Novel Phenoxazinones as potent agonist of PPAR-α: Design, synthesis, molecular docking and in vivo studies

Article abstract of DOI:10.1186/s12944-018-0764-y

Background: The use of statin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for the treatment of dyslipidemia has been associated with dose limiting hepatoxicity, mytotoxicity and tolerability due to myalgias thereby necessitating the synthesis of new drug candidates for the treatment of lipid disorder. Methods: The reaction of appropriate benzenesulphonamide with substituted phenoxazinone in the presence of phenylboronic acid gave the targeted compounds. The molecular docking study were carried out using autodock tool against peroxisome proliferator activated receptor alpha. The in vivo lipid profile were assayed using conventional methods. The kidney and liver function test were carried out to assess the effect of the derivatives on the organs. The LD₅₀ of the most active derivatives were determined using mice. Results: The targeted compounds were successfully synthesized in excellent yields and characterized using spectroscopic techniques. The results of the molecular docking experiment showed that they were good stimulant of peroxisome proliferator activated receptor alpha. Compound 9f showed activity at Ki of 2.8 nM and binding energy of 12.6 kcal/mol. All the compounds tested reduced triglyceride, total cholesterol, low density lipoprotein cholesterol and very low density lipoprotein cholesterol level in the mice model. Some of the reported compounds also increased high density lipoprotein cholesterol level in the mice. The compounds did not have appreciable effect on the kidney and liver of the mice used. The LD₅₀ showed that the novel compounds have improved toxicity profile. Conclusion: The synthesis of fifteen new derivatives of carboxamides bearing phenoxazinone and sulphonamide were successful. The compounds possessed comparable activity to gemfibrozil. The reported compounds had better toxicity profile than gemfibrozil and could serve as a replacement for the statins and fibrate class of lipid agents.

Full text of DOI:10.1186/s12944-018-0764-y

Ugwu et al. Lipids in Health and Disease (2018) 17:120
https://doi.org/10.1186/s12944-018-0764-y
RESEARCH
Open Access
Novel Phenoxazinones as potent agonist of
PPAR-α: design, synthesis, molecular
docking and in vivo studies
1
,2*
1
2
3
David I. Ugwu , Uchechukwu C. Okoro , Narendra K. Mishra and Sunday N. Okafor
Abstract
Background: The use of statin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for the treatment of
dyslipidemia has been associated with dose limiting hepatoxicity, mytotoxicity and tolerability due to myalgias
thereby necessitating the synthesis of new drug candidates for the treatment of lipid disorder.
Methods: The reaction of appropriate benzenesulphonamide with substituted phenoxazinone in the presence of
phenylboronic acid gave the targeted compounds. The molecular docking study were carried out using autodock
tool against peroxisome proliferator activated receptor alpha. The in vivo lipid profile were assayed using
conventional methods. The kidney and liver function test were carried out to assess the effect of the derivatives on
the organs. The LD50 of the most active derivatives were determined using mice.
Results: The targeted compounds were successfully synthesized in excellent yields and characterized using
spectroscopic techniques. The results of the molecular docking experiment showed that they were good stimulant
of peroxisome proliferator activated receptor alpha. Compound 9f showed activity at Ki of 2.8 nM and binding
energy of 12.6 kcal/mol. All the compounds tested reduced triglyceride, total cholesterol, low density lipoprotein
cholesterol and very low density lipoprotein cholesterol level in the mice model. Some of the reported compounds
also increased high density lipoprotein cholesterol level in the mice. The compounds did not have appreciable
effect on the kidney and liver of the mice used. The LD50 showed that the novel compounds have improved
toxicity profile.
Conclusion: The synthesis of fifteen new derivatives of carboxamides bearing phenoxazinone and sulphonamide
were successful. The compounds possessed comparable activity to gemfibrozil. The reported compounds had
better toxicity profile than gemfibrozil and could serve as a replacement for the statins and fibrate class of lipid
agents.
Keywords: Carboxamide, Cardiac disease, Cholesterol, Molecular docking, Sulphonamides, Synthesis
Background
the deaths in developed countries. High levels of total
High level of low density lipoprotein cholesterol (LDLC), cholesterol (TC), triglycerides (TG), and low density
triglycerides and low levels of high density lipoprotein lipoprotein-cholesterol (LDLC) have been implicated as
cholesterol (HDLC) are considered to be among the pre- contributing risk factors in progress of CHD and athero-
dominant risk factor in coronary heart disease [1]. sclerosis [3]. Accordingly, different lipid lowering drugs
Hypercholesterolemia is a common disorder which is have been applied for the treatment of hypercholesterol-
known as main cause of coronary heart disease (CHD) emia [4]. Statins are the effective hypocholesterolemic drugs
[
2]. This disease is recognized as cause of the most of which competitively inhibit the activity of 3-hydroxy-3-
methylglutaryl-coenzyme A (HMG-CoA) reductase, the
*
Correspondence: izuchukwu.ugwu@unn.edu.ng
rate limiting enzyme in cholesterol biosynthesis [5]. Heart
disease is a major cause of mortality and morbidity in the
world. The main risk factors, such as family history and age
1
Medicinal Chemistry unit, Department of Pure and Industrial Chemistry,
University of Nigeria, Nsukka 410002, Nigeria
2
Department of Chemistry, Indian Institute of Technology, Kanpur, India
Full list of author information is available at the end of the article
©
The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 2 of 13
cannot be changed. However, other risk factors including, and reduce the risk of coronary heart disease in patients
obesity, diabetes, smoking, diet, high blood pressure, total with low HDLC levels [15]. Clofibrate was the first fibrate
cholesterol (TC), low-density lipoprotein cholesterol to be developed. Researches targeted on development of
(
LDLC), and low levels of high-density lipoprotein choles- lipid lowering agent were revolutionized following the dis-
terol (HDLC) can be changed or treated [6, 7]. These risk covery of the other fibrates such as ciprofibrate, bezafi-
factors can be controlled and corrected by diet, exercise, brate, fenofibrate and gemfibrozil (Fig. 2). However, the
hypolipidemic drugs and herbal medicine [8]. Currently, reported proliferation of peroxisome leading to hepato-
the most common method to treat dyslipidemia is the use megaly and formation of tumour in the liver of rodents as-
of statins typified by simvastatin and atorvastatin (Fig. 1) sociated with fibrates is worrisome [16–20].
which are HMG-CoA reductase inhibitors. The widespread
To address the problems of toxicities associated with
clinical use of the statins is accompanied by potential dose- statins and fibrates, ezetimibe was launched in 2002 as a
limiting hepatoxicity and mytotoxicity [9]. Cerivastatin, one new drug which inhibits cholesterol absorption in the in-
of the second generation statins was withdrawn from the testine without affecting the absorption of triglycerides
world market in 2001 due to its adverse effects [10]. The [21]. Ezetimibe inhibits free cholesterol uptake from the
work of Ference et al. on Mendelian randomized trials fur- intestine and as such reduces plasma LDLC levels. How-
ther highlights the need for new drugs for the treatment of ever, the activity of ezetimibe was not comparable to the
coronary heart disease linked to cholesterol [11].
statins and fibrates, thereby necessitating the develop-
The fibrate classes of lipid lowering drugs typified by ment of new class of lipid lowering agent that will retain
gemfibrozil (Fig. 2) are selective activators of α-isotype of the high activity of fibrates and statins and an improved
the receptors peroxisome proliferator activated receptor toxicity profile.
(PPAR) [12, 13]. PPAR-α agonists lower triglyceride levels
Although there is no report to our knowledge on sul-
and increase HDLC level in hyperlipidemic patients [14] phonamides used as lipid lowering agents, the reported
H C
3
F
CH3
O
O
NH
O
H C
CH3
3
N
OH
O
H C
3
OH
OH
O
H C
HO
O
3
CH3
atorvastatin
simvastatin
H C
3
H C
CH3
3
O
H C
3
H C
3
O
N
O
OH
CH3
O
OH
OH
CH3
HO
F
OH
O
H C
3
HO
OH
pravastatin
cerivastatin
Fig. 1 Statins

