1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole derivatives blockade P2X7 receptor in?vitro and inflammatory response in?vivo

Article abstract of DOI:10.1016/j.ejmech.2017.08.034

Fifty-one 1,2,3-triazole derivatives were synthesized and evaluated with respect to P2X7 receptor (P2X7R) activity and its associated pore. These triazoles were screened in vitro for dye uptake assay and its cytotoxicity against mammalian cell types. Seven 1,2,3-triazole derivatives (5e, 6e, 8h, 9d, 9i, 11, and 12) potently blocked P2X7 receptor pore formation in vitro (J774.G8 cells and peritoneal macrophages). All blockers displayed IC₅₀ value inferior to 500 nM, and they have low toxicity in either cell types. These seven selected triazoles inhibited P2X7R mediated interleukin-1 (IL-1β) release. In particular, compound 9d was the most potent P2X7R blocker. Additionally, in mouse acute models of inflammatory responses induced by ATP or carrageenan administration in the paw, compound 9d promoted a potent blocking response. Similarly, 9d also reduced mouse LPS-induced pleurisy cellularity. In silico predictions indicate this molecule appropriate to develop an anti-inflammatory agent when it was compared to commercial analogs. Electrophysiological studies suggest a competitive mechanism of action of 9d to block P2X7 receptor. Molecular docking was performed on the ATP binding site in order to observe the preferential interaction pose, indicating that binding mode of the 9d is by interacting its 1,2,3-triazole and ether moiety with positively charged residues and with its chlorobenzene moiety orientated toward the apolar end of the ATP binding site which are mainly composed by the Ile170, Trp167 and Leu309 residues from α subunit. These results highlight 9d derivative as a drug candidate with potential therapeutic application based on P2X7 receptor blockade.

Full text of DOI:10.1016/j.ejmech.2017.08.034

Accepted Manuscript
1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole derivatives blockade P2X7 receptor in vitro
and inflammatory response in vivo
Daniel Tadeu Gomes Gonzaga, Leonardo Braga Gomes Ferreira, Thadeu Estevam
Moreira Maramaldo Costa, Natalia Lidmar von Ranke, Paulo Anastácio Furtado
Pacheco, Ana Paula Sposito Simões, Juliana Carvalho Arruda, Luiza Pereira Dantas,
Hércules Rezende de Freitas, Ricardo Augusto de Melo Reis, Carmen Penido,
Murilo Lamim Bello, Helena Carla Castro, Carlos Rangel Rodrigues, Vitor Francisco
Ferreira, Robson Xavier Faria, Fernando de Carvalho da Silva
PII:
S0223-5234(17)30637-2
10.1016/j.ejmech.2017.08.034
EJMECH 9678
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 May 2017
Revised Date: 2 August 2017
Accepted Date: 15 August 2017
Please cite this article as: D.T.G. Gonzaga, L.B.G. Ferreira, T.E. Moreira Maramaldo Costa, N.L. von
Ranke, P. Anastácio Furtado Pacheco, A.P. Sposito Simões, J.C. Arruda, L.P. Dantas, Hé.Rezende.
de Freitas, R.A. de Melo Reis, C. Penido, M.L. Bello, H.C. Castro, C.R. Rodrigues, V.F. Ferreira, R.X.
Faria, F.d.C. da Silva, 1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole derivatives blockade P2X7 receptor in
vitro and inflammatory response in vivo, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.034.
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ACCEPTED MANUSCRIPT

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1-Aryl-1H- and 2-aryl-2H-1,2,3-triazole derivatives blockade P2X7
receptor in vitro and inflammatory response in vivo
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a,b
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Daniel Tadeu Gomes Gonzaga, Leonardo Braga Gomes Ferreira, Thadeu Estevam Moreira
d,e
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Maramaldo Costa, Natalia Lidmar von Ranke, Paulo Anastácio Furtado Pacheco, Ana Paula
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Sposito Simões, Juliana Carvalho Arruda, Luiza Pereira Dantas, Hércules Rezende de Freitas,
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Ricardo Augusto de Melo Reis, Carmen Penido, Murilo Lamim Bello, Helena Carla Castro,
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Carlos Rangel Rodrigues, Vitor Francisco Ferreira, Robson Xavier Faria, * Fernando de Carvalho
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da Silva *
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Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos, Farmanguinhos - Fiocruz,
Departamento de Síntese de Fármacos Manguinhos, CEP 21041-250, Rio de Janeiro-RJ, Brazil.
b
Universidade Federal Fluminense, Departamento de Química Orgânica, Instituto de Química,
Campus do Valonguinho, CEP 24020-150, Niterói-RJ, Brazil.
c
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Inflamação, Avenida Brasil
4365, Manguinhos, CEP 21045-900, Rio de Janeiro-RJ, Brazil.
d
Fundação Oswaldo Cruz, Laboratório de Farmacologia Aplicada, Farmanguinhos - Fiocruz, CEP
21041-250, Rio de Janeiro-RJ, Brazil.
e
Fundação Oswaldo Cruz, Centro de Desenvolvimento Tecnológico em Saúde, Instituto Nacional
de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN), Rio de Janeiro-RJ,
Brazil.
f
Universidade Federal Fluminense, Laboratório de Antibióticos, Bioquímica, Ensino e Modelagem
Molecular – LABiEMol, CEP 24020-150, Brazil.
g
Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, Departamento de Fármacos e
Medicamentos, Laboratório de Modelagem Molecular e QSAR - ModMolQSAR, CEP 21941-902,
RJ, Brazil.
h
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Toxoplasmose e outras
Protozooses, Avenida Brasil 4365, Manguinhos, CEP 21045-900, Rio de Janeiro-RJ, Brazil.
i
Universidade Federal do Rio de Janeiro, Instituto de Biofísica, Laboratório de Neuroquímica, CEP
21941-902, RJ, Brazil.

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Corresponding authors
Chemistry: Fernando de Carvalho da Silva, Universidade Federal Fluminense, Departamento de
Química Orgânica, Instituto de Química, Campus do Valonguinho, CEP 24020-150, Niterói-RJ,
Brazil; E-mail: gqofernando@vm.uff.br
Pharmacology: Robson Xavier Faria, Fundação Oswaldo Cruz, Instituto Oswaldo Cruz,
Laboratório de Comunicação Celular, Avenida Brasil 4365, Manguinhos, CEP 21045-900 - Rio de
Janeiro-RJ, Brazil.; E-mail: robson.xavier@gmail.com
*Both authors contributed equally to this work.
Abbreviations
ATP, adenosine 5′-triphosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate; IC , half-maximum
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inhibitory concentration; IL, interleukin; LPS, lipopolysaccharide; ESI, electrospray ionization;
NMR, nuclear magnetic resonance, LDH, lactate dehydrogenase; EGTA, ethylene glycol tetraacetic
+
acid; PBS, phosphate-buffered saline; NAD , Nicotinamide adenine dinucleotide; IL-1β, Interleukin
1β; PI, Propidium iodide; BBG, Brilliant Blue G; DMEM, Dulbecco's Modified Eagle's Medium;
IR, infrared spectroscopy; TMS, tetramethylsilane; TLC, Thin Layer Chromatography; ROS,
Reactive oxygen species; iNOS, inducible nitric oxide synthase; PPADS (pyridoxalphosphate-6-
azophenyl-2',4'-disulfonic
acid);
A740003,
N-[1-[[(Cyanoamino)(5-
quinolinylamino)methylene]amino]-2,2-dimethylpropyl]-3,4-dimethoxybenzeneacetamide;
A804598, AZ 10606120 dihydrochloride, N-Cyano-N"-[(1S)-1-phenylethyl]-N'-5-quinolinyl-
guanidine; N-[2-[[2-[(2-Hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-
1-ylacetamide
dihydrochloride;
A438079,
3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-
yl]methyl]pyridine hydrochloride; AZ11645373, 3-[1-[[(3'-Nitro[1,1'-biphenyl]-4-yl)oxy]methyl]-
3-(4-pyridinyl)propyl]-2,4-thiazolidinedione; THP-1 cell, human monocytic cell line derived from
an acute monocytic leukemia patient; and SAR, Structure-activity relationship.

