Synthesis of highly potent anti-inflammatory compounds (ROS inhibitors) from isonicotinic acid

Article abstract of DOI:10.3390/molecules26051272

In search of anti-inflammatory compounds, novel scaffolds containing isonicotinoyl motif were synthesized via an efficient strategy. The compounds were screened for their in vitro antiinflammatory activity. Remarkably high activities were observed for isonicotinates 5-6 and 8a-8b. The compound 5 exhibits an exceptional IC₅₀ value (1.42 ± 0.1 μg/mL) with 95.9% inhibition at 25 μg/mL, which is eight folds better than the standard drug ibuprofen (11.2 ± 1.9 μg/mL). To gain an insight into the mode of action of anti-inflammatory compounds, molecular docking studies were also performed. Decisively, further development and fine tuning of these isonicotinates based scaffolds for the treatment of various aberrations is still a wide-open field of research.

Full text of DOI:10.3390/molecules26051272

molecules
Communication
Synthesis of Highly Potent Anti-Inflammatory Compounds
(ROS Inhibitors) from Isonicotinic Acid
Sana Yaqoob 1, Nourina Nasim 1, Rahila Khanam 1, Yan Wang 1, Almas Jabeen 2, Urooj Qureshi 1,
Zaheer Ul-Haq 1,2 , Hesham R. El-Seedi 3,4,5,
* *
, Zi-Hua Jiang 6 and Farooq-Ahmad Khan 1,
1
Third World Center for Science and Technology, H.E.J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan;
sana.yaqoob@iccs.edu (S.Y.); nourina@iccs.edu (N.N.); rahila.khanam@iccs.edu (R.K.);
yan.wang@iccs.edu (Y.W.); urooj.qureshi@iccs.edu (U.Q.); zaheer.qasmi@iccs.edu (Z.U.-H.)
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi 75270, Pakistan; almas@iccs.edu
International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University,
S-106 91 Stockholm, Sweden
2
3
4
5
6
Department of Chemistry, Faculty of Science, Menoufia University, Menoufia 32511, Egypt
Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada;
zjiang@lakeheadu.ca
*
Correspondence: hesham.elseedi@su.se (H.R.E.-S.); farooq.khan@iccs.edu (F.-A.K.);
Tel.: +46-700-434-343 (H.R.E.-S.); +92-21-111-222-292 (F.-A.K.)
Abstract: In search of anti-inflammatory compounds, novel scaffolds containing isonicotinoyl motif
were synthesized via an efficient strategy. The compounds were screened for their in vitro anti-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
inflammatory activity. Remarkably high activities were observed for isonicotinates
The compound exhibits an exceptional IC50 value (1.42 0.1 g/mL) with 95.9% inhibition at
25 g/mL, which is eight folds better than the standard drug ibuprofen (11.2 1.9 g/mL). To
gain an insight into the mode of action of anti-inflammatory compounds, molecular docking studies
were also performed. Decisively, further development and fine tuning of these isonicotinates based
scaffolds for the treatment of various aberrations is still a wide-open field of research.
5–6 and 8a–8b.
Citation: Yaqoob, S.; Nasim, N.;
Khanam, R.; Wang, Y.; Jabeen, A.;
Qureshi, U.; Ul-Haq, Z.; El-Seedi,
H.R.; Jiang, Z.-H.; Khan, F.-A.
5
±
µ
µ
±
µ
Synthesis of Highly Potent
Anti-Inflammatory Compounds (ROS
Inhibitors) from Isonicotinic Acid.
Molecules 2021, 26, 1272. https://
doi.org/10.3390/molecules26051272
Keywords: anti-inflammatory; ROS inhibitors; isonicotinic acid; pyridine carboxylic acid; molecular
docking studies
Academic Editor: Bogumil E. Brycki
Received: 13 January 2021
Accepted: 23 February 2021
Published: 26 February 2021
1. Introduction
Inflammation is a complex defense-related response caused by invading microbes or
physical injuries [
are associated with the progression of inflammatory response cycle [
described as partially reduced oxygen-containing metabolites with strong oxidising capa-
bilities [ ]. These molecules are highly reactive to various intracellular components due to
the presence of unstable oxygen atom in them [ ]. Recent studies explicitly demonstrate
1
]. Reactive-oxygen species (ROS) are the key signaling molecules, which
2]. They are broadly
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
3
4
that ROS instigates several signaling cascades within immune cells, which ultimately leads
to the activation of innate and acquired immunity. Thus, the enhanced production of ROS
plays an important role for the development and progression of inflammatory disorders [
Therefore, the suppression of ROS overproduction is a promising approach for the pro-
phylaxis and treatment of various chronic inflammation-related aberrations [ ]. Lately,
many isonicotinic acid derived compounds are being patented due to their astounding
inhibitory activities against certain enzymes, such as myeloperoxidase (MPO), [ ] urease,
acetylcholinesterase, [ ] cyclooxygenase-2 (COX-2), [ ] Bcr-Abl tyrosine kinase [ ], and his-
tone demethylase [ 10]. In the market, Isoniazid (anti-tuberculosis), Enisamium (antiviral),
3].
Copyright:
© 2021 by the authors.
5
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
6
6
7
8
9,
Ethionamide (anti-tuberculosis), and Dexamethasone isonicotinate (anti-inflammatory and
anti-allergic) exemplify isonicotinic acid-derived drugs (Figure 1) [11].
Molecules 2021, 26, 1272. https://doi.org/10.3390/molecules26051272
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1272
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Figure 1. Drugs in the market having isonicotinoyl cores.
Comprehensive and vivid literature is available that highlight the therapeutic value
of this pyridine derivative [10 12 18]. Notably, Gilani et al. showed that isonicotinic
,
–
acid-derived 1,3,4-oxadiazoles exhibited an interesting profile of anti-inflammatory ac-
tivity, which was superior to the standard drug Naproxen [19]. The close congener, 2,6-
disubstituted isonicotinic acid hydrazides, also demonstrated an enormous anti-inflammatory
profile [20]. Inspired by these anecdotes, we synthesized isonicotinates of lipophilic na-
ture and studied their anti-inflammatory potential. Herein, we report exceptional anti-
inflammatory activities of several isonicotinic acid derivatives.
2. Results and Discussion
To discover novel scaffolds, our molecular design was based on the documented
activity of isonicotinic acid as a pharmacophoric moiety. In this context, isonicotinates
were considered interesting target molecules, since isonicotinamide derivatives are mostly
associated with antimicrobial activities in the literature. Isonicotinates were synthesized by
employing meta- and para-linkers; whereas alkyl chains of varying lengths were used to
augment or abridge the lipophilic behavior of these compounds (Figure 2).
Figure 2. Design rationale of isonicotinates.
To synthesize the target compounds, isonicotinic acid was initially reacted with meta- or
para-nitro phenol using N,N’-dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) in dimethylformamide (DMF), which afforded
1 and 2 with 36% and 31% yields,
respectively (Scheme 1). When these products were subjected to catalytic hydrogenation
(Pd/C, 1–6 bar H2) to reduce the nitro group, hydrolysis of the reduced product returned
isonicotinic acid and the corresponding aminophenol. This was presumably due to the
presence of strong electron withdrawing effect of nitro group on the linker, which renders
the carbon-oxygen bond in
1 and 2 considerably weak and labile under the catalytic
hydrogenation conditions. An alternative strategy was adopted involving protection–
deprotection method (Scheme 2). An equimolar mixture of meta- or para-substituted
aminophenol was treated with di-tert-butyl pyrocarbonate to afford amine protected
intermediates. This was confirmed by the presence of a signal (
δ ~1.51 ppm) due to
C(CH3)3 group in the 1H-NMR spectrum. The N-Boc-protected aminophenols were then
reacted with isonicotinic acid using DCC/DMAP in DMF to afford
yields (41% and 36%, respectively).
3 and 4 in satisfactory

