Synthesis and anti-inflammatory activity of diversified heterocyclic systems

Article abstract of DOI:10.1111/cbdd.13576

In continuation with our research program on the development of novel bioactive molecules, we report herein the design and synthesis of a series of diversified heterocycles (4–22). The synthesized compounds were evaluated for their anti-inflammatory activity. The chemical structures of the newly synthesized compounds have been confirmed by NMR, FTIR, and microanalysis.

Full text of DOI:10.1111/cbdd.13576

DR AHMED BARI (Orcid ID : 0000-0002-9447-2361)
Article type : Research Article
Title: Synthesis and anti-inflammatory activity of diversified heterocyclic systems
Running Title: Heterocyclic systems as potential drug candidates
List of Authors:
Ahmed Bari1,2*, Daniel Grenier3, Jabrane Azelmat3 Saeed Ali Syed1,2, Abdulrahman M. Al-Obaid1, Eric
C. Hosten4
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box:
2457, Riyadh, 11451, Saudi Arabia.
*abari@ksu.edu.sa
Tel: +966533977946, Fax: +96614676220
2Research Center, College of Pharmacy, King Saud University, P.O. Box: 2457, Riyadh, 11451, Saudi
Arabia.
3Oral Ecology Research Group, Faculty of Dentistry, Université Laval, Quebec City, QC, Canada, G1V
0A6.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13576
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4Nelson Mandela Metropolitan University, Department of Chemistry, PO Box 77000, Port Elizabeth
6031, South Africa.
Corresponding Author:
Ahmed Bari, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box: 2457, Riyadh, 11451, Saudi Arabia.
*abari@ksu.edu.sa
Tel: +966533977946, Fax: +96614676220
Abstract:
In continuation with our research program on the development of novel bioactive molecules, we
report herein the design and synthesis of a series of diversified heterocycles (4-22). The synthesized
compounds were evaluated for their anti-inflammatory activity. The chemical structures of the
newly synthesized compounds have been confirmed by NMR, FTIR and micro analysis.
1. Introduction:
The prevalence of inflammatory diseases has increased rapidly over the past decades and affects
more than 6 percent of world population. Although public awareness about the cause and treatment
of inflammation is still growing, it remains a major health concern worldwide. Inflammatory disease,
often associated with pain, fever and limited mobility, may lead to depression, hence affect the
quality of life (Beekman et al., 2013). Many naturally occurring and synthetic non-steroidal anti-
inflammatory drugs are based on heterocyclic core structure. These drugs are widely used to reduce
pain and inflammation but given their troublesome side effects, such as nephrotoxicity and gastritis,
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they are not suitable for everyone (Sharma et al., 2005).. Therefore, there is a need to develop more
potent and selective. Moreover, the limited efficacy of the current therapies for inflammatory
disorders supports the need for the development of alternative with less toxicity and more potency.
Despite the progress made on non-streoidal anti-inflammatory drugs, the development of new
therapy methods which have both pharmacological profile and therapeutic safety represents a
major concern for many scientists. Heterocyclic chemistry has generated intensive interest due to
their pharmacological properties. Over the last years, several reports on the synthesis of heterocyclic
molecules with anti-inflammatory potential have been published (Muralidharan et al., 2018;
Fernandes et al., 2017; Rani et al., 2004). Therefore, there is a need to identify the core moiety
responsible for such effects. The current paper describes the synthesis of various heterocyclic
systems along with their anti-inflammatory activity in view to identify the potent nucleus.
Phenazone is one of the most commonly used non-steroidal anti-inflammatory drugs (NSAID) known
as cyclooxygenase and lipoxygenase inhibitors (Herxheimer et al., 1961). Formyl group attached to
the heterocyclic system can also serve as a versatile tool to synthesize various biologically active
analogues. Published data have revealed that the conversion of various aromatic or heterocyclic
aldehyde groups into pyrazolo[b]quinolines is of considerable interest and often increases bioactivity
both in vivo and in vitro. Furthermore, hydrazides also constitute an important class of heterocycles
associated with a variety of biological properties, including antimicrobial and anticancer activities.
Thiourea derivatives can be used as an intermediate for various therapeutic drugs and can also be
used as an intermediate to synthesize heterocycles. Interestingly, several papers in the literature
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have been published on the structural modification of thiourea (Bari et al., 2014; Fattah et al., 2010;
Quiroga et al., 2001).