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Abstract: Fifty-one 1,2,3-triazole derivatives were synthesized and evaluated with respect to P2X7
receptor (P2X7R) activity and its associated pore. These triazoles were screened in vitro for dye
uptake assay and its cytotoxicity against mammalian cell types. Seven 1,2,3-triazole derivatives (5e,
6e, 8h, 9d, 9i, 11, and 12) potently blocked P2X7 receptor pore formation in vitro (J774.G8 cells
and peritoneal macrophages). All blockers displayed IC value inferior to 500 nM, and they have
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low toxicity in either cell types. These seven selected triazoles inhibited P2X7R mediated
interleukin-1 (IL-1β) release. In particular, compound 9d was the most potent P2X7R blocker.
Additionally, in mouse acute models of inflammatory responses induced by ATP or carrageenan
administration in the paw, compound 9d promoted a potent blocking response. Similarly, 9d also
reduced mouse LPS-induced pleurisy cellularity. In silico predictions indicate this molecule
appropriate to develop an anti-inflammatory agent when it was compared to commercial analogs.
Electrophysiological studies suggest a competitive mechanism of action of 9d to block P2X7
receptor. Molecular docking was performed on the ATP binding site in order to observe the
preferential interaction pose, indicating that binding mode of the 9d is by interacting its 1,2,3-
triazole and ether moiety with positively charged residues and with its chlorobenzene moiety
orientated toward the apolar end of the ATP binding site which are mainly composed by the Ile170,
Trp167 and Leu309 residues from α subunit. These results highlight 9d derivative as a drug
candidate with potential therapeutic application based on P2X7 receptor blockade.
Keywords: Purinergic receptors; antagonist; anti-inflammatory; synthetic products; ATP; pore
formation.
1. Introduction
The physiology and pharmacology of the ligand-gated P2X family of ion channels have
been studied broadly in terms of their biophysical, pharmacological and physiological aspects [1-4].
As a function of its wide expression in cells of hematopoietic origin, the ATP-sensitive P2X7
receptor (P2X7R) has acquired a respectable amount of attention because recent data suggest that it
has a role in acute and chronic inflammation [5-8].
P2X7R is found on native or cell lineages of macrophages [9-12]. Activation of P2X7R with
extracellular ATP promotes ion flux [13] and the formation of a reversible cell membrane pore that
is associated with lysis and cell death [14-16]. In addition, this receptor has also been linked to
inflammatory conditions that activate and release interleukin-1 β [17,18], among other substances,
such as glutamate [19].
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The cooperation between P2X7R stimulation of glutamate metabolism and cytokines is part
of the functional rationale for its action in the amelioration and progression of important disorder
states or conditions involving inflammation [20], neurodegeneration [21], and neuropathic pain
[22,23]. In the P2X7 knockout mice, there is a decrease in the development of inflammatory and
neuropathic pain and rigor symptoms in an arthritis model [24,25].
Although there are a large number of P2X7R antagonists commercially available, research
of novel molecules with antagonist and therapeutic action on this receptor is necessary. First-
generation P2X7R antagonists (Suramin, PPADS, BBG, KN-62 and Reactive blue-2) are non-
selective inhibitors, acting also on other P2Rs [26] or in proteins related to P2X7R pore formation
mechanism [6]. The second generation of P2X7R antagonists includes JNJ-47965567 [27],
A740003 [28], GSK314181 [29], triazole derivatives A438079 [30], A839977 [31], AZ11645373
[81], AZ10606120 [32] and AZD9056 [33]. Characterization of their mechanisms of action and
pharmacologic properties in vivo are largely unknown. In some cases, they exhibit reduced
availability and variable potency according to the species studied [26].
Clinical trials using P2X7R antagonists against rheumatoid arthritis indicated clinical
efficacy and safety of the P2X7R antagonists AZD9056 or CE-224,535 [33,34]. In contrast, both
trials did not exhibit therapeutic benefit [80,81]. A possible explanation is associated to studies
related to differential pharmacological sensibility in P2X7R genotype function, as observed by
McHugh and collaborators in vitro [35]. This scenario leaves open a possibility to search and
develop novel P2X7R antagonists.
Numerous types of P2X7R antagonists have been identified [36,37], however most are
inappropriate for therapeutic use. In recent years, our scientific group has concentrated on making
P2X7R antagonist molecular low weight compounds for treating inflammation and pain. In this
context, the 1,2,3-triazoles are synthetic five members heterocyclic aromatic compounds containing
three nitrogen atoms [38]. These compounds have diverse biological activities [39] such as anti-
HIV [40], β-lactamase inhibitors [41], antiepileptic activities [42], anti-platelet agents [43,44],
dopamine D2 receptor ligands (related to Schizophrenia) [45], anti-inflammatory [46], antimicrobial
[47-50], anti-herpes simplex virus (HSV) [51], trypanocidal [52], antileishmanial agents [53],
antifungal agents [54] and glycosidase inhibitors [55,56].
Some heterocyclic compounds containing 1,2,3-triazole structure core have been described
as P2X7R antagonists [57,58]. In 2007, Carrol and colleagues promoted substitutions in a tetrazole
core inserting triazole isostere. Triazole-based P2X7 antagonists showed potency (pIC 6.43-7.12)
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and physiochemical properties improved in comparison to tetrazole analogues [59]. Based on assays
above, Florjancic and collaborates used SARs to search the aminotriazole activity at both human

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and rat P2X7R. In consequence, they observed drugs with pIC value in turn of 7.5 to block both
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receptors [60].
Honore [31] demonstrated in vitro and in vivo the inhibitory activity of a structurally novel
P2X7R antagonist, 1-(2, 3-dichlorophenyl)-N-[2-(pyridin-2-yloxy) benzyl]-1H-tetrazol-5-amine (A-
839977) in mice. A-839977 inhibited BzATP-evoked calcium influx at recombinant human, rat and
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mouse P2X7Rs. The IC values varied from 20-150 nM for Ca assay, pIC = 8.18 ± 0.03 for dye
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uptake and pIC = 7.43 ± 0.13 to IL-1β release assay.
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However, phenyl triazoles were not test against P2X7R antagonistic activity. Then, the aim
of this study was to evaluate the blocking action and cytotoxicity of 1-Phenyl-1H- and 2-phenyl-
2H-1,2,3-triazol derivatives (Fig. 2) on P2X7R activity and its associated pore.
2. Experimental Section
2.1. Chemistry
The reagents were purchased from Sigma-Aldrich Brazil and were used without further
purification. Column chromatography was performed with silica gel 60 (Merck 70-230 mesh).
Analytical thin layer chromatography was performed with silica gel plates (Merck, TLC silica gel
60 F254), and the plots were visualized using UV light or aqueous solutions of ammonium sulfate.
The indicated yields refer to chromatographically and spectroscopically homogeneous materials.
Melting points were obtained on a Fischer-Johns apparatus and were uncorrected. Infrared spectra
were measured with KBr pellets on a Perkin-Elmer model 1420 FT-IR Spectrophotometer, and the
-1
spectra were calibrated relative to the 1601.8 cm absorbance of polystyrene. NMR spectra were
recorded on a Varian Unity Plus VXR (500 MHz) instrument in DMSO-d or CDCl solutions. The
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chemical shift data were reported in units of d (ppm) downfield from tetramethylsilane or the
solvent, either of which was used as an internal standard; coupling constants (J) are reported in
hertz and refer to apparent peak multiplicities. CHN elemental analyses were performed on a
Perkin-Elmer 2400 CHN elemental analyzer.
2.1.1. General procedure for preparing 1,2,3-triazoles
The protocols for preparing all of the 1,2,3-triazoles and the physical and spectroscopic data
for 5a-e, 6a-b, 7a, 8a-k, 9a, 9c-d, 9f, 9i-n, 10, 11, 12, 13a-d, 14a and 14c were previously reported
in our studies [47,56].