Molecules 2021, 26, 1272
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Scheme 1. (a) DCC, DMAP, and DMF, 36% for 1 and 31% for 2. (b) Pd/C, H2, CHCl3/MeOH.
Scheme 2.
(a) 3-Aminophenol or 4-Aminophenol, Di-tert-butyl pyrocarbonate, Et3N; >99%.
(
b
)
Pyridine-4-carboxylic acid, DCC, DMAP, and DMF, 41% for
3
and 36% for
4
.
◦
(c
) CH2Cl2 + TFA (1:1), 0 C to room temperature (RT), >99% for
5–
6. (
d) CH3OH, (CnH2n+1CO)2O,
7a: 58%, 7b: 50 %, 7c: 43%, 7d: 83%, 7e: 82%, 8a: >99%, 8b: 44%, 8c: 76%, 8d: 64%, and 8e: 94%.
These intermediates were then treated with trifluoroacetic acid (TFA) and dichloromethane
(1:1) mixture at 0 ◦C to get free amines
5
and
mixture of different acid anhydrides of varying alkyl chains to afford lipophilic isonicotinate
derivatives 7a 7e and 8a 8e in appreciable yields, thus rendering this protocol practical
6, which were then reacted with equimolar
–
–
and straightforward to implement. The structures of the synthesized compounds were
confirmed by various spectroscopic techniques. Spectral data of all the active compounds
is presented in the Supplementary Materials.
The amassed collection of compounds was assayed for anti-inflammatory activity.
The oxidative burst assay was performed using chemiluminescence technique to evaluate
their anti-inflammatory potential [21]. Briefly, the compounds were incubated with human
blood (25 µL) diluted with Hank’s Balanced Salt Solution (HBSS) in 96-well half-area plates
in the thermostatic chamber of luminometer, and serum-opsonized zymosan (SOZ) and
luminol were then added. The inhibitory effect of these compounds on the production
of reactive oxygen species by human blood cells was measured, and their half-maximal
inhibitory concentration (IC50) values are presented in Table 1. The in vitro results demon-
strate that both compounds
anti-inflammatory activity. Compound
is the most potent compound among all the synthesized compounds. An IC50 value
of 1.42 ± 0.1 µg/mL for was eight-fold better than that of standard drug Ibuprofen
(IC50 = 11.2 1.9 g/mL). When a lipophilic acyl chain is introduced into the nitrogen
atom of the aminophenol linker, a significant change in their potency was observed. For
compounds with a meta-aminophenol type linker (7a 7e), only a weak activity was ob-
served. On the other hand, for compounds with a para-aminophenol linker (8a 8e), a
wider range of anti-inflammatory potency was observed. Compound 8b with a butyryl
group has an IC50 of 3.7 1.7 g/mL, which is three-fold better than Ibuprofen, while
compound 8d with an octanoyl group is devoid of any activity. It is interesting to note that
compounds and 8a (with an acetyl group) show comparable values of % inhibition at
a dose of 25 µg/mL; however, has a much lower IC50 value (8.6 0.5 g/mL) than 8a
(19.6 3.4 g/mL). The cogent manipulation of lipophilicity in our synthesized compounds
results is a positive contribution towards anti-inflammatory activity of isonicotinates con-
5
and
6, which are devoid of lipophilic chain, exhibit high
5
is an isonicotinate of meta-aminophenol and
5
±
µ
–
–
±
µ
6
6
±
µ
±
µ

