Antioxidant activity
The DPPH radical scavenging assay was used to measure the
ability of the compounds to scavenge the stable free radical
of DPPH spectrophotometrically [36, 37]. DPPH radical
(1 mM) solution in methanol was prepared. Stock solutions
were prepared for all test compounds (2 mg/mL in DMSO/
methanol [1:1]). In 96-well plates, 15 mL of each compound
was added in triplicate, followed by the addition of 41.7 mL
of DPPH radical and 243.3 mL of methanol to complete the
volume up to 300 mL. A final concentration of 100 mg/mL of
each compound was obtained within each well. The blank
used was 15 mL of methanol and DMSO (1:1) with 41.7 mL of
DPPH radical solution and 243.3 mL methanol. After 30 min
of incubation in the dark at room temperature,
the absorbance was recorded at 517 nm using Biotek
ELX800 plate reader (USA). Lower absorbance values of
reaction mixture indicate higher free radical scavenging
activities. Ascorbic acid was used as the standard reference
at six different concentrations (0.8, 4.2, 8.3, 12.5, 16.7,
20.8 mg/mL) to calculate its half maximal inhibitory concen-
tration (IC50) value.
Induction of hyperlipidemia in experimental rats
Hyperlipidemia was induced experimentally using a single
intraperitoneal (i.p.) injection of 300 mg/kg Triton WR-1339
dissolved in water. Dose was selected based on literature
recommendations where Triton WR-1339 has been reported
to be used in doses ranging from 200 mg/kg to 400 mg/kg
intraperitoneally for hyperlipidemia induction [18–21, 27–29,
32, 33]. Triton WR-1339 group was compared with a control
group of six healthy rats receiving i.p. water injection only.
Acute toxicity study and selection of the experimental
dose
The selection of the experimental dose was based on previous
acute toxicity study that was conducted by the same research
team on related compounds “N-(benzoylphenyl)pyridine-
3-carboxamide derivatives” at doses from 200 mg/kg to
1000 mg/kg on Balb/c mice [16].
Due to similarity in structure between these compounds
and our carboxamide derivatives, a dose–response curve was
established for C6 compound at four different doses (20, 30,
50, and 100 mg/kg). This was conducted to select the dose
with significant efficacy to be used in the subsequent
experimentation.
The capability of scavenging the DPPH radical was
calculated by using the following formula:
% Inhibition ¼½ðAB ꢃ AAÞ=ABꢄ ꢅ 100
where AB is the absorption of the blank and AA is the
absorption of the test compounds. All the tests were
performed in triplicates and the results were averaged.
Anti-hyperlipidemic activity of N-(benzoylphenyl)-
pyridine-4-carboxamide derivatives
Statistical analysis
The experimental rats were randomly divided into 13 groups
(each consisting of six rats). All groups except the control
group (CG) were injected intarperitonially with 300 mg/kg
Triton WR-1339 dissolved in water, while rats in control group
received i.p. water injection.
Animals in the CG and the hyperlipidemic group (HG) were
gavaged with 6% DMSO/corn oil, while animals in the
remaining 11 groups received an intragastric administration
All data were entered and analyzed using SPSS© 19 (SPSS, Inc.,
Chicago, IL, USA) to evaluate the potential antihyperlipidemic
effect of the studied compounds. 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.
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Regardingtheantioxidantexperiment,alldatawereenteredin
Excel to calculate the DPPH radical scavenging effect for each
compound. XY plot was constructed using GraphPad Prism-5
between concentration of ascorbic acid in mg/mL (X axis) and %
DPPH radical inhibition (Y axis), then non-linear regression
analysis was used to calculate IC50 of ascorbic acid.
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