Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.04.079

The series of new 1,2,4-triazole derivatives with methacrylic acid moiety were synthesized and characterized by NMR and IR spectroscopy as well as X-ray crystallography. The influence of newly synthesized compounds on the inflammation on the level of cyto

Full text of DOI:10.1016/j.bmcl.2015.04.079

Accepted Manuscript
Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives
Renata Paprocka, Malgorzata Wiese, Andrzej Eljaszewicz, Anna Helmin-Basa,
Andrzej Gzella, Bozena Modzelewska-Banachiewicz, Jacek Michalkiewicz
PII:
S0960-894X(15)00411-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.04.079
BMCL 22662
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
8 January 2015
21 April 2015
23 April 2015
Please cite this article as: Paprocka, R., Wiese, M., Eljaszewicz, A., Helmin-Basa, A., Gzella, A., Modzelewska-
Banachiewicz, B., Michalkiewicz, J., Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.04.079
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Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives
Renata Paprockaa#, Malgorzata Wieseb#, Andrzej Eljaszewiczc, Anna Helmin-Basab, Andrzej
Gzellad, Bozena Modzelewska-Banachiewicza, Jacek Michalkiewiczb
# those authors contributed equally to the work
aDepartment of Organic Chemistry, Faculty of Pharmacy, Nicolaus Copernicus University in
Torun, Jurasza 2, 85-089 Bydgoszcz, Poland
bDepartment of Immunology, Faculty of Pharmacy, Nicolaus Copernicus University in Torun,
M. Curie-Sklodowska 9, 85-094 Bydgoszcz, Poland
cDepartment of Regenerative Medicine and Immune Regulation, Medical University of
Bialystok, Waszyngtona 13, 15-269 Bialystok
dDepartment of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6,
60-780 Poznan
Corresponding author: Renata Paprocka, renata.bursa@cm.umk.pl, Department of Organic
Chemistry, Faculty of Pharmacy, Nicolaus Copernicus University in Torun, Jurasza 2, 85-089
Bydgoszcz, tel. (+48) 52-585-3905.
Keywords:
amidrazone; triazole; proliferation; TNF-α; IL-6; IL-10; PBMC; ibuprofen
Abstract
The series of new 1,2,4-triazole derivatives with methacrylic acid moiety were synthesized
and characterized by NMR and IR spectroscopy as well as X-ray crystallography. The
influence of newly synthesized compounds on the inflammation on the level of cytokine
production and the proliferation of human peripheral blood mononuclear cells (PBMC) were
experimentally evaluated. Obtained triazoles showed antiproliferative activity and diverse
effects on cytokine production. Two compounds demonstrated potentially anti-inflammatory
activity and comparable effects with ibuprofen.
Inflammation is a common state during infections and a number of diseases from hay fever,
periodontitis, atherosclerosis, rheumatoid arthritis to cancer. Commonly used non-steroidal
anti-inflammatory drugs (NSAIDs) provide analgesic and antipyretic effects as well as anti-
inflammatory in higher doses. The therapeutics are prevalent, however they seriously increase