With the objective to develop new therapeutic agents with better anti-inflammatory activity and less
toxicity, we focused our efforts to synthesize thiazolidinone derivatives. Due to the low solubility of
thiosemicarbazone moiety, there are certain limitations that can be improved pharmaceutically by
converting thiourea group with thiazolidinone ring which is well known for anti-inflammatory
activity. There are several several reports on the synthesis of thiazole heterocycles with a protected
C-4 group of the ring, however, due to the high cytotoxicity associated with the molecule, we
decided to synthesize thiazolidinone moiety, which is slightly
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different from thiazole, due to the presence of carbonyl group ( Singh et al., 2008) at C-4.
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Typically, the indolinone nucleus is a well known natural product and is extensively used as key
intermediate in organic synthesis. Moreover, quinazoline-4-one is a fused heterocyclic compound
and its derivatives have been extensively studied by researchers for a wide range of biological
activities. In the current investigation, 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-
carbaldehyde 2 and pyridine-3-carboxaldehyde 3 were used as starting materials to synthesize
indolin-2-one and quinazoline-4-one derivatives which are often associated with antitubercular,
anticancer and anti-inflammatory properties and which were previously (Kumar & Rajput, 2009)
reported.
Fig. 1: Crystal structure of compound 5
2.0 Methods and Materials:
2.1 Biology:
2.1.1 Effects of compounds on cell viability
Prior to evaluating the anti-inflammatory properties of the compounds, we determined their
cytotoxic effect on the U937 3xκB-LUC cell line to exclude the possibility that toxicity related to the
compounds might cause a decrease in NF-κB activation. The cells were treated for 6.5 h with two-
fold serial dilutions of the compounds, starting at 1000 µM. Thereafter, a colorimetric MTT cell
viability assay (Roche Diagnostics, Germany) using 3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide as the substrate was performed according to the manufacturer’s protocol. Untreated
control cells were assigned a value of 100%.
2.1.2 Activation of the NF-κB transcription factor
The human monoblastic leukemia cell line U937 3xκB-LUC, a subclone of the U937 cell line stably
transfected with a luciferase gene coupled to a promoter of three NF-κB-binding sites, was used to
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investigate the effect of the compounds on lipopolysaccharide (LPS)-induced NF-κB activation. Cells
were routinely cultivated in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum
(FBS), 100 µg/ml of penicillin G/streptomycin, and 75 µg/ml of hygromycin B at 37°C in a 5% CO2
atmosphere. To induce activation of the NF-κB transcription factor, the U937 3xκB-LUC cells (106
cells/well) were placed into wells of a black wall, black bottom 96-well microplate (Greiner Bio-One
North America Inc.) and were stimulated for 6 h with Aggregatibacter actinomycetemcomitans ATCC
20522 LPS (1 µg/ml). Thereafter, to investigate the effect of the compounds on LPS-induced NF-κB
activation, U937 3xκB-LUC cells were pre-incubated with non-cytotoxic concentrations of the
compounds (in RPMI containing 1% FBS) for 30 min and were then stimulated for 6 h with LPS. Wells
with no LPS or no compounds were used as controls to measure basal NF-κB activity. An assay using
the commercial inhibitor BAY-11-7082 (25 µM) was used as a positive control for inhibition of NF-κB
signaling pathway. NF-κB activation was quantify by measuring luciferase activity following the
addition Bright-Glo™ reagent (Promega Corporation, Madison, WI, USA) in accordance with
manufacturer’s protocol. Luminescence was monitored using a Synergy 2 microplate reader.
Fig. 4: Crystal structure of compound 22
2.2 Chemistry:
All solvents and reagents were purchased from Aldrich Chemical Co and were used as obtained. The
reactions were carried out under air atmosphere unless stated otherwise. Melting points measured
with a Thermo Scientific 9100 apparatus and are uncorrected. IR spectra were recorded with a
1
Bruker Alpha spectrometer using ATR accessory. H- and 13C-NMR spectrum was recorded on a
1
13
Bruker instrument at 500 and 700 MHz for H and 125 MHz for C at 295 K in DMSO-d6 and CDCl3.
The mass spectrometric experiments were carried out on a Jeol JMS-700 mass spectrometer. Thin-
layer chromatography (TLC) was performed on a fluorescent aluminium backed silica gel HF254 plates
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(Merck) and was viewed under UV 254 and 265 lights and charring with EtOH/H2SO4. Micro analysis
was carried out on a Perkin Elmer CHNS 2400 instrument. Merck silica gel 60 (230-400 mesh) was
used for column chromatography separations.