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 3 of 13
Cl
CH3
O
CH3
OH
H
N
O
Cl
H C
3
CH3
O
O
Cl
O
CH3
HO
O
O
H C
ciprofibrate
3
O
bezafibrate
H C
3
CH3
H C
O
3
O
Cl
CH3
O
H C
O
3
CH3
HO
Cl
H C
H C
3
CH3
O
3
clofibrate
O
fenfibrate
gemfibrozil
Fig. 2 Fibrates
wide biological activities of sulphonamide aroused the using electrospray ionization-time of flight (ESI-TOF)
curiosity in synthesizing novel sulphonamides and test- mass spectrometer (Aerodyne Research Inc. USA), so-
ing them for lipid lowering activity. Again, most of the dium formate was used as the calibrant. All experiments
statins and fibrate class of lipid lowering agents contains were carried out at Prof. Sandeep Verma’s Laboratory,
a carboxamide and/or carboxylic acid group which un- department of Chemistry, Indian Institute of Technol-
derscores the importance of carboxamide functionality ogy, Kanpur. Melting points were determined using
in lipid lowering agents.
digital melting point apparatus (Stuart, SMP20) and
We therefore report herein the first class of phenoxa- were uncorrected. The biological studies were carried
zinone derivatives bearing sulphonamide and carboxa- out at the Department of Biochemistry, University of
mide functionalities as potential lipid lowering agents. Nigeria, Nsukka under the guidance of Mr. OGB.
This research exploited the relative stability of sulphona-
mides, their wide pharmacological applications and ease Synthesis of substituted benzenesulphonamides (3a-p)
of synthesis in combination with the reported lipid low- Sodium carbonate (Na CO , 1.590 g, 15 mmol) was added
2
3
ering activity of carboxamides. The need for new lipid to a solution of amino acids (2a-h, 12.5 mmol) in water
lowering agents with comparable activity with the statins (15 mL) with continuous stirring until all the solutes had
and fibrates, ease of synthesis and low toxicity profile dissolved. The solution was cooled to − 5 °C and the ap-
motivated this research.
propriate benzenesulphonyl chloride (1a-c, 15 mmol) was
added in four portions over a period of 1 h. The slurry
was further stirred at room temperature for about 4 h.
The progress of the reaction was monitored using TLC
Methods
Instrumentation
All reactions requiring inert atmosphere were carried (MeOH/DCM, 1:9). Upon completion, the mixture was
out under nitrogen atmosphere. Drying of solvents was acidified using 20% aqueous hydrochloric acid to pH 2.
achieved using molecular sieve for 48 h. All reagents The crystals was filtered via suction and washed with
were purchased from commercial suppliers, Aldrich, pH 2.2 buffer. The pure products (3a-x) were dried over
Merck, Fluka, Avra, SD fine and Alfa Aesar. Thin layer self-indicating fused silica gel in a desiccator [22].
chromatography was carried out using silica plates pur-
chased from Avra. The plates were visualized under UV Synthesis of N-benzoyl derivatives of
light (popular India). FT-IR spectroscopy of the com- Benzenesulphonamides (5a-f and i-m)
pounds was run in PerkinElmer Spectrum version 10.03. Appropriate benzenesulphonamides (3a-f and i-m, 1.
06 and the bands presented in wavenumber. Proton and 0 mmol) was dissolved in NaOH (10%, 10 mL) in a
carbon-13 NMR spectroscopy were run in DMSOd and 50 mL round bottom flask. Benzoyl chloride (4, 1.
6
CD OD, unless otherwise stated on either Jeol 500 MHz 1 mmol, 0.2 mL) was transferred into the solution of ap-
3
or 400 MHz. The chemical shifts were reported in part propriate benzenesulphonamides and stirred at room
per million with reference to tetramethylsilane. High temperature. The reaction progress was monitored by
Resolution Mass spectrometry (HRMS) was carried out TLC (3% MeOH/CH Cl ) to the disappearance of the
2
2