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2.1.1.1. 1-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (6c). Brown solid, 84% yield; m.p.
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131-132ºC; IR (KBr, cm ): ν 3133, 2969, 1688, 1607, 1518, 1459, 1299, 1255, 1206, 1168, 1027,
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827, 777, 614; H NMR (DMSO-d , 500 MHz): 3.96 (3H, s), 7.27-7.30 (2H, m), 7.98-8.02 (2H, m),
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9.55 (1H, s), 10.22 (1H, s); C NMR (DMSO-d , 125 MHz APT): 55.7, 115.0, 122.4, 125.9, 129.3,
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147.5, 159.9, 184.9.
2.1.1.2. 1-(3,5-dichlorophenyl)-1H-1,2,3-triazole-4-carbaldehyde oxime (7b). White solid, 52%
-1
yield; m.p. 111-112ºC; IR (KBr, cm ): ν 3098, 1588, 1477, 1437, 1337, 1234, 1122, 1053, 987,
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932, 854, 812, 695, 665; H NMR (DMSO-d , 500 MHz): 7.89 (1H, t, J 2.0 Hz), 7.88 (2H, d, J 2.0
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Hz), 8.36 (1H, s), 9.30 (1H, s), 11.63 (1H, s); C NMR (DMSO-d , 125 MHz APT): δ 118.9, 120.9,
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128.2, 135.2, 138.0, 139.8, 142.6. Anal. Calcd for C H Cl N O: C, 42.05; H, 2.35; N, 21.79. Found:
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C, 41.95; H, 2.45; N, 21.65.
2.1.1.3. (1-(2,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl nonanoate (8l). White solid, 95%
-1
yield; m.p. 35-36ºC; IR (KBr, cm ): ν 3664, 2919, 2852, 1741, 1588, 1489, 1451, 1377, 1285,
1
1252, 1210, 1168, 1102, 1074, 1043, 1018, 874, 822, 722, 651; H NMR (DMSO-d , 500 MHz):
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0.97 (3H, t, J 7.0 Hz), 1.30-1.41 (12H, m), 1.59-1.67 (2H, m), 2.46 (2H, t, J 7.0 Hz), 5.36 (2H, s),
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7.85 (2H, dd, J 2.0 and 8.0 Hz), 7.93 (1H, d, J 8.0 Hz), 8.01 (1H, d, J 2.0 Hz), 8.42 (1H, s); C
NMR (DMSO-d , 125 MHz APT): δ 13.9, 22.0, 24.4, 28.4, 28.6, 28.8, 31.3, 33.4, 33.7, 56.7, 126.7,
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127.5, 128.1, 131.5, 131.9, 132.5, 135.3, 142.3, 172.6. Anal. Calcd for C H Cl N O : C, 57.29; H,
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6.33; N, 10.55. Found: C, 57.00; H, 6.25; N, 10.65.
2.1.1.4. (1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl benzoate (8m). Brown solid, 40%
-1
yield; m.p. 85-86ºC; IR (KBr, cm ): ν 3149, 3089, 1707, 1584, 1484, 1451, 1277, 1108, 1053,
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1013, 970, 855, 804, 712, 665; H NMR (DMSO-d , 500 MHz): 5.63 (2H, s), 7.64-7.68 (2H, m),
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7.78-7.81 (1H, m), 7.87 (1H, t, J 2.0 Hz), 8.12-8.14 (2H, m), 8.21 (2H, d, J 2.0 Hz), 9.20 (1H, s);
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C NMR (DMSO-d , 125 MHz APT): 57.8, 118.8, 123.3, 128.1, 128.8, 129.3, 129.3, 133.5, 135.2,
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138.1, 143.5, 165.4. Anal. Calcd for C H Cl N O : C, 55.19; H, 3.18; N, 12.07. Found: C, 55.35;
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H, 3.08; N, 12.02.
2.1.1.5. (1-(3,5-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl acetate (8n). Brown solid, 87% yield;
-1
m.p. 74-75ºC; IR (KBr, cm ): ν 3136, 3096, 2361, 1710, 1588, 1477, 1439, 1387, 1367, 1284,