2.2.1 General procedure for the synthesis of 2a, 2b and 3a, 3b:
To a solution of pyridine-3-carboxaldehyde or phenazone-4-carboxaldehyde (1 mmol) in absolute
ethanol was added respective thiosemicarbazide (1.2 mmol) followed by few drops of acetic acid.
The reaction mixture was refluxed with stirring for 3 hrs followed by the addition of ice. The solid
separated was collected by filtration and washed several times with water.
2.2.2 General procedure for the synthesis of compound 5-10, 16, 17:
To a solution of aldehyde 1 (2 mmol) in absolute ethanol (50 mL) was added respective hydrazine
(2.2 mmol) and catalytic amount of acetic acid. The resulting mixture was refluxed for 3 h with
stirring followed by the evaporation of solvent in vacuo. The solid obtained was washed with cold
water several times and recrystallized with ethanol.
2.2.3 General procedure for the synthesis of compound 11:
To a mixture of thiourea derivatives 2a (2 mmol) in acetonitrile (30 mL) was added maleic anhydride
slowly in 10 mins time at room temperature. The reaction mixture was than refluxed for 5 h with
stirring followed by the addition of ice. The obtained precipitate was collected by filtration, washed
with water and recrystallized with ethanol afforded cyclized product in good yield.
2.2.4 General procedure for the synthesis of compound 12, 13, 20:
To a solution of thiourea derivatives 2a, 3a or 2b (2 mmol) in glacial acetic acid was added anhydrous
sodium acetate (2.2 mmol) and monochloroacetic acid (2.2 mmol). The mixture was refluxed for 4 h
with stirring followed by the addition of ice. The obtained precipitate was collected by filtration,
washed with water and recrystallized with cold ethanol.
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2.2.5 General procedure for the synthesis of compound 18, 19:
A mixture of thiourea derivatives 2a (2 mmol) in aceticanhydride (30 mL) was refluxed for 5 h with
stirring followed by the addition of ice. The obtained precipitate was collected by filtration, washed
with water and recrystallized with ethanol afforded cyclized products in good yields.
2.2.6 General procedure for the synthesis of compound 15:
To a solution of aldehyde 2 (2 mmol) in absolute ethanol (50 mL) was added dimedone (4.2 mmol)
and ammoniumacetate (2.2 mmol). The resulting mixture was stirred at room temperature for 10
min than PPA/SiO2 (50 mg) was added. The reaction mixture was refluxed for 4 h, filtered and
evaporation of solvent in vacuo resulted in the precipitate. The solid obtained was washed with cold
water several times afforded compounds 2 and 3.
2.2.7 General procedure for the synthesis of compound 14, 21, 22:
To a solution of aldehyde 1 (2 mmol) in absolute ethanol (50 mL) was added dimedone (3 mmol),
ammoniumacetate (2.2 mmol) and appropriate amine (2.2 mmol). The resulting mixture was cooled
in an ice bath and sodium metal (2.2 mmol) in ethanol was added dropwise. After complete
addition, the mixture was refluxed for 4 h, filtered and evaporation of solvent in vacuo
resulted in the precipitate. The solid obtained was washed with cold water several times, dried and
purified by column chromatography (1:1 EtOAc-Hexane) afforded compounds 2 and 3.
Fig. 5: NF-kB inhibition and cytotoxicity data
Fig. 6: NF-kB inhibition and cytotoxicity data
2.3 X-ray Crystallographic Data: Crystals of compound 45, 13, 14a and 22 were obtained
by allowing slow solvent evaporation. Crystallographic data have been deposited at the
Cambridge Crystallographic Data Center (CCDC Nr for compound 5 is1896597, CCDC
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Nr for compound 13 is 1896596, CCDC Nr for compound 14a is 1896365 and CCDC Nr
for compound 22 is 1896364).
3
Results and Discussion:
3.1 Chemistry:
For the synthesis of compounds 2a and 3a, 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-
carbaldehyde 2 and pyridine-3-carboxaldehyde 3 were treated with chosen thiosemicarbazides in
refluxing ethanol with few drops of acetic acid. The precipitate obtained was washed several times
with water and used without further purification (scheme 1).