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 4 of 13
benzenesulphonamides spot. Upon completion, the solu- reaction mixture was filtered and cooled in ice overnight
tion was transferred into a beaker containing crushed ice and filtered to obtain a solid. Analytical sample was ob-
and then acidified to pH of 3 with concentrated hydro- tained by column chromatography using benzene as the
chloric acid. The solid was collected via suction filtration eluting solvent followed by recrystallization to obtain the
and transferred into a beaker containing CCl (10 mL) target compound.
4
and covered with watch glass boiled for 10 min. The
mixture was allowed to cool slightly and then filtered.
The products (5a-f and i-m) obtained were washed with
Phenylboronic acid catalysed Amidation reaction of
1
0–20 mL of CCl and dried over fused self-indicating carboxylic acid and 4-amino-11-chloro-2-sulfanyl-10H-12-
4
silica gel in a dessicator [22].
oxa-1,3,5-triazatetraphen-10-one derivatives (9a-o)
N-benzoylated-substituted-benzenesulphonamides
(1 mmol), was dissolved in dry toluene (50 mL). Phenyl-
boronic acid (0.1 mmol) was added to the above solution
Synthesis of 4-amino-11-chloro-2-sulfanyl-10H-12-oxa-
1
,3,5-triazatetraphen-10-one (8)
5
,6-Diamino-2-sulphanylpyrimidin-4-ol (6, 2.47 g), 2,3- and then 4-amino-11-chloro-2-sulphanyl-10H-12-oxa-
dichloro-1,4-dihydronaphthalen-1,4-dione (7, 2.27 g), so- 1,3,5-triazatetraphen-10-one (1 mmol) was added. The
dium acetate (0.36 g) and benzene (60 mL) mixed with mixture was dissolved using ultrasonication and then
dimethyl formamide (30 mL) were charged into 250 mL refluxed for an average of 10 h using Dean-Stark appar-
two-necked round bottomed flask fitted with short mag- atus for azeotropic removal of water. Upon completion
netic stirring bar and a reflux condenser. The mixture of reaction (TLC monitored), the mixture was allowed
was stirred while heating on a water bath at 70–75 °C to cool to ambient temperature and the amides precipi-
for 8 h. The colour of the reaction mixture changed tated via the addition of 30 mL of n-hexane. The crystals
from light brownish green to yellowish red and intense were washed with n-hexane after filtration using suction.
red as the reaction progressed. At the end of 8 h, the The products were dried over fused silica gel.
O
HO
O
O
N
N
Cl
O
O
N
N
Cl
S
NH
O
O
O
S
NH
O
O
N
N
N
N
SH
HS
O
HC3
O
N
N
HS
9
h
HC3
O
9
g
N
SH
S
NH
O
8
8
O
N
Cl
O
O
O
N
N
HC3
O
N
8
9a
S
NH
O
O
N
Cl
O
O
HC3
CH3
8
HC3
O
N
R
SH
S
9
f
8
O
O
HO
O
N
N
O
O
HC3
N
5
a-h
S
NH
O
SH
8
O
N
Cl
O
O
8
O
N
N
HC3
O
N
8
S
NH
O
9
b
O
HC3
N
Cl
O
SH
HC3
O
CH3
O
N
N
O
N
S
NH
O
9
e
SH
O
N
Cl
O
O
N
N
HC3
O
N
NH
O
S
NH
O
O
CH3
N
Cl
O
HC3
CH3
9c
9
d
Fig. 3 4-methylbenzenesulphonamides derivatives (9a-h)