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1238, 1120, 1023, 990, 951, 899, 856, 831, 795, 670, 638; H NMR (DMSO-d , 500 MHz): 2.20
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(3H, s), 5.34 (2H, s), 7.87 (1H, t, J 2.0 Hz), 8.19 (2H, d, J 2.0 Hz), 9.09 (1H, s); C NMR (DMSO-
d6, 125 MHz APT): 20.6, 56.9, 118.7, 123.2, 128.1, 135.3, 138.1, 143.6, 170.1. Anal. Calcd for
C H Cl N O : C, 46.18; H, 3.17; N, 14.69. Found: C, 46.06; H, 3.08; N, 14.32.
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2.1.1.6. (1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl benzoate (8o). White solid, 65% yield;
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m.p. 95-96ºC; IR (KBr, cm ): ν 3126, 2921, 1708, 1599, 1518, 1448, 1379, 1268, 1189, 1099,
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1035, 941, 824, 771, 708; H NMR (DMSO-d , 500 MHz): 3.95 (3H, s), 5.61 (2H, s), 7.25-7.26
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(2H, m), 7.65 (2H, m), 7.79 (1H, m), 8.11-8.13 (2H, m), 8.95 (1H, s); C NMR (DMSO-d , 125
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MHz APT): 55.6, 57.9, 114.9, 121.9, 123.0, 128.8, 129.3, 129.4, 130.0, 133.5, 142.9, 159.4, 165.5.
Anal. Calcd for C H N O : C, 66.01; H, 4.89; N, 13.58. Found: C, 66.21; H, 4.85; N, 13.60.
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2.1.1.7. (1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl acetate (8p). Brown solid, 82% yield;
-1
m.p. 60-61ºC; IR (KBr, cm ): ν 3143, 1722, 1610, 1517, 1440, 1375, 1303, 1247, 1191, 1111,
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1027, 976, 921, 829, 759, 632; H NMR (DMSO-d , 500 MHz): 2.18 (3H, s), 3.95 (3H, s), 5.32
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(2H, s), 7.24-7.27 (2H, m), 7.90-7.93 (2H, m), 8.83 (1H, s); C NMR (DMSO-d , 125 MHz APT):
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20.6, 55.5, 57.0, 114.9, 121.8, 122.8, 130.0, 142.9, 159.4, 170.1. Anal. Calcd for C H N O : C,
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58.29; H, 5.30; N, 16.99. Found: C, 58.49; H, 5.25; N, 16.89.
2.1.1.8. (1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl pentanoate (8q). Yellow oil, 55% yield;
-1
IR (KBr, cm ): ν 2932, 2870, 1734, 1611, 1518, 1462, 1304, 1253, 1164, 1109, 1034, 989, 832,
1
770; H NMR (DMSO-d , 500 MHz): 0.96 (3H, t, J 7.4 Hz), 1.35-1.40 (4H, m), 1.52 (2H, p, J 7.4
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Hz), 2,45 (2H, t, J 7.4 Hz), 3.95 (3H, s), 5.33 (2H, s), 7.25 (2H, d, J 9.0 Hz), 7.91 (2H, d, J 9.0 Hz),
1
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8.82 (1H, s); C NMR (DMSO-d , 125 MHz APT): 13.7, 21.7, 24.1, 30.6, 33.3, 55.6, 56.9, 114.9,
6
121.8, 122.8, 130.0, 142.9, 159.4, 172.6. Anal. Calcd for C H N O : C, 63.35; H, 6.98; N, 13.85.
1
6
21
3
3
Found: C, 63.05; H, 7.12; N, 13.89.
-1
2.1.1.9. 1-phenyl-4-(propoxymethyl)-1H-1,2,3-triazole (9b). Yellow oil, 62% yield; IR (KBr, cm ):
1
ν 2961, 2933, 2872, 1730, 1598, 1503, 1465, 1377, 1339, 1229, 1095, 1040, 989, 814, 757, 690; H
NMR (DMSO-d , 500 MHz): 0.87 (3H, t, J 7.0 Hz), 1.54 (2H, sex, J 7.0 Hz), 3.45 (2H, t, J 7.0 Hz),
6
1
3
4.58 (2H, s), 7.75 (1H, t, J 7.7 Hz), 7.59 (2H, t, J 7.7 Hz), 7.89 (2H, d, J 7.7 Hz), 8.76 (1H, s); C
NMR (DMSO-d , 125 MHz APT): 10.6, 22.5, 63.3, 71.5, 120.2, 122.2, 128.8, 130.0, 136.8, 145,6.
6
Anal. Calcd for C H N O: C, 66.34; H, 6.96; N, 19.34. Found: 66.54; H, 7.12; N, 18.89.
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2.1.1.10. 1-(4-chlorophenyl)-4-(propoxymethyl)-1H-1,2,3-triazole (9e). Yellow solid, 45% yield;
-1
m.p. 75-76 ºC; IR (KBr, cm ): ν 3160, 2968, 2932, 2871, 1721, 1563, 1502, 1456, 1341, 1229,
1
1194, 1092, 984, 954, 837, 817, 783, 737, 697, 753; H NMR (DMSO-d , 500 MHz): 0.99 (3H, t, J
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7.0 Hz), 1.66 (2H, sex, J 7.0 Hz), 3.56 (2H, t, J 7,0 Hz), 4.70 (2H, s), 7.77-7.79 (2H, m), 8.05-8.07
1
3
(2H, m), 8.90 (1H, s); C NMR (DMSO-d , 125 MHz APT): 10.6, 22.5, 63.3, 71.5, 121.9, 122.2,
6
129.9, 133.1, 135.6, 145.7. Anal. Calcd for C H ClN O: C, 57.26; H 5.61; N, 16.69. Found: C,
1
2
14
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57.42; H, 5.82; N, 16.37.
2.1.1.11. 1-(2,5-dichlorophenyl)-4-(ethoxymethyl)-1H-1,2,3-triazole (9g). Yellow oil, 50% yield; IR
-1
(KBr, cm ): ν 3141, 2975, 2868, 1732, 1588, 1487, 1448, 1376, 1231, 1097, 1038, 874, 809, 698,
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671, 651; H NMR (DMSO-d , 500 MHz): 1.27 (3H, t, J 3.9 Hz), 3.67 (2H, q, J 3.9 Hz), 4.70 (s,
6
1
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2H), 7.84 (1H, dd, J 1.5 and 5.4 Hz), 7,92 (1H, d, J 5.4 Hz), 8.02 (1H, d, J 1.5 Hz), 8.63 (1H, s); C
NMR (DMSO-d , 125 MHz APT): 14.9, 62.8, 65.0, 125.8, 127.4, 128.0, 131.2, 131.8, 132.4, 135.5,
6
144.4. Anal. Calcd for C H Cl N O: C, 48.55; H 4.07; N, 15.44. Found: C, 48.63; H, 4.02; N,
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15.37.
2.1.1.12. 1-(2,5-dichlorophenyl)-4-(propoxymethyl)-1H-1,2,3-triazole (9h). Yellow oil, 50% yield;
-1
IR (KBr, cm ): ν 2962, 2872, 1588, 1486, 1450, 1369, 1231, 1096, 1037, 1000, 874, 811, 760, 694,
1
651; H NMR (DMSO-d , 500 MHz): 0.99 (3H, t, J 7.0 Hz), 1.67 (2H, p, J 7.0 Hz), 3.58 (2H, t, J
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7.0 Hz), 3.61 (2H, t, J 6.9 Hz), 4.72 (2H, s), 7.85 (1H, dd, J 2.5 and 9.0 Hz), 7.92 (1H, d, J 9.0 Hz),
13
8.01 (1H, d, J 3.0 Hz); 8.66 (1H, s); C NMR (DMSO-d , 125 MHz APT): 10.5, 22.4, 63.0, 71.3,
6
125.9, 127.5, 128.2, 131.4, 131.9, 132.5, 135.5, 144.4. Anal. Calcd for C H Cl N O: C, 50.37; H
1
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13
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4.58; N, 14.68. Found: C, 50.24; H, 4.52; N, 15.30.
2.1.1.13. 1-(3,5-dichlorophenyl)-4-(ethoxymethyl)-1H-1,2,3-triazole (9j). Yellow solid, 54% yield;
-1
m.p. 49-50ºC; IR (KBr, cm ): ν 2974, 2927, 2865, 1728, 1585, 1475, 1440, 1374, 1334, 1278,
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1094, 1039, 851, 807, 666; H NMR (DMSO-d , 500 MHz): 1.28 (3H, t, J 6.5 Hz), 3.67 (2H, t, J
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1
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6.5 Hz), 4.71 (2H, s), 7.86 (1H, t, J 2.0), 8.19 (1H, d, J 2.0 Hz), 9.03 (1H, s); C NMR (DMSO-d ,
6
125 MHz APT): 15.0, 63.0, 65.1, 118.6, 122.4, 127.9, 135.2, 138.3, 145.7. Anal. Calcd for
C H Cl N O: C, 48.55; H 4.07; N, 15.44. Found: C, 48.66; H, 3.98; N, 15.40.
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2.1.1.14. 1-(3,5-dichlorophenyl)-4-(propoxymethyl)-1H-1,2,3-triazole (9k). Yellow solid, 54%
-1
yield; m.p. 49-50ºC; IR (KBr, cm ): ν 3142, 3048, 2922, 2872, 1585, 1476, 1436, 1365, 1337,
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1228, 1088, 1036, 1004, 955, 889, 853, 811, 667; H NMR (DMSO-d , 500 MHz): 0.88 (3H, t, J
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7.5 Hz), 1.55 (2H, sex, J 7.5 Hz), 3.45 (2H, t, J 7.5 Hz), 4.58 (2H, s), 7.74 (2H, t, J 2.0 Hz), 8.07
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(2H, d, J 2.0 Hz), 8.91 (1H, s); C NMR (DMSO-d , 125 MHz APT): 10.5, 22.4, 63.2, 71.4, 118.5,
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122.3, 127.8, 135.2, 138.2, 145.7. Anal. Calcd for C H Cl N O: C, 50.37; H 4.58; N, 14.68.
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Found: C, 50.44; H, 4.59; N, 14.34.
2.1.1.15. 1-(4-methoxyphenyl)-4-(propoxymethyl)-1H-1,2,3-triazole (9m). Yellow oil, 65% yield;
-1
IR (KBr, cm ): ν 2873, 1611, 1518, 1461, 1377, 1303, 1253, 1190, 1094, 1037, 989, 832, 768, 695;
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H NMR (DMSO-d , 500 MHz): 0.99 (3H, t, J 7.0 Hz), 1.67 (2H, sex, J 7.0 Hz), 3.56 (2H, t, J 7.0
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Hz), 3.95 (3H, s), 4.69 (2H, s), 7.23-7.26 (2H, m), 7.90-7.94 (2H, m), 8.78 (1H, s); C NMR
(DMSO-d , 125 MHz APT): 10.5, 22.4, 55.6, 63.3, 71.3, 114.9, 121.7, 122.0, 130.1, 145.1, 159.3.
6
Anal. Calcd for C H N O : C, 63.14; H 6.93; N, 16.99. Found: C, 63.44; H, 5.72; N, 16.55.
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2.1.1.16. 4-(butoxymethyl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (9n). Brown solid, 54% yield;
-1
m.p. 63-64ºC; IR (KBr, cm ): ν 2932, 2866, 1610, 1517, 1462, 1375, 1304, 1254, 1190, 1095,
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1039, 989, 831, 767, 695; H NMR (DMSO-d , 500 MHz): 0.99 (3H, t, J 6.9 Hz); 1.41-1.48 (2H,
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m), 1.60-1.67 (2H, m), 3.60 (2H, t, J 6.6 Hz), 3.95 (OCH ), 4.68 (2H, s), 7.25 (2H, d, J 9.2 Hz),
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7.92 (2H, d, J 9.2 Hz), 8.78 (1H, s); C NMR (DMSO-d , 125 MHz APT): 13.8, 18.9, 31.2, 55.6,
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63.3, 69.4, 114.9, 121.8, 122.0, 130.2, 145.2, 159.3. Anal. Calcd for C H N O : C, 64.35; H 7.33;
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N, 16.08. Found: C, 64.44; H, 6.89; N, 16.35.
-
2.1.1.17. 2-phenyl-4-(propoxymethyl)-2H-1,2,3-triazole (14b). Yellow oil, 98% yield; IR (KBr, cm
1
1
): ν 2926, 2873, 1598, 1498, 1462, 1414, 1356, 1312, 1100, 1047, 965, 910, 850, 754, 690, 665; H
NMR (DMSO-d , 500 MHz): 1.00 (3H, t, J 7.0 Hz), 1.67 (2H, sex, J 7.0 Hz), 3.57 (2H, t, J 7.0 Hz),
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1
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4.75 (2H, s), 7.51-7.56 (1H, m), 7.66-7.70 (2H, m), 8.10-8.13 (2H, m), 8.18 (1H, s); C NMR
(DMSO-d , 125 MHz APT): 10.5, 22.4, 63.1, 71.6, 118.3, 127.4, 127.6, 135.6, 139.2, 147.3. Anal.
6
Calcd for C H N O: C, 66.34; H, 6.96; N, 19.34. Found: C, 66.49; H, 7.02; N, 19,12.
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2.2.1. In Vitro experiments