vascular and gastrointestinal risks.1 Because heart failure risk was roughly doubled by all
NSAIDs cardiovascular risk needs to be taken into account when prescribing any medicine of
this group.2 Over one hundred years after inventing aspirin there is still a need for a safe anti-
inflammatory drug.
Searching for new potentially active substances, we combined the methacrylic acid with
1,2,4-triazole core. Compounds possessing triazole ring are known for their wide range of
biological activities, and anti-inflammatory properties among them.3-6 Methacrylic acid
resembles propanoic acid present in the structure of some NSAIDs like ibuprofen, naproxen
and ketoprofen. Anti-inflammatory activity of compounds containing methacrylic acid moiety
has been reported.7 However, 1,2,4-triazoles combined with this acid were never examined as
anti-inflammatory agents.
In present work we describe the syntheses of new 1,2,4-triazoles substituted with methacrylic
acid and their influence on the inflammation on the level of proliferation of peripheral blood
mononuclear cells (PBMC) and cytokine production.
The series of novel 1,2,4-triazole derivatives 8-14 containing methacrylic acid moiety were
synthesised in reaction of N3-substituted amidrazones 1-78 with itaconic anhydride (Figure
1). All compounds were obtained with satisfactory yields (50-91 %), except derivative 14
possessing 2-pyridyl and 4-nitrophenyl groups (about 15 % yield). According to the method
described previously9 we obtained triazole 14 with better efficiency by cheating crude
precipitate obtained in general reaction in 2 % NaOH solution. It is noteworthy that the
reaction of amidrazones 6-7 with itaconic anhydride conducted by the same procedure leaded
to complete isomerisation to (E)-3-(4,5-diaryl)-4H-1,2,4-triazol-3-yl)-2-methylacrylic acids
15-16. In our study compound 14 was obtained in alkaline solution with 56 % yield and
isomerisation product was not found. Obtained derivatives were characterised by
spectroscopic techniques: 1H NMR, 13C NMR, IR, MS, the structures of 1010, 1211, 1312 and
1611 were also confirmed by single-crystal X-ray study. Purities of compounds 8-16 were
proved by RP-TLC chromatography. In reverse-phased system triazoles 8-16 showed single
spots without the presence of impurities and degradation products for all used mobile phases.
The effect of different concentrations of compounds 8-16 on the viability of peripheral blood
mononuclear cells (PBMC) at rest, and activated by monoclonal antibody anti-CD3 (aCD3),
phytohemaglutinin P (PHA) or E. coli lipopolisacharide (LPS) was assessed. Compounds 8-
10 and 13-16 induce no apoptosis or necrosis of analyzed cells at any concentration alone or

in the presence of used stimulators (data shown in supplementary materials). Derivatives 11-
12 showed the highest toxicity alone (50-100 µg/mL) so they were excluded from further
biological assays.
Afterwards the effect of compounds 8-10, 13-16 on mitogens (PHA and aCD3)-induced
PBMC proliferation in short term culture (72h) was investigated. Reference compound
potently inhibited proliferation induced by anty-CD3 antibody and PHA in the medium and
high doses (50 µg/mL and 100 µg/mL). We found that compounds 14 and 16 significantly
suppressed proliferation of anty-CD3 and PHA-induced PBMC, regardless of the used
compounds concentration. However, the strongest inhibition was observed at 100 µg/mL.
Compound 10 showed similar to triazole 16 suppression of proliferation in almost every dose
excluding the middle dose in culture stimulated by aCD3. Additionally, compound 15 inhibits
proliferation effects in low and high doses while derivative 8 in middle and high doses. (Fig.
2a-b).
It is noteworthy that compounds 8-10, 13-16 did not markedly alter the cell cycle profile.
Thus, based on our data, we initially hypothesize that reduction on mitogen-activated PBMC
proliferation by analyzed compounds is caused by their anti-inflammatory properties. It
should be noted that several anti-inflammatory drugs including NSAIDs were shown to
inhibit proliferation of colorectal cancer cell lines, mucosal cells, endometrial cancer cell lines
and mitogen-induced lymphocytes, among others.13
Having found reduced proliferation of mitogen–activated PBMC in the presence of
compounds 8-10, 13-16, next we assessed their effect on LPS-induced cytokine production.
Notably, LPS is an endotoxin of Gram-negative bacteria, used extensively for inducing an
immune response in vitro.14-16 It induces cytokine production by PBMC including pro-
inflammatory TNF-α and IL-6 and anti-inflammatory IL-10.17-18 Therefore we studied above
mentioned cytokines in compounds and LPS-stimulated PBMC culture. In fact substantial
increase in TNF-α, IL-6 and IL-10 levels in cell culture supernatants was observed after 24h
stimulation. However, we found that compounds 10, 15 and 16 at 10, 50 and 100 µg/mL
significantly reduced TNF-α release of LPS-induced PBMC (Fig. 3a). The four consecutive
compound 8, 9, 13 and 14 worked differently to ibuprofen because they induced higher levels
of TNF-α (p<0.05). Interestingly, similar activity to reference compound in TNF-α production
was observed after stimulation with compounds 10 (only at 10 and 100 µg/mL) 15 and 16 (in
three doses).