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 5 of 13
Molecular docking experiment
Preparation of ligands
Molecular docking experiments
In our effort to identify potential lipid lowering lead(s)
ACD/ChemSketch 2015 (Ref: ACD/Structure Elucidator, among compounds 9a–o, we carried out docking calcu-
version 15.01, Advanced Chemistry Development, Inc., lations using Autodock v4.0.1 into the 3D structure of
Toronto, ON, Canada, www.acdlabs.com, 2015.) was the catalytic sites of 2p54,. The Gasteiger charge calcula-
used to draw the structures of compounds 9a-o (Figs. 3 tion method was used and partial charges were added to
and 4) and also convert them to 3D formats.
the ligand atoms prior to docking. The Lamarckian gen-
etic algorithm (LGA), which is available in Auto Dock,
was employed [23]. Finally, Auto Dock was used to cal-
Preparation of protein targets
The 3D structure of the peroxisome proliferator-activated culate the binding free energy of a given inhibitor con-
receptor alpha (PPAR-α, pdb code 2p54) was retrieved formation in the macromolecular structure.
from the RCSB Protein Data Bank (PDB) (www.rcsb.org/
pdb/home/home.do). All bound ligands, cofactors, and
water molecules were removed from the proteins using In vivo lipid lowering activities determination
Discovery Studio Visualizer v16. 1.0. 15,350.
Male mice were kept on a 12 h light/dark cycle at a
All file conversions required for the docking study temperature of 22 ± 1 °C. After acclimatizing for one week
were performed using the open source chemical toolbox in a cage, animals were randomly divided into three
Open Babel version 2.3.2 (www.openbabel.org).
groups (n = 6):
O
HO
O
O
N
O
Cl
N
O
S
NH
O
O
N
Cl
N
O
S
NH
O
N
N
O
SH
O
N
N
HS
O
N
N
O
N
9
o
HS
S
NH
O
9
n
8
8
O
N
Cl
O
SH
8
9i
O
O
N
N
O
O
N
S
NH
CH3
O
8
O
N
R
O
O
HC3
N
Cl
O
S
SH
8
O
HO
O
O
N
N
5i-p
O
N
9m
8
S
NH
O
O
8
N
Cl
O
SH
O
O
N
N
SH
9
j
O
N
O
S
NH
O
O
N
N
O
HC3
O
N
Cl
O
N
S
NH
O
CH3
O
N
Cl
9
l
NH
O
9
k
Fig. 4 benzenesulphonamides derivatives (9i-o)

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 6 of 13
1
R
1
O
R
R
O
O
OH
O
1
Cl
O
R
O
S
S
N
H
OH
2
H N
COOH
o
2a-f
CO
S
OH
N
2
g-h
O
o
i. Na
2
3
, 0
C
NH
i. Na
2
CO
3
, 0
C
O
R
ii. HCl
O
R
ii. HCl
3a-f, 3i-m
1
1
R
COCl
3g-h, 3n-o
NH2
N
O
4
N
ii. HCl
O
R
HS
N
O
O
8
S
Cl
i.phenylboronic acid (15 mol%)
N
O
toluene, reflux,
ii. n-hexane
12 h
O
1
R
OH
5a-f, 5i-m
1
R
O
8
phenylboronic acid (15 mol%)
toluene, reflux, 12 h
ii. n-hexane
O
N
N
Cl
O
S
NH
O
O
SH
O
N
N
O
N
N
O
N
HS
S
NH
O
R
O
9g-h, 9n-o
CH
3
N
Cl
O
H C
3
9a-f, 9i-m
R= CH in 3a-h, 5a-f,and 9a-h and H in 3i-o, 5i-mand 9i-o
3
1
R = H in a and i; C H in b and j; C H in c and k; C H in d and l;
7
7
9
7
5 11
1
R = C H in e; C H9 in f and m; OH in g and n; H in h and o
5
11
4
Scheme 1 Synthesis of the target compounds (9a-o)
Group 1: hypercholesterolemic group (received chow
2% w/w cholesterol + 0.5% w/w cholic acid) [24].
High density lipoprotein cholesterol (HDLC), triglycer-
ide (TG) and total cholesterol (TC) levels were at the
+
Group 2: carboxamide (received hypercholesterolemic baseline prior to the experiment and were similar be-
diet + 0.001% w/w carboxamide).
Group 3: control (chow only).
tween animals. New carboxamides (9a-o) were dissolved
in physiological saline and mixed with animal’s diet. An
equivalent amount of saline was added to the diet of hy-
percholesterolemic and control groups. At the end of
four weeks, mice were fasted for 12 h, and then
Fig. 5 binding mode of 2p54_9f
Fig. 6 binding mode of 2p54_9 k

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 7 of 13
anesthetized using CO and sacrificed. Blood was then Determination of triacylglycerol concentration
2
collected from the heart and serum was separated by The concentration of triacylglycerol (TG) was determined
centrifugation at 3000 g for 10 min. All the dead mice by the method of Allain et al. [24] using Randox kit.
were disposed in bio-safety containers in accordance
with local standard protocols.
Liver function tests (LFTs)
The liver function tests were carried out using aspartate
aminotransferase (AST), alanine transaminase (ALT)
and alkaline phosphatase (ALP) as biomarkers employ-
Determination of Total cholesterol concentration
Total cholesterol concentration was determined by the ing the methods of Reitman and Franke 1957 [28].
method of Allain et al. [25] using Randox kit.
Renal or kidney function test
Kidney function tests were carried out on the two most
Determination of high-density lipoproteins (HDL)-cholesterol
concentration
active derivatives (9f and 9 k) using serum urea, creatin-
ine and uric acid as biomarkers. The method reported
The concentration of high-density lipoprotein (HDL) by Kaplan and Teng 1982 [29] was used in the determin-
was determined by the method of Albers et al. [26] ation of urea and creatinine while that reported by
using Randox kit.
Ochie and Kolhattar 2000, was used for the determin-
ation of uric acid [30].
Determination of low-density lipoprotein (LDL)–cholesterol
concentration
Determination of LD50 for the active anti-inflammatory
compounds
The concentration of Low density lipoprotein-Cholesterol Male mice were divided into various groups and test
was determined based on the Polyvinylsulphate method compounds were administered in various doses intraperi-
described by Demacker et al. [27].
toneally. Following treatments, the animals were
Fig. 7 Types of interaction between the 9 k and 2P54