Molecules 2021, 26, 1272
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taining a para-aminophenol linker. Lipophilic chains of moderate length were found more
effective as compared to the shorter or longer chains. For example, compound 8b having a
butyryl group had five-fold better anti-inflammatory activity than 8a with an acetyl group.
These results overall demonstrate the indispensable significance of suitably positioned
linkers in isonicotinates.
Table 1. Anti-inflammatory activities of isonicotinamides and isonicotinates.
Anti-Inflammatory Activity
Compounds
% Inhibition *
IC50 (µg/mL) **
5
6
7a
7b
7c
7d
7e
8a
95.9
67.3
29.3
25..8
15.7
21.6
13.2
66.6
85.4
44.6
-7.7
5.8
1.42 ± 0.1
8.6 ± 0.5
na
na
na
na
na
19.6 ± 3.4
3.7 ± 1.7
na
8b
8c
8d
na
na
8e
Ibuprofen
73.2
11.2 ± 1.9
* % inhibition at 25 µg/mL; ** half-maximal inhibitory concentration; na: not applicable.
In order to gain insight into the binding mode of the potent isonicotinates and look
for clues to assist in the lead-to-candidate optimization, a docking study using Molecular
Operating Environment (MOE) version 2019.01 builder module that allows examining a
potential binding mode to the enzyme was carried out. Heteroatom number designation
(Figure 3) in isonicotinates was generated by a webserver, as shown below:
Figure 3. Heteroatom number designation in isonicotinates.
Reactive oxygen species (ROS) or free radicals is a prominent factor of producing the
inflammation. Cyclooxygenase-2 (COX-2) inhibition by nonsteroidal anti-inflammatory
drugs (NSAIDs), like Ibuprofen, can relieve the symptoms of inflammation and pain. We
therefore assume that the anti-inflammatory activity of these isonicotinoyl compounds
may be correlated to their inhibitory effect on COX-2 enzyme. Hence, in silico molecular
docking of isonicotinic acid derivatives was conducted on the COX-2 enzyme cavity to
decipher their binding potential. Benchmarking of the docking software was done with
the cognate ligand Ibuprofen (Figure 4), and docking complex images of the enzyme with
compounds 5, 6, 8a, and 8b are shown in Figure 5.

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Figure 4. The redock alignment pose with interactions of Ibuprofen (orange) with the initial pose
(cyan) in the binding pocket of COX-2 enzyme; 3D mode (left), 2D mode (right).
Figure 5. Binding interactions of isonicotinates 5–6 and 8a–8b in the pocket of the COX-2 enzyme.
The overall binding affinities of the active compounds,
5–6 and 8a–8b, are in the range
of −7.26 to −6.56 (see Table 2). These values signify strong-to-moderate interactions.