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2.2.1.1. Mice peritoneal macrophages. Mice peritoneal macrophages were harvested from male
Swiss mice through the lavage of their peritoneal cavity with 10 mL of Dulbecco's modified Eagle's
medium (DMEM) medium. Our protocols adhered to the Ethical Principles in Animal
Experimentation adopted by the Brazilian College of Animal Experimentation and were approved
by the FIOCRUZ Research Ethics Committee (number LW-033/12). The isolated cells were
centrifuged and resuspended. Aliquots (0.5 mL) of cell suspension were added to microplate wells
and placed in a humidified atmosphere (37 °C, 5% CO ) for 30 minutes for cell adhesion. Non-
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adherent cells were removed by washing with DMEM medium containing 10% fetal bovine serum
(FBS) and gentamycin (1 µL/mL). Firmly adhering cells were re-suspended in phenol red-free
DMEM medium and used for subsequent experimental procedures.
2.2.1.2. HEK-293 cells transfected with P2X7R. HEK-293 cells expressing P2X7R were maintained
in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics
(50 U/ml penicillin and 50 mg/ml streptomycin) in a humidified 5% CO atmosphere at 37 °C.
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After diluting to 2.5 X 10 cells/ml, 80 mL of the cell suspension was added to each well of 96-well
culture plates. The 9d derivative was incubated for 10 minutes and then ATP was added, and the
cells were incubated for 20 minutes in a humidified 5% CO2 atmosphere at 37 °C. After incubation,
a Gemini fluorescence plate reader was used to measure the absorbance at an excitation wavelength
of 530 nm and an emission wavelength of 620 nm. The inhibition (percent) of ethidium ion uptake
was expressed as a relative value of the maximum accumulation when stimulated with ATP. To
calculate IC50 values, we calculated a series of dose-response data using nonlinear regression
analysis (i.e., percentage accumulation of ethidium bromide vs compound concentration).
2.2.1.3. Spectrofluorometric Measurement of Dye uptake. Peritoneal macrophages expressing the
6
P2X7Rs were resuspended at 2.5 x 10 cells/mL in assay buffer composed of 10 mM HEPES, 5
mM KCl, 1 mM MgCl , 1 mM CaCl and 140 mM NaCl (pH 7.4). The 1,2,3-triazole derivatives or
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Brilliant Blue G (BBG) (as a standard inhibitor) was added to each sample, followed by the P2X7R
agonist ATP or BzATP. The plates were incubated at 37 °C for 30 minutes, and the cellular
accumulation of propidium iodide (PI) to peritoneal macrophages assays and ethidium bromide
(EB) to HEK-293 trasfected cells was determined by measuring the fluorescence with a Molecular
Devices SpectraMax M5 fluorescent plate reader (excitation wavelength 530 nm; emission
wavelength 590 nm).