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 8 of 13
13
observed for up to 4 h continuously and were then kept NMR, C NMR and MS. Worthy of mention in the
−
1
under observation for 72 h. All behavioral changes and FTIR is the absorptions between 3405 and 3312 cm
−
1
deaths during the observation periods were recorded. due to NH stretch, 2559–2468 cm due to SH stretch,
due to C=O of ketone, the bands
culated, converted to probits and the LD50 (μM/kg) between 1637 and 1619 cm due to C=N stretch, the
−
1
The percentage of death at each dose level was then cal- 1763–1714 cm
−
1
−
1
values were calculated [31]. To observe the health status band between 756 and 743 cm due to C-Cl stretch.
of the mice, they were monitored 4 times a day. Humane These bands are indicative of successful coupling of
endpoints were used when the animal shows sign of 4-amino-11-chloro-2-sulfanyl-10H-12-oxa-1,3,5-triaza-
weight loss, weakness accompanied by inability to get tetraphen-10-one. In the proton NMR, the peak be-
food, complete anorexia and convulsion for 24 h. For the tween 11.9–11.8 was indicative of the presence of SH
purpose of ameliorating the suffering of the dying mice, proton. In the carbon-13 NMR spectra of the 4-
CO euthanasia was applied. All the dead mice were dis- amino-11-chloro-2-sulfanyl-10H-12-oxa-1,3,5-triazate-
2
posed in bio-safety containers in accordance with local traphen-10-one Derivatives (9a-o), the appearance of
standard protocols. The mortality rate in each group was the ketonic carbonyl peak between 178.4–172.7, the
calculated according to the formula:
azomethine carbon peak between 170.0–151.1 ppm
Mortality rate (%) = (the number of dead mice/the seriously indicated the successful formation of the
number of mice in the group) × 100.
target molecules. The spectral analysis above in
addition to the molecular ion peaks showed that
there was successful coupling of the amino group of
4-amino-11-chloro-2-sulfanyl-10H-12-oxa-1,3,5-triazatetra-
phen-10-one with the carboxylic acid of the benzenesul-
Results and discussion
Synthesis of the target compounds
All the 4-amino-11-chloro-2-sulfanyl-10H-12-oxa-1,3,5- phonamides. The summaries of the 4-amino-11-chloro-
triazatetraphen-10-one derivatives (9a-o, Figs. 3 and 4) 2-sulfanyl-10H-12-oxa-1,3,5-triazatetraphen-10-one deriva-
1
showed similar pattern of absorption in the FTIR, H tives (9a-o) are presented in Scheme 1.
Fig. 8 distance of interaction between 2P54 and 9 k in Å

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 9 of 13
Molecular docking
into the binding cavity of the receptor, 2p54 resulting in
We undertook molecular docking studies to gain more the observed binding energies. Because of these interac-
insight into the binding interactions between these com- tions, 9f and 9 k can elicit its pharmacological actions on
pounds and the receptor (2p54).
the organisms as it competitively antagonizes the normal
From Table 2, all the compounds showed significant biochemical processes in the receptor. It can also be seen
binding affinities with the receptor used. The result re- from Table 2, as also indicated in Figs.7 and 8 that four
vealed that compound 9f showed higher binding affinities different chemical interactions were involved namely:
with the receptor than the other compounds assessed. hydrogen bonding, van der Waal, pi-sigma and pi-alkyl in-
+
The binding energy of 9f with 2p54 is given as − 12. teractions. Figs. 7 and 8 and a Ligplot (Figs. 9 and 10)
6
kcal/mol with an activity constant (Ki) of 2.8 nM. There- clearly suggest that a total of 17 active amino acid residues
fore, we carried out further study on 9f and 9 k to show of 2p54 interacted with different atoms/groups of
its binding modes in the receptor, 2p54 (Figs. 5 and 6) compound 9f and 9 k respectively. They are MET:355,
and to identify the different chemical interactions between SER:280, MET:330, THR:283, LEU:321, ILE:317, VAL:324,
9f and 9 k with different amino acid residues of the recep- MET:320, THR:279, VAL:332, TYR:334, GLY:335, MET:
tor (Figs. 7 and 8). The ligand, 9f and 9 k were well fitted 220, LEU:331, ASP:219, ALA:333 and CYS:276 for 2p54_
+
Fig. 9 Ligplot results for 2p54_9f complex, showing all the 17 amino acid residues of active pocket

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 10 of 13
+
Fig. 10 Ligplot results for 2p54_9 k complex, showing all the 17 amino acid residues of active pocket
9
f and ILE:354, LEU:344, MET:355, MET:330, ALA:333, Lipid lowering activities
VAL:332, MET:220, MET:320, LEU:321, THR:279, ILE: Cardiovascular disease (CVD) includes conditions such as
17, CYS:275, CYS:276, HIS:440, SER:280, PHE:318 and heart failure, congenital heart disease, coronary heart dis-
3
GLN:277 for 2p54_7k. Figure 11 shows the binding of ease, arrhythmia, stroke, heart valve disease and hyperten-
compound 9 k in the pocket of peroxisome proliferator sion. Many epidemiological studies have shown that a
activated receptor alpha (2p54).
direct correlation exists between lipid abnormalities
Table 1 Binding energy (ΔG - kcal/mol) of compounds with
target proteins (2p54)
Comp
ΔG (kcal/mol) 2P54
Ligand efficiency
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
−8.4
0.19
0.15
0.14
0.16
0.18
0.27
0.19
0.21
0.22
0.17
0.20
0.19
0.18
0.19
0.21
− 7.9
− 7.8
− 7.5
− 8.4
− 12.6
− 7.7
− 8.2
− 9.6
− 8.4
− 10.8
− 8.9
− 8.3
− 7.5
− 7.8
g
h
i
j
k
l
m
n
Fig. 11 9 k ligand inside the protein pockets showing regions of
H-Bond donors and acceptors
9o