Ibuprofen was described to activate or quiescent IL-6 production in HUVEC (human
umbilical vein endothelial cells) culture.19 However we obtained no statistically significant
differences in IL-6 level after stimulation with any of the compounds (p<0.05).
Finally and not less noteworthy, compounds 9 and 14 possessing nitro group were shown to
significantly increase IL-10 release of LPS-induced PBMC at 100 µg/mL (p<0.05, Fig 3c).
Different results were observed after stimulation with compounds 10, 15 (at 50, 100 µg/mL)
and compound 16 (at 100 µg/mL). These compounds reduced production of IL-10 (p<0.05 in
both cases). Interestingly reference compound also reduces the concentration of IL-10
compared to LPS but only at the highest dose (p<0.05).
It should be noted that monocytes are the main source of TNF-α, IL-6, IL-10 in blood.
Moreover monocytes serve as a target for LPS, which biological activity is associated with
CD14 interaction.20-22 CD14 is a membrane glycoprotein mostly present on the surface of
monocytes and macrophages. Consequently, PBMC stimulation by LPS resulted
predominantly in monocyte/macrophage activation. Therefore, observed anti-inflammatory
effect of analyzed compounds 8-10, 13-16 may be associated with modulation of monocytes
activation. However future studies are needed to clarify the mechanisms involved in this
effect.
Tacking together, seven new 1,2,4-triazole derivatives 8-16 were synthesized in reaction of
N3-substituted amidrazones with itaconic anhydride. Here we revealed that all tested
compounds 8-10, 13-16 possess immunomodulatory activity. Among these, compounds 10,
15-16 seems to have the strongest anti-inflammatory potential. Notably triazoles 15-16 (3-
substituted-2-methylacrylic acid derivatives) showed lower toxicity than their isomers 11-12
and were more active than all 2-substituted-methylacrylic acid derivatives except of 10.
However, further studies are warranted to elucidate the role of analyzed new 1,2,4-triazole
derivatives, in particular compound 10, 15-16, as a potential novel non-steroid anti-
inflammatory drug.
The authors state no conflict of interest.
Supplementary data
Details of synthesis, spectral data, bioassays and cytograms are available as Supplementary data.

References and notes
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Index of Figures
Figure 1. The synthesis and structures of compounds 8-16
N
N
COOH
diethyl
ether
R1
N
8-14
R2
O
N
O
NH2
N
N
N
2% NaOH,
reflux, 2h
diethyl
ether
NH
COOH
+
COOH
O
R1
R1
NH
R2
N
14
R1
NH
R2
1-7
R2
O
2% NaOH,
reflux, 2h
N
N
COOH
R1
N
15-16
R2
1, 8
2, 9
5, 12, 16
3, 10
7, 14
N
No.
R1
4, 11, 15
6, 13
N
N
N
N
N
R2
NO2
NO2
CH3
CH3
a)

b)
Figure 2. The effect of compound 8-10, 13-16 on proliferation of human peripheral
blood mononuclear cells (PBMC) induced by the anti-CD3 antibody (a) or PHA (b).
Cells were treated with anti-CD3 antibody (4 µg/mL) or PHA (0.5 µg/mL) and
compounds 8-10, 13-16 at concentrations 10, 50, 100 µg/mL. Ibuprofen (IBU) was
used as reference drug, negative control (-) – no additions. After 72/h incubation, the
3
proliferation of PBMC were measured using H thymidine incorporation assay. The
results were shown as percentage of positive control (anti-CD3 antibody or PHA).
Values expressed as medians from five independent experiments and interquartile
ranges [Q1-Q3], * - indicates significant difference compared to anti-CD3 antibody
alone (a) or PHA alone (b) at p < 0.05; # - indicates significant difference compared to
ibuprofen at p < 0.05.

a)
b)
c)
Figure 3. The effect of compound 8-10, 13-16 on the LPS-induced production of
cytokines: TNF-α (a), IL-6 (b) and IL-10 (c) in PBMC cultures. Cells were treated
with LPS (1 µg/mL) and compounds 8-10, 13-16 at concentrations 10, 50, 100 µg/mL.

Ibuprofen (IBU) was used as reference drug, negative control (-) – no additions. After
24/h incubation, the concentration of cytokines were measured in the supernatants
using the immunoensymatic test. The results were shown as percentage of positive
control (LPS). Values expressed as medians from five independent experiments and
interquartile ranges [Q1-Q3], * - indicates significant difference compared to LPS
alone at p<0.05; # - indicates significant difference compared to ibuprofen at p<0.05.

Graphical abstract