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2.2.1.4. LDH Release Assay. The presence of LDH in the media was detected in all of the
experiments using a cytotoxicity detection kit (Sigma kit for LDH) according to the manufacturer’s
+
instructions. The cell supernatants were tested for LDH, which reduces NAD , which in turn
converts tetrazolium dye into a soluble, colored formazan derivative. In this assay, we treated the
cells for 1 h, 24 h and 72 h with 1,2,3-triazole derivatives.
2.2.1.5. Dye uptake assay. Cell permeabilization was visualized by the differential uptake of
propidium iodide (696 Da). Macrophages were incubated with 1 mM ATP or 100 µM BzATP, with
or without P2X7R antagonists or 1,2,3-triazole analogs for 25 minutes at 37 °C. PI (0.05 mg/mL in
PBS) or ethidium bromide (750 ng/mL in PBS) was added during the last 5 minutes of the
incubation. Microplate wells were washed with saline solution (150 mM KCl, 5 mM NaCl, 1 mM
MgCl , 0.1 mM EGTA and 10 mM HEPES, pH 7.4) or PBS, pH 7.4 and observed under a
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fluorescence microscope (Nikon) equipped with rhodamine (546/FT 580/LP 590) and fluorescein
(450-490/FT 510/LP 520) filters. The fluorescence pattern was also analyzed by flow cytometry.
Dead cells and cellular debris were excluded based on low forward and side scatters and an
extremely high fluorescence profile. Simultaneously, samples with 1 mM ATP, with or without
P2X7R antagonists or 1,2,3-triazole analogs were incubated at 37 °C for 25 minutes; the PI was
added during the final 5 minutes and the samples were analyzed immediately. The 1,2,3-triazole
analog doses varied from 0.01 ng/mL to 10 µg/mL. P2X7R antagonists (BBG or A740003) were
used as the control.
2.2.1.6. Electrophysiological measurements. A whole-cell configuration was set up as described by
Faria and coworkers [61]. The series resistance was 5-11 MΏ for all of the experiments in standard
saline (bath and pipette solutions), and no compensation was applied for currents less than 1500 pA.
Above this level, the currents were compensated by 90%. Measurements were discarded when the
series resistance was increased substantially. Macrophage (mean ± s.d., 13.2 ± 4.44 pF; n = 106)
cell capacitance was measured by applying a 20 mV hyperpolarizing pulse from a holding potential
of 20 mV; the capacitive transient was then integrated and divided by the amplitude of the voltage
step (20 mV). All recordings were obtained in a holding potential of -60 mV at 37 °C.
2.2.1.7. Saline solutions for electrophysiology. Different saline solutions were used in the pipette or
the bath, depending on the protocol. The bath solution (in mM) consisted of the following: 150
NaCl, 5 KCl, 1 MgCl , 1 CaCl , and 10 HEPES (pH, 7.4); the pipette solution (in mM) consisted of
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the following: 150 KCl, 5 NaCl, 1 MgCl , 10 HEPES, and 0.1 EGTA (pH, 7.4).
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2.2.1.8. Drug application. Patch clamp experiments were performed under perfusion (RC-24
chamber, Warner Instrument Corp.) at a rate of 1 mL/min to confirm the data obtained by
micropipette application. All of the drugs were dissolved in saline solution immediately before use.
Ion currents were studied by applying 1 mM ATP (for 300 s) and adding or not adding 1,2,3-
triazole derivatives or BBG.
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2.2.1.9. IL-1β production by THP-1 macrophages and mice peritoneal macrophages. THP-1 cells
were maintained in Roswell Park Memorial Institute 1640 medium (RPMI medium) supplemented
with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) in a humidified 5% CO2
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atmosphere at 37 °C. Cells were plated at 2 X 10 cells/well in 96-well culture plates, and 500
ng/ml of phorbol 12-myristate 13-acetate (PMA) and 10 ng/ml of IFN-γ were co-treated for 24 h to
differentiate human monocytic THP-1 cells into macrophage-like cells. PMA-differentiated human
THP-1 macrophages were treated with BBG, A740003 and 9d derivative for 30 minutes at 37 °C,
followed by stimulation with LPS (1 µg/ml) for 4 h. THP- 1 cells received 1 mM ATP for 30
minutes at 37 °C. Cells were centrifuged at 1000 rpm for 5 minutes at 4 °C, and the supernatants
were collected and stored at -70 °C. IL-1β release was measured using a Human IL-1 beta ELISA
Kit (ab46052 -ABCAM, Cambridge).
Peritoneal macrophages were primed 4 hours with LPS 100 ng/mL. After washes with PBS,
these macrophages were treated with 1 mM ATP for 30 min at 37 ºC. In some assays, BBG,
A740003 and 9d derivative were added 15 min before the addition of LPS or ATP. The mature
form of IL- released from macrophages was quantified by sandwich ELISA following
manufacturers’ protocols (eBioscience (San Diego, CA, USA).
2.2.1.10. Caco-2 cells culture and incubations. Caco-2 cells were seeded in 96-well polyester
®
Corning Costar® transwell plates (Sigma-Aldrich, St. Louis, MO, USA) at a density of 3 × 105
cells/well with medium (DMEM with 10% fetal calf serum. Caco-2 cells cultured for up to 21 days
in a humidified incubator maintained at 37 °C in an atmosphere of 5% CO . The medium and
2
monolayer integrity were checked every 3 days by measuring the Lucifer Yellow. On the day of
experiment, working solutions of the 9d, vinblastine (poor permeability control), and propranolol
(high permeability control) at 100 µM were prepared in transport buffer (HBSS and 25 mM
HEPES) at pH 7.4 or 6.5 and 0.5% (v/v) DMSO. Cells were washed with prewarmed (37 ºC)
transport buffer at the corresponding pH. To equilibrate the cells with the transport buffer, 0.3 mL
of transport buffer were added to the wells. Feeder tray was substituted for a 24- well Enhanced

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Recovery Plate with 1 mL of transport buffer at pH 7.4. These plates are treated with a hydrophilic
covalent coating to resist nonspecific adsorption of compounds. Then, cells were incubated for 30
minutes at 37 °C and 5% CO . After this time, transport buffer in the apical wells was removed and
2
0.3 mL of the corresponding 9d, vinblastine or propranolol solution was added. The cells were
placed again in the incubator for 60 minutes. Then the plates were separated, and LY concentrations
were measured in the donor and acceptor wells and initial solution. For lucifer yellow, fluorescence
was measured at plate reader M5 (molecular probes) at excitation 485 nm and emission 530 nm.
2.2.1.11. pH Dependent Solubility of 9d. Kinetic solubility 9d was assessed from 1 to 250 µM by
spiking DMSO stock solutions (5 µL, in triplicate) into 995 µL buffer (pH 2.0-hydrochloride, 4.0-
100 mM citrate buffer and 7.4-100 mM phosphate buffer) in a 96-well plate and placing at room
temperature for 2 h. Calibration standards were constructed by spiking 5 µL of DMSO stock
solutions into 995 µL acetonitrile/buffer (1:1) mixture. After centrifugation (10,000 rpm, 10 min, 25
ºC) the reaction samples were diluted 1:1 with acetonitrile.
2.2.1.12. Distribution coefficient (Log D) in octanol/PBS pH 7.4. Octanol and PBS pH 7.4 with
ratio 1:1 (v/v) taken in a flask and shaken mechanically for 24 hours to pre-saturate PBS with
octanol and octanol with PBS. Pre-saturated solvents were used for the present study in according
to [62] methodogy. Triazole 9d in a volume of 4 µL and concentration of 25 mM added in 396 µL
PBS undergo partitioning with different volumes of octanol (100- 400 µl). After 2 hours with
vigorous shaking for mixture, centrifugation at 3000 rpm for 5 minutes to separate, followed by 1
hour standing without disturbance. PBS layer was taken out. Acetonitrile (100 µl) added to a 100 µl
PBS aliquot had its absorbance measured (396 µL PBS containing 4 µL of 25 mM 9d + 400 µL
acetonitrile). Two standard compounds were similarly studied to validate the assay.
2.2.1.13. In vitro stability assays in liver microsomes. Liver microsomes from male mouse and
human with final protein concentration of 0.5 mg/ml in 0.1 M phosphate buffer at pH 7.4. Triazole
9d with final concentration of 1 µM and DMSO concentration of 0.5 µM were pre-incubated at 37
°C before NADPH addition with final concentration of 1mM to initiate the reaction. The final
incubation volume was 50 µl. Buffer containing 0.1 M phosphate at pH7.4 was used as control
replacing NADPH (minus NADPH). Diazepam for mice , and verapamil for human were incubated
as positive control. Triazole 9d and controls were incubated for 0, 5, 15, 30 and 45 min. Negative
control (minus NADPH) was incubated for 45 min. only. Methanol (50 µL) was used to stop