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Table 2 in vivo lipid lowering activities
Page 11 of 13
S/N
TG (mg/dL)
148.2
156.3
153.6
153.6
165.6
144.6
157.7
157.3
165.9
149.9
146.4
159.3
157.3
152.3
157.7
145.2
173.2
TC (mg/dL)
179.7
176.2
178.3
153.5
171.2
173.1
166.6
179.7
169.8
185.3
163.8
159.9
171.2
179.7
166.6
174.6
206.3
HDLC (mg/dL)
23.9
LDLC (mg/dL)
126.2
99.8
VLDLC (mg/dL)
29.6
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
45.2
31.3
30.9
116.7
22.6
30.7
100.1
52.6
30.7
85.5
33.1
112.8
85.6
31.4
28.9
g
49.4
31.6
h
55.2
93.0
31.5
i
j
23.8
112.8
136.1
2.1
33.2
19.2
29.9
k
132.4
45.3
29.3
l
82.7
31.9
m
73.9
65.9
31.5
n
o
65.0
84.2
30.5
85.6
49.4
31.6
gemfibrozil
control
110.1
70.7
35.5
29.1
101.0
34.6
particularly elevated levels of total cholesterol, triglycer-
ides and low density lipoprotein cholesterol, and the risk
of coronary vascular disease [2]. The in vivo lipid lowering
activities (Table 1) showed that all the compounds pre-
pared reduced the triglyceride (TG) and total cholesterol
(C) content after 1 week. In some case, an increment in
high density lipoprotein cholesterol (HDLC) were ob-
served whereas there were observed decreased in the
HDLC level in some cases as against the expectation of a
good heart drug. The very low density lipoprotein choles-
terol (VLDLC) values showed total decrease when com-
pared with the control, although in some cases, the
decrease was not significantly different. Some of the deriv-
atives showed fascinating features of good heart drugs.
Compounds 9a, 9f, 9j and 9 k showed marked decrease
in the triglycerides contents from 176.2 mg/dL in the con-
trol to 148.2, 144.6, 149.9 and 146.4 mg/dL respectively.
The total cholesterol decreased from 206.3 mg/dL in the
control to 176.7, 173.1, 185.3 and 163.8 mg/dL respect-
ively for compounds 9a, 9f, 9j and 9 k. Like the results of
triglycerides and total cholesterol, there was marked de-
crease in the low density lipoprotein cholesterol and very
low density lipoprotein cholesterol contents from 101.0
and 34.6 mg/dL in the control to 31.4 and 28.9 mg/dL for
compound 9f and 2.1 and 29.3 mg/dL for compound 9 k
respectively. Compounds 9a and 9j showed an increased
LDLC value thereby marking them bad candidate for
treatment of heart disease. As was the case for LDLC, only
compounds 9f and 9 k showed marked increase in the
HDLC level.
Table 3 Percentages of lipid profile of the new derivatives
S/N
TG
TC
HDLC
–
LDLC
−24.9
1.3
VLDLC
14.4
9.8
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
14.4
9.7
12.9
14.6
13.6
25.6
17.0
16.1
13.3
12.9
17.7
10.2
20.6
22.5
17.0
12.9
19.3
15.4
–
11.3
11.3
4.4
–
−15.5
77.6
15.3
68.9
25.1
7.9
11.3
11.3
4.4
41.7
–
16.5
10.5
9.2
59.6
1.9
–
16.5
10.5
9.2
g
h
i
j
4.2
–
−11.6
−34.7
97.9
18.2
34.8
16.7
51.1
64.9
4.2
13.4
15.5
8.0
–
13.4
15.5
8.0
k
87.4
–
l
m
9.2
4.6
–
9.2
n
o
9.2
12.1
8.9
8.9
21.2
55.8
gemfibrozil
16.1
16.1
TG implies triglyceride; TC implies total cholesterol; HDLC imply high density
lipoprotein cholesterol; LDLC implies low density lipoprotein cholesterol;
VLDLC implies very low density lipoprotein cholesterol. The percentage
triglyceride were calculated using TG (%) = [TG (control)-TG (sample)/TG
(
control)]X 100. This was also used for percentage total cholesterol, high
density lipoprotein cholesterol, low density lipoprotein and very low density
lipoprotein cholesterol

