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: DOI: Reference:

S0960-894X(15)00411-4 http://dx.doi.org/10.1016/j.bmcl.2015.04.079 BMCL 22662

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: 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., ModzelewskaBanachiewicz, 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 a

Department of Organic Chemistry, Faculty of Pharmacy, Nicolaus Copernicus University in

Torun, Jurasza 2, 85-089 Bydgoszcz, Poland b

Department of Immunology, Faculty of Pharmacy, Nicolaus Copernicus University in Torun,

M. Curie-Sklodowska 9, 85-094 Bydgoszcz, Poland c

Department of Regenerative Medicine and Immune Regulation, Medical University of

Bialystok, Waszyngtona 13, 15-269 Bialystok d

Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6,

60-780 Poznan Corresponding author: Renata Paprocka, [email protected], 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 antiinflammatory 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 antiinflammatory 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 1-7 8 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: 1 H NMR,

13

C NMR, IR, MS, the structures of 10 10, 12 11, 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 810 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 1112 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 proinflammatory 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

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

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-r...
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