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reactions at the appropriate time points. Samples incubated in plates were centrifuged at 1640 × g
for 20 min. at 4 °C to aid protein precipitation.
In vitro intrinsic clearance (CLint mic) for the metabolism of 9d in mouse and human liver
microsomes was calculated using equations below:
Half life (t ) (min) = 0.693/k (1)
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/2
V(µL/mg) = volume of incubation (µL)/protein in the incubation (mg) (2)
Intrinsic Clearance (CL ) (µL/min/ mg protein) = V x 0.693/ t1/2 (3)
int
in according to Biosystem instructions.
2.2.2. In Vivo experiments
Male Swiss Webster mice (18 to 20 g) provided by Oswaldo Cruz Foundation breeding unit
(Fiocruz, Rio de Janeiro, Brazil) were used. Mice were caged with free access to food and fresh
water in a room with temperature ranging from 22 to 24 °C and a 12 h light/dark cycle at Helio &
Peggy Pereira vivarium experimental animal facility. All experimental procedures were performed
according to Oswaldo Cruz Foundation's Committee on Ethical Use of Laboratory Animals
(number LW-58/14).
2.2.2.1. Paw edema. The in vivo efficacy of the novel P2X7R antagonists in the 1,2,3-triazole series
was evaluated using a paw edema inflammatory model. In these experiments, 1,2,3-triazole were
administered oral (for gavage) or intraperitoneally 60 minutes prior to the intrathecal administration
of a 1 mM ATP saline suspension. Thirty minutes later, the paw edema was measured and the
animals were euthanized by CO inhalation, and their peritoneal cavities were lavaged (2 x 15 mL)
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with ice-cold phosphate-buffered saline (w/o Ca and Mg ). For cytokine determinations, the
samples were spun at 10000 X g in a refrigerated microfuge (4 °C). The supernatants were removed
and frozen until the IL-1β levels were determined by ELISA technique.
2.2.2.2. LPS-induced pleurisy. Male Swiss mice received intrathoracic (i.t.) injection of 0.1 mL of
LPS (250 ng/cavity, from E. coli serotype 0127:B8) or vehicle (control group) using an adapted
needle (13 x 0.45 mm) carefully inserted at a depth of 1 mm into the right side of the thoracic cavity
of mice. Twenty four hours after the stimulus, the animals were killed in a CO chamber, the
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thoracic cavity was opened and washed with 1 mL of heparinized saline (10 UI ml ). Pleural wash
aliquots were collected and diluted in Turk solution (2% acetic acid) for total leucocyte count in
Neubauer chambers. Differential leucocyte analysis was performed on cytocentrifuged smears
stained by the May-Grunwald-Giemsa method Sterile saline (0.9%)-injected animals constituted the
control group.
2.2.2.3. Leukocyte counts. Total leukocyte counts were made in Neubauer chamber, under an
optical microscope, after dilution in Türk fluid (2% acetic acid). Differential counts of mononuclear
cells, neutrophils and eosinophils were made by using stained cytospins (Cytospin 3, Shandon Inc.,
Pittsburgh, PA) by May-Grünwald-Giemsa method. Counts are reported as numbers of cells per
cavity.
2.2.2.4. Statistical analyses. Statistical comparisons were expressed as the mean ± SD (standard
deviation) as indicated in the text. The statistical significance of the differences between means was
tested by one-way ANOVA followed by Tukey’s test. A bicaudal p < 0.05 was considered
significant.
2.3. In Silico evaluation
2.3.1. ADMET properties. The prediction of the compounds druglikness such as pharmacokinetic
and toxicological profile are important parameters for drug design. Thus, the screening of the
®
compound 9d toxicity profile was performed using Osiris program from Actelion Pharmaceuticals
Ltda. (http://www.organic-chemistry.org/prog/peo/) and the compound 9d pharmacokinetic profile
®
was performed by ADMET Predictor (Simulation Plus).
2.3.2. Ligand preparation. The inhibitors (9d, 8h and 12) molecular structure was built by
Spartan´10 v.1.0.1. Thus, the conformer distribution was applied to obtain the local energy
minimum conformers using MMFF force field [63]. A selected conformer was submitted to
equilibrium geometry applying the RM1 (Recife Model 1) semi-empirical method [64]. Finally, the
single point energy calculation was performed by the Density Function Theory (DFT) using
B3LYP/6-31G** quantum basis sets [65-67].
2.3.3. Molecular docking. The molecular docking was carried out by AutoDock Vina program
[68,69]. For this purpose the apo closed state human trimeric protein P2X7 structure was retrieved

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from the Protein Data Bank (PDB ID: 5U1L) [70]. Both receptor and ligand were previously
prepared by AutoDock tools 4.2.6 which included the addition of hydrogen atoms as well as
Gasteiger charges. The grid box of dimension 16x16x16 Å was centered around the eight conserved
residues known to be involved in ATP binding cavity such as: Lys64, Lys66, Phe188 and Thr189
from one subunit, and Asn292, Phe293, Arg294 and Lys311 from an adjacent subunit [71,72]. For
supporting this docking approach a re-docking was performed by using the crystal complex P2X7-
JNJ47965567 recently deposited in the Protein Data Bank by the code 5U1X. The re-docking
clearly identifies that the ligand JNJ47965567 docked preferably in the allosteric site, which
comprises a groove formed between two neighboring subunits. In addition, a further re-docking
centered on the allosteric site reproduced the main interaction performed by the ligand in the crystal
structure. The superposition of the ligand JNJ47965567 crystal structure and conformation
generated by the re-docking is presented in Fig. 1.
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17
18
5
Fig. 1. superposition of the ligand JNJ47965567 into the crystal P2X7 structure (PDB ID: 5U1X).
In green is depcted the crystal binding pose and in pink is depicted the best docking obtained from
AutoDock Vina program
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3. Results
3.1. Chemistry
3.1.1. Synthesis of 1H -1,2,3-triazoles [73]

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The method used to prepare the 1H-1,2,3-triazole was based on a variant of the Huisgen 1,3-
dipolar cycloaddition protocol [74] in which a reaction of aryl azides (from anilines 1a-e) and
propargylic alcohol was catalyzed by Cu(I), providing only regioisomer 1,4-disubstituted 5a-e at
high yields (55-82%). The partial oxidation of 5 generated the 4-carboxaldehyde-1H-1,2,3-triazoles
(6a-c) with yields ranging from 64-84%, and afterwards, treating with NH OH·HCl in caustic
2
solution yielded oximes (7a,b). The esterified (8a-q) and etherified (9a-n) derivatives were made
from a nucleophilic substitution reaction between the alcohol (5) and acid chlorides or alkyl
bromides in basic medium, respectively (Fig. 2).
The 2H-1,2,3-triazole series was obtained starting from Fischer’s method to obtain
glucoseosazone 3 from
D-glucose, followed by oxidative cyclization by Hudson’s method
(refluxing in an aqueous solution of CuSO ). This method generated the osotriazole 4, which by
4
treatment with aqueous NaIO afforded the 3-carboxaldehyde-2H-1,2,3-triazoles (10). In sequence,
4
we synthesized 2H-1,2,3-triazole alcohol (12) by reduction with NaBH , which was obtained at a
4
quantitative yield. The oxime (11), the esters (13a-d) and the ethers (14a-c) were prepared using the
same protocol as above with yields ranging from 30% to quantitative (Fig. 2).