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 12 of 13
Table 4 LFT, KFT and LD50 results of the most active derivatives
Biomarker
AST (IU/L)
41 ± 2.00
42 ± 1.83
40 ± 1.65
ALT (IU/L)
18.4 ± 0.80
18.9 ± 0.42
20.0 ± 0.75
ALP (IU/L)
0.8 ± 0.016
0.7 ± 0.020
0.8 ± 0.014
Urea (IU/L)
Creatinine (IU/L)
1.6 ± 0.33
Uric acid (IU/L)
6.6 ± 0.02
LD50 μM/kg
8.0
9
9
f
17.7 ± 0.008
18.1 ± 0.002
19.1 ± 0.008
k
1.4 ± 0.11
6.6 ± 0.03
7.6
control
1.5 ± 0.05
6.4 ± 0.005
8.8 [32]
All data were expressed as mean ± SE (standard error). AST means aspartate aminotransferase, ALT means alanine transaminase and ALP means
alkaline phosphatase
The percentage lipid lowering activities of the new car- gemfibrozil. In general, the KFT, LFT and LD50 values of
boxamides were presented in Table 2. Compounds 9a, the reported lipid lowering agents suggest that they are
9f and 9 k showed percentage reduction in triglyceride relatively safe toxicologically.
level of 17.3, 16.5 and 15.5% respectively which is com-
parable with gemfibrozil (16.1%, Table 3). Compounds Conclusion
9
d, 9e, 9f, 9i, 9o, 9 l, 9 m and 9 k reduced the total Fifteen new derivatives of phenoxazinone bearing
cholesterol level at 25.6, 17.0, 16.1, 17.7, 19.3, 22.5, 17.0, sulphonamide and carboxamide pharmacophores have
0.6% respectively which were comparable with percent- been described in this work. All the compounds showed
age reduction of 15.7% of gemfibrozil. Compounds 9d, good binding with PPAR alpha (2p54) with binding ener-
f and 9 k increased the high density lipoprotein gies ranging from − 7.5 to 12.6 kcal/mol. Compound 9f
2
9
cholesterol level in the range of 41.7, 59.6 and 87.4% showed Ki of 2.8 nM and binding energy of 12.6 kcal/mol.
respectively comparable to 55.8% reduction from All the derivatives showed lipid lowering activity in the in
gemfibrozil. Compound 9 k had 97.9% reduction of vivo experiment but only two derivatives 9f and 9 k
the low density lipoprotein cholesterol level against showed characteristics of a lipid lowering agent compar-
gemfibrozil (64.90%). Compound 9f reduced very low able to statins and fibrates. The results of the in vivo ex-
density lipoprotein cholesterol level at 16.5% which periment were in agreement with the in silico experiment
was comparable with gemfibrozil (16.1%). The pro- as compounds 9f and 9 k were found to be the most ac-
line derivatives 9f, 9 h, 9n and 9o were the more tive derivatives in both experiment. Although compound
active than the acyclic derivatives. The structure- 9 k was more active than 9f in the in vivo test contrary to
activity relationship study showed that methyl group the result obtained from the in silico experiment, this ob-
para to the benzenesulphonamide is significant in servation is in agreement with the experimental error of
the lipid lowering activity. In exception of trypto- docking procedure. The structure-activity relationship
phan derivatives (9c and 9 k), the para substituted study showed that the 4-methylbenzenesulphonamide de-
derivatives showed better lipid lowering activity than rivatives were better lipid lowering agent than the benze-
the derivatives without substitution (9i-o). This find- nesulphonamide derivatives in exception of compound
ing is also supported by the molecular docking ex- 7 k which was more active than its corresponding 4-
periment which showed an additional interaction methylbenzenesulphonamide derivative 7c. The kidney
between the methyl group and an amino acid in the function tests, liver function tests and LD50 showed that
protein (2p54) as shown in Fig. 9. Among the trypto- the compounds were safe as there was no remarkable
phan derivatives 9 k and 9c, the additional presence of change in the biomarkers of the mice fed with the com-
NH group is suggested to have affected the lipid lowering pounds and the control.
activity possibly by positioning compound 9 k for better
binding in a preferred orientation than the para
Abbreviations
ALP : Alkaline phosphatase; ALT : Alanine transaminase; AST : Aspartate
substituted 9c which is in agreement with the molecular
docking results.
aminotransferase; CHD : Coronary heart disease; CVD : Cardiovascular disease;
ESI-TOF : Electrospray ionization-time of flight; FTIR : Fourier Transform
Infrared; HDLC : High density lipoprotein cholesterol; HMG-CoA : 3-hydroxy-
3-methylglutaryl-coenzyme A; HRMS : High resolution mass spectrometer;
IIT : Indian Institute of Technology; KFT : Kidney function test; LD50 : Lethal
Dose at 50%; LDLC : Low density lipoprotein cholesterol; LFT : Liver Function
test; LGA : Lamarckian genetic algorithm; NMR : Nuclear magnetic resonance;
PDB : Protein data bank; PPAR-α : Peroxisome proliferator-activated receptor
alpha; RCSB : Royal Chemical Society bank; TC : Total Cholesterol; TG
Toxicity analysis
The result obtained from kidney function test (KFT) and
liver function test (LFT) as presented in Table 4 showed
that at 500 mg/kg administration, the new derivatives
showed no significant change in the concentration of the
biomarkers on comparison with the control. The LD50
values (Table 4) of the two most active carboxamide de-
rivatives (7f and 7 k) showed that they the new com-
pounds had better toxicity profile when compared to
:
Triglycerides; VLDLC : Very low density lipoprotein cholesterol
Acknowledgements
Authors wish to acknowledge Indian Institute of Technology, Kanpur for
providing the equipment and instruments used in the synthesis and
characterization. David Ugwu wish to acknowledge the University of Nigeria
for the study leave that allowed me visit IIT, Kanpur for the experiment.
Authors wish to thank the Department of Biochemistry University of Nigeria