Reaction of carboxaldehydes 2 and 3 with various substituted hydrazines afforded schiff bases 4-10,
16 and 17 in acidic medium. Reaction with various isatin derivatives are of utmost importance
because indoline-2-one molecules are well known for their antimicrobial, antiviral and anticancer
properties. Disappearance of formyl proton and a presence of a singlet at around 8 ppm for CH and a
broad peak in the downfield region for NH confirm the postulated structure. Moreover, IR and mass
spectra are also in accordance with the structures. The compound 5 was crystallized in ethanol
afforded crystals suitable for x-ray crystallography (fig 1).
In the current paper, construction of thiazolidine heterocyclic system was carried out by reaction of
substituted thiosemicarbazone with monochloroacetic acid and maleicanhydride in acetic acid
affording 11, 12, 13 and 20 in excellent yields (scheme 3 and 4). More constrained thiazolidine ring
system was selected due to their involvement in molecules reported as anti-inflammatory ( Gupta,
Sundaram & Reuter, 2010).
Fig. 2: Crystal structure of compound 13
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Structure of compound 13 was also confirmed by x-ray crystallography which was obtained by slow
evaporation of ethanol (fig 2). On the basis of reaction time and the corresponding yields that the
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intermediate thiourea derivatives with phenyl group is less reactive than the other one because of
the steric effect of phenyl group that reduces the ability of NH undergo nucleophilic cyclization.
Same was observed when the intermediate (NH) was reacted with acetic anhydride 18 and 19. The
NH2 due to strong nucleophilicity may facilitate substitution with acetic anhydride. However, the
resonance effect between NH and the phenyl group of intermediate reduces the nucleophilic
substitution with the steric effect of phenyl group further plays a role in reducing yields (Muscia &
Buldain, 2014). The reaction of 2a and 2b with acetic anhydride was unsuccessful even after
prolonged reaction time and reflux. Appearance of CH of the thiazolidine ring at around 4 ppm and
NH in the downfield region (11 to 12 ppm) confirms the ring transformation reaction. Mass, FTIR and
elemental analysis also confirm the postulated structures. All the synthesized compounds were
washed with water and cold ethanol prior to characterization by spectroscopic method and
elemental analysis.
In the synthesis of quinazoline-4-one (10) type of molecules, more time was required due to the
steric hindrance caused by the phenyl group making the amino group less available for cyclization.
However, the quinazoline-5-one ring system which is of considerable importance due to their
biological activity was also synthesized. Several reports for the synthesis of such systems involved
Friedlander approach but due to the formation of various side products there is a need to develop a
more efficient approach with higher yields (Kumar, Kaur & Kumari, 2012). Therefore, for the
synthesis of quinazoline-5-one heterocyclic system 14a, 15, 21 and 22, silica gel supported
polyphosphoric acid (PPA-SiO2) as a green and reusable heterogeneous catalyst was utilized. In the
course of the
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synthesis of 14a, cyclohexane-1,3-dione 14b was obtained as by product which is assumed to be due
to the Knoevenageal condensation which is a initial step in the cyclocondensation reaction. The NMR
spectroscopic data is also consistent with the proposed structures, for example compounds 14a, 21
and 22, exhibit in a 1H NMR spectrum, the two relatively sharp singlets which are readily assigned as
exchangeable NH-groups. Moreover another two couples of doublets appear around 2-3 ppm
corresponding to the diastereoscopic CH2 groups respectively. Proposed molecule is further
confirmed by x-ray crystallographic studies of compound 14a (fig 3) and 22 (fig 4) which were
obtained in ethanol-dimethylsulfoxide mixture. However, when 2 were treated with two equivalents
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of dimedone, in the presence of ammonium acetate, compound 15 was obtained in high yield. The
structures were assigned using 13C, DEPT, HMBC and HSQC experiments.
Fig. 3: Crystal structure of compound 14a
3.2 Biology:
In the present study, we used the U937-3xκB-LUC human monocytic cell line, which contains a
luciferase reporter possessing three NF- κB binding sites, to evaluate the ability of the designed
molecules to inhibit activation of the NF-κB signaling pathway. This model is very convenient to
rapidly evaluate the potential of new compounds to block activation of the transcription factor NF-κB,
an important regulator of many pro-inflammatory pathways (Cardinal et al., 2017; Lagha & Grenier,
2016; Lagha, LeBel & Grenier, 2018). First, to exclude the possibility that cell toxicity might have
been responsible for any decrease in NF-κB activation, the viability of the U937-3xκB-LUC cells
treated with the molecules was evaluated using an MTT test. Table 1 reports the highest
concentration of the tested molecules for which no significant toxicity was found. On the one hand,
some molecules were found to be cytotoxic at low concentrations; for instance the highest non-
cytotoxic concentration for 16 was 0.24 µM while for 6, 19, and 22 was 1.95 µM. On the other hand,
some other molecules were much less cytotoxic; for instance the highest non-cytotoxic concentration
for 2a, 3a, 7, and 17 was 500 µM while for 11, and 18 was 250 µM.