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Table 2. Binding score and the key interactions of highly potent isonicotinates in the active site of cycloxygenase-2
(COX-2) enzyme.
Hydrogen Bonding
Residue
Binding Energy
Compound
Ibuprofen
Hydrophobic Interaction
Ligand
(Kcal/mol)
Distance (Å)
Val350, Leu353, Tyr356,
Leu360, Tyr386, Trp388,
Val524, Ala528
−
Lig:CO2 Salt
Tyr356:OH
Arg121
1.97
3.55
−7.61
bridge
+
Lig:N(1)
Lig:N(1)
Lig:N(3)
Lig:N(1)
Lig:N(1)
Lig:N(3)
Lig:O(3)
Lig:N(3)
Arg121:NH2
Tyr356:OH
Met523:CO
Arg121:NH2
Tyr356:OH
3.05
2.32
2.45
3.00
2.33
2.87
2.19
2.82
Val350, Leu353, Tyr386,
Val524, Ala528
−6.66
−6.56
−6.86
5
6
+
Val350, Leu353, Tyr386,
Val524, Ala528
Val117, Val350, Leu353,
Tyr356, Phe519, Val524,
Ala528, Trp388
+
Arg121:NH2+
8a
Arg121:NH2
Tyr356:OH
Phe206, Phe210, Val345,
Val350, Leu353, Phe382,
Tyr386, Val524, Ala528
Lig:N(1)
Lig:N(3)
Arg121:NH
Ser531:OH
3.03
2.39
8b
−7.26
Binding energy value for Ibuprofen is
active molecule and co-crystal ligand outlines the interaction pattern with the essential
residues in the cavity. Key interactions for all compounds ( 8a and 8b) in the bind-
−
7.61. Comprehensive visual inspection of each
5, 6,
ing pocket of the enzyme are listed in Table 2. For comparative analysis, the following
observations were noted to interpret the active nature of isonicotinates: (i) The binding
orientation (dock poses) is focused on isonicotinoyl moiety (see Figure 5); (ii) Backbone
residues with prominent interaction are Val350, Leu353, Tyr356, Leu360, Tyr386, Trp388,
Val524, Ala528 (hydrophobic residues), whereas Arg121 and Tyr356 are involved in hy-
drogen bonding, just like in Ibuprofen (Figure 4); (iii) Crucial residues are involved in
different types of interaction, such as π-π and hydrogen bonding; (iv) Addition of an acyl
group on the amino group of the linker decreases the electron density on the pyridine
nitrogen, which in turn may reduce its capacity as a hydrogen bond acceptor and affect
the orientation of the molecule in the binding pocket. For example, the pyridine nitrogen
in 6 forms hydrogen bonding with Arg121 and Tyr356. In contrast, the pyridine residue
in 8a (having an acetyl group) is located further away from the Arg121 and Tyr356, while
the pyridine nitrogen in 8b (having a butyryl group) is hydrogen-bonded only to the inner
NH of Arg121. Briefly, the docking study confirms that all active compounds show good
binding affinity with COX-2 enzyme and are potentially good inhibitors of the enzyme,
which correlates well with their potent anti-inflammatory activity demonstrated by the
in vitro oxidative burst assay.
3. Conclusions
In conclusion, isonicotinates were synthesized by adopting efficient synthetic strate-
gies. Isonicotinates with the least lipophilicity demonstrate exceptionally high anti-
inflammatory/ROS inhibitory activities among the series. The obtained data of the anti-
inflammatory activity indicate that compound
5 is a valuable candidate for further studies.
Further studies are in progress to show the anti-inflammatory potential of these compounds
in in vivo models.
Supplementary Materials: 1. Protocol for anti-inflammatory assay, 2. Docking methodology for
1
anti-inflammatory activity, 3. NMR spectra of the active compounds, Figure S1: H-NMR spectrum
of N-(3-Aminophenyl) isonicotinamide (
5
), Figure S2: 1H-NMR spectrum of N-(4-Aminophenyl)
1
isonicotinamide (
6), Figure S3: H-NMR spectrum of N-(4-Acetamidophenyl) isonicotinamide (12a),
1
Figure S4: H-NMR spectrum of N-(4-Butyramidophenyl) isonicotinamide (12b).

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Author Contributions: Conceptualization, F.-A.K.; organic syntheses, S.Y., N.N., and R.K.; purifica-
tion and characterization of the compounds, S.Y., N.N., R.K., and Y.W.; anti-inflammatory activities,
A.J.; computational studies, U.Q. and Z.U.-H.; resources, F.-A.K. and H.R.E.-S.; data curation, F.-A.K.,
H.R.E.-S., and Z.-H.J.; writing—original draft preparation, S.Y., N.N., and U.Q.; writing—review and
editing, F.-A.K., H.R.E.-S. and Z.-H.J.; and supervision, F.-A.K., H.R.E.-S. and Z.-H.J. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was supported by the Swedish Research Council Vetenskapsrådet (VR Grant
2016–05885).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or the supplementary data file.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the corresponding author
@ 250 $ per 10 mg, excluding the shipping charges.
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