Ugwu et al. Lipids in Health and Disease (2018) 17:120
Page 13 of 13
and Biological Science and Bioengineering, IIT, Kanpur for the in silico and in
vivo lipid lowering activity analysis.
18. Gray TJ, Beamand JA, Lake BG, Foster JR, Gangolli SD. Toxicol Letter. 1982;
10:273–9.
1
9. Reddy JK, Krishnakantha TP. Science. 1975;190:787–9.
Funding
20. Leighton F, Coloma L, Koenig C. J Cell Biol. 1975;67:281–309.
21. Rao MS, Subbarao V, Reddy JK, Natl Cancer J. Inst. 1986;77:951–6.
There is no funding available for this publication.
2
2. Ugwu DI, Okoro UC, Ukoha PO, Okafor S, Ibezim A, Kumar NM. Eur J Med
Availability of data and materials
Chem. 2017;135:349–69.
All data relating to the manuscript are provided in the manuscript and
supporting document and are available without constraint.
23. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ.
J Comput Chem. 1998;19(14):1639–62.
2
4. Mohammadi A, Norouzian P, Jamshidi M, Najafi N, Oshaghi EA. J Adv Chem.
Authors’ contributions
2013;5:784–91.
The design, synthesis and biological evaluation was carried out by DIU. The
project design and manuscript writing was supervised by UCO. The
molecular docking studies and manuscript editing was carried out by SNO.
The spectral characterization and synthetic materials were provided by NKM.
The writing of the manuscript was done by DIU. The implementation of the
project was monitored by UCO. All authors read and approved the final
manuscript.
25. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Clin Chem. 1974;20:470–5.
26. Albers JJ, Warnick GR, Chenng MC. Lipids. 1978;13:926–32.
27. Demacker PN, Hijmans AG, Brennink BJ, Jansen BP, Vantlaar A. Clin Chem.
1984;30:1797–800.
28. Reitman S, Frankel S. Am J Clin Pathol. 1957;28:56–63.
29. Kaplan A, Teng LL. In selected methods of clinical chemistry, Ed. By W.R.
Faulkner and S. Meites, Washington: AACC. 1982;9:357–63.
3
0. Ochei J, Kolhatkar A. Medical Laboratory Science. Theory and Practice. 2nd
Ethics approval
Edition. New Delhi: Tata Mcgraw-Hill; 2000. p. 331–49.
The use and care for animal used in the study was approved by the
University of Nigeria ethical committee.
31. Ghosh M. Fundamentals of experimental pharmacology scientific book
agency. Calcutta. 1984:153–8.
3
2. Pfizer Material Safety Data Sheet (2006) 4 www.pfizer.com/files/products/
material_ safety_data/500.pdf assessed on 09/08/2017. Accessed 9 Aug 2017.
Consent for publication
All the authors approved the submission of the manuscript to Lipid in
Health and Disease for onward publication after peer-review processes.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Medicinal Chemistry unit, Department of Pure and Industrial Chemistry,
2
University of Nigeria, Nsukka 410002, Nigeria. Department of Chemistry,
Indian Institute of Technology, Kanpur, India. Department of Pharmaceutical
3
and Medicinal Chemistry, University of Nigeria, Nsukka, Nigeria.
Received: 3 December 2017 Accepted: 30 April 2018
References
1
.
Sarwar N, Danesh J, Eiriksdottir G, Sigurddson G, Wareham N, Bingham S,
Boekholdt SM, Khaw KT, Gudnason V. Circulation. 2007;115:450–8.
Stone GW, Maehara A, Lansky AJ, de Bruyne B, Cristea E, Mintz GS, Mehran
R, McPherson J, Farhat N, Marso SP, Parise H, Temphin B, White R, Zhang Z,
Serruys PW. N Engl J Med. 2011;364:226–35.
2
.
3.
4.
5.
Steinberg D. J Lipid Res. 2006;47:1339–51.
Pahan K. Cell Mol Life Sci. 2006;63:1165–78.
Jick H, Zornberg GL, Jick SS, Seshadri S, Drachman DA. Lancet. 2000;356:
1627–31.
6
7
.
.
Anderson KM, Odell PM, Wilson PW, Kannel WB. Am Heart J. 1991;121:293–8.
Malik S, Wong ND, Franklin SS, Kamath TV, L'Italien GJ, Pio JR. Circulation.
2004;110:1245–450.
8
9
1
1
.
.
Malloy MJ, Kane JP. Adv Intern Med. 2001;47:111–36.
Thompson PD, Clarkson P, Karas RH. JAMA. 2003;289:1681–90.
0. Furberg CD, Pitt B. Curr Control Trials Cardiovasc Med. 2001;2:205–7.
1. Ference BA, Majeed F, Penumetcha R, Flack JM, Brook RD. J Am Coll Cardiol.
2015;65:1552–61.
12. Zynolebadi N, Moradi MN, Ghasemi H, Totonchi A, Goodarzi M, Oshaghi EA.
Res Pharm Sci. 2012;7:S757.
1
1
3. Elisaf M. Curr Med Res Opin. 2002;18:269–76.
4. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC.
Circulation. 1998;98:2088–93.
15. Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPARs: from orphan
receptors to drug discovery. J Med Chem. 2000;43(4):527–50.
16. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH,
Linares E, Schaefer EJ, Schectman G. N Engl J Med. 1999;341:410–8.
17. Lazarow PB, Shio H, Leroy-Houyet MA, Lipid J. Res. 1982;23:317–26.