Two-fold serial dilutions of the highest non-cytotoxic concentration of each molecule were then tested
for their ability to inhibit the LPS-induced activation of NF-κB signaling pathway (Fig 5, 6). While
some molecules did not show any inhibition (4, 9, 12, 16, 19, 20, and 21), others significantly and
dose-dependently inhibited LPS-induced NF-κB activation. The most effective molecules were 2a,
3a, 6, 11, and 15 and their IC50 values calculated were as below:
2a : 183.7 ± 4.6 µM
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3a : 115.4 ± 4.6 µM
6 : 0.5 ± 0.2 µM
11 : 107.4 ± 7.6 µM
15 : 43.3 ± 12.9 µM
Given that the transcription factor NF-κB controls the expression of a large array of genes involved in
inflammation, some of the novel molecules by inhibiting NF-κB activation shows potential to reduce
the host inflammatory response.
Thiosemicarbazide of both starting materials 2a and 3a bearing a free NH2 group showed excellent
inhibition as compared to the phenylthiosemicarbazide derivatives 2b and 3b. This is assumed to be
due to the steric hindrince caused by the phenyl group which plays a major role in reducing
cytotoxicity.
The schiff base of phenazone carboxaldehyde 6 enhances inhibition in comparision with
benzothiazole schiff base of pyridine 16. This could be due to the biological activity associated with
phenazone moeity itself while the benzothiazole bearing a fluoro group also plays a role in
increasing biological activity. The cyclization of thiosemicarbazono group to the oxathiozolidine
bearing exocyclic aceticacid 11 also showed excellent inhibition. It was anticipated that the presence
of the elongated substituent in the thiazolidine ring at C-5 enhance the inhibition considerably.
Moreover, the impact of phenazone group in the overall activity is also taken in account.
Hexahydroacridine 15 having phenazone group at C-9 also showed marked inhibition as compared to
other analogues suggesting that the two diketone groups attached to acridine nucleus seems more
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favourable for constructing an antiinflammatory agent than the case of substitution with electron
withdrawing group.
Conclusion:
In conclusion, the newly synthesized compounds having phenazone group were found to be more
potent than the corresponding pyridine analogues. But, additional studies are required to investigate
the stability of hydrazones such as 11 in biological systems.
Acknowledgement:
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University
for funding this work through research group no RG-1440-009.
Conflict of Interest:
The authors declare no competing financial interest.
Supplementary Information:
1
Supplementary information (spectroscopic data, H NMR and Mass spectra) is available free
of charge as PDF file.
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Cytotoxicity
Cytotoxicity
‡ : Considered as non-cytotoxic
Inh : NF-κB inhibitor (BAY 11-7082)
* : significantly different from LPS-stimulated cells at p ‹ 0.01
† : significantly different from LPS-stimulated cells at p ‹
0.05
Fig. 5: NF-kB inhibition and cytotoxicity data
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Cytotoxicity
Cytotoxicity
‡ : Considered as non-cytotoxic
Inh : NF-κB inhibitor (BAY 11-7082)
* : significantly different from LPS-stimulated cells at p ‹ 0.01
† : significantly different from LPS-stimulated cells at p ‹ 0.05
Fig. 6: NF-kB inhibition and cytotoxicity data
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Table 1. Cytotoxicity of the molecules towards the U937-3xκB-LUC human monocytic cell line, as
determined with a MTT colorimetric assay.
Molecule
Highest concentration showing no cytotoxicity (µM)
2a
3a
4
500
500
15.62
7.81
1.95
500
5
6
7
8
62.5
7.81
31.25
250
9
10
11
12
13
14
15
16
17
18
19
20
21
22
62.5
125
125
125
0.24
500
250
1.95
15.62
7.81
1.95
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Fig. 1: Crystal structure of compound 5
Fig. 2: Crystal structure of compound 13
Fig. 3: Crystal structure of compound 14a
Fig. 4: Crystal structure of compound 22
Fig. 5: NF-kB inhibition and cytotoxicity data
Fig. 6: NF-kB inhibition and cytotoxicity data
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Fig 1
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Fig 2
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Fig 3
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Fig 4
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