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Fig. 2. Synthetic routes to 1H-1,2,3- and 2H-1,2,3-triazole series. The reagents and conditions were
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as follows: i) NaNO , HCl 50%, 0-5 ºC then NaN , H O; ii) Propargylic alcohol, CuSO , ascorbic
2
(aq)
3
2
4
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acid, tBuOH, H O; iii) R-Br, THF, NaH, reflux; iv) RCOCl, CH Cl , Pyridine, DMAP, rt; v) IBX,
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DMSO, rt; vi) NH OH.HCl, CH Cl , Pyridine, rt; vii) PhNHNH .HCl, H O, reflux; viii) CuSO ,
2 2 2 2 2 4
H O, reflux; xix) NaIO , H O, rt and x) NaBH , CH OH, rt.
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3.2. Biology
3.2.1. In Vitro
P2X7R function was measured using whole cell experiments and dye uptake assays in mice
peritoneal macrophages (MPM) and HEK 293 cell transfected with hP2X7R.
Triazoles screnning activity against mice P2X7R functionality. ATP-induced pore formation
in MPM measure with a Fluorescent Imaging Plate Reader (FLIPR). We screened the antagonistic
1,2,3-triazole analogs inhibitory activity using 10 µM as cutoff. MPM in 96-well plates were treated
with 10 µM 1,2,3-triazole analogs in the presence of 1 mM ATP for 15 minutes. We grouped
compounds according to their chemical groups to facilitate the understanding of the results. In
parallel, we measured LDH release induced by 1,2,3-triazoles alone in the concentration of 10 µM
after 60 minutes of continuous exposition. As criterions to select the promising triazoles, we
considered the compounds with percentage inhibition higher than 75%, when compared to ATP
response alone and toxicity less than 20%.
As shown in Table 1, when we applied esters triazoles in MPM, the molecules 8f, 8g, 8i, 8k,
8l, 8m, 8n, and 8q did not block ATP-induced dye uptake. Analogues 8a, 8c, 8p, 13c and 13d
modestly inhibited ATP-induced dye uptake. Analogues 8b, 8d, 8e, 8h, 8j, 13a and 13b inhibited
P2X7R pore formation in comparison with treatments with 1 mM ATP alone. When we studied the
toxicity caused by these compounds (Table 1), triazoles 8a, 8b, 8d, 8e, 8g, 8i, 8j, 8k, 8m, 8n, 8q,
13a, 13b, 13c and 13d released more than 20% of LDH, and they were considered toxic in
comparison with the negative control. Among the esters compounds, analog 8j inhibited ATP-
induced pore formation in a manner similar to that of BBG (Table 1), but it exhibited considerable
toxicity relative to the negative control (Table 1, compare the second with fourth column). Merely,
ester containing compound 8h inhibited ATP-induced dye uptake and showed low toxicity to
mammalian cells.
Table 1. Effects of ester triazoles in P2X7R antagonistic activity
Compound
% Inhibition (a) % LDH release (b)
8
8
a (X = H, R = Ph)
b (X = H, R = Me)
32.1 ± 2.02
83.95 ± 2.93
58.1 ± 4.86
36.39 ± 11.02

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c (X = H, R = pentyl)
44.6 ± 0.46
59.99 ± 8.34
60.61 ± 5.78
23.18 ± 6.75
11.1 ± 4.52
76 ± 2.9
11 ± 10.44
76.6 ± 2.95
20.65 ± 8.26
9.86 ± 4.87
14.21 ± 4.83
65.18 ± 1.88
24.38 ± 3.94
29.44 ± 2.99
26.55 ± 4.22
81.3 ± 2.91
73.5 ± 1.95
29.6 ± 0.37
29.5 ± 4.02
78.89 ± 3.81
15.83 ± 1.3
d (X = H, R = nonyl)
e (X = 4-Cl, R = Ph)
f (X = 4-Cl, R = Me)
g (X = 4-Cl, R = pentyl)
h (X = 4-Cl, R = nonyl)
i (X = 2,5-diCl, R = Ph)
j (X = 2,5-diCl, R = Me)
k (X = 2,5-diCl, R = pentyl)
l (X = 2,5-diCl, R = nonyl)
m (X = 3,5-diCl, R = Ph)
n (X = 3,5-diCl, R = Me)
o (X = 4-OMe, R = Ph)
p (X = 4-OMe, R = Me)
q (X = 4-OMe, R = pentyl)
3a (R = Ph)
42.53 ± 0.4
45.73 ± 1.71
19.66 ± 0.6
34.19 ± 2.01
8 ± 0.6
26.66 ± 1.2
32 ± 1
22.66 ± 0.3
10 ± 0.7
24.02 ± 1.7
45.33 ± 1.4
10.66 ± 0.5
15 ± 1.9
46.3 ± 5.12
36.94 ± 10.6
38.5 ± 6.2
43.04 ± 5.01
42.5 ± 1.04
13.22 ± 2.66
3b (R = Me)
3c (R = pentyl)
3d (R = nonyl)
BBG (c)
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5
5
5
5
5
5
5
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84
85
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(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of propidium
stimulated by 1 mM ATP only. Data values are expressed as means ± SDs. All experiments were repeated at least 3
and 5 times.
(b) % LDH release values at 10 µM were expressed as a percentage, relative to maximum LDH release caused
by 0.05% Triton X- 100 only. Data values are expressed as means ± SDs. All experiments were repeated at least 3 and 4
times.
(c) BBG concentration of 750 nM.
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Alcohol containing triazoles derivatives 5a-d did not diminish ATP-induced dye uptake
(Table 2), however compounds 5e and 12 led to inhibition with low cytotoxic. For this reason, we
ruled out 5a, 5b, 5c and 5d triazoles.
Four synthesized aldehydes are shown in the Table 2. Analogs 6b and 10 alone were not
able to inhibit ATP-induced pore formation. Other analogs, namely 6a and 6c, impaired ATP-
induced dye uptake. They showed low cytotoxic when applied for 60 minutes. Additionally,
compound 6a effectively diminished ATP-induced dye uptake compared with ATP treatment alone
and 6c partially reduced.
Table 2. Effects of alcohol, aldehyde and oxime triazoles in P2X7R antagonistic activity
Compound
% Inhibition (a)
% LDH release (b)
5
a (X = H)
10.87 ± 3.96
22.52 ± 1.86
28.33 ± 1.7
11.36 ± 1.89
20.98 ± 0.2
15.57 ± 3.2
12.09 ± 0.01
30.55 ± 5.81
5
b (X = 4-Cl)
c (X = 2,5-diCl)
d (X = 3,5-diCl)
Alcohol
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e (X = 4-OMe)
86.31 ± 1.03
84.16 ± 1.91
9.7 ± 2.1
9.18 ± 0.4
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a (X = H)
b (X = 2,5-diCl)
c (X = 4-OMe)
84.6 ± 1.19
36.74 ± 1.21
44.26 ± 7.33
31.49 ± 1.98
10.51 ± 0.82
13.73 ± 0.72
17.92 ± 6.61
41.65 ± 1.62
6
6
Aldehyde
Oxime
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7
a (X = H)
57.99 ± 0.86
57.99 ± 6.39
83 ± 0.58
28.59 ± 0.5
29.47 ± 0.7
12.66 ± 0.2
7b (X = 3,5-diCl)
1
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BBG (c)
78.89 ± 3.81
13.22 ± 2.66
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(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of propidium
stimulated by 1mM ATP only. Data values are expressed as means ± SDs. All experiments were repeated at least 3 and
5 times.
(b) % LDH release values at 10 µM were expressed as a percentage, relative to maximum LDH release caused
by 0.05% Triton X- 100 only. Data values are expressed as means ± SDs. All experiments were repeated at least 3 and 4
times.
(c) BBG concentration of 750 nM.
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As shown in Table 2, we evaluated oximes 7a, 7b and 11. Three oximes 7a, 7b and 11
inhibited the ATP-induced pore formation. However, compounds 7a and 7b had a discreet
cytotoxicity (Table 2). Only analog 11 diminished ATP-induced dye uptake, and it did not exhibit
toxicity (Table 2).
Ether triazole effects were tested on ATP-induced dye uptake. 9b, 9h, 9k, 9m and 14b
triazoles did not inhibit ATP- induced dye uptake (Table 3). Analogs 9a, 9e, 9f, 9j, 9l, 9n, 14a and
14c partially inhibited ATP-induced uptake, and all of them exhibited modest cytotoxicity (Table
3). Conversely, analogs 9c, 9d, 9g, and 9i effectively blocked the ATP action via P2X7R activation
(Table 3). However, 9c and 9g displayed cytotoxicity against MPM. Analogs 9d and 9i showed low
toxic and blocked ATP-induced pore formation (Table 3).
Table 3. Effects of ether triazoles in P2X7R antagonistic activity
Compound
% Inhibition (a)
% LDH release (b)
9
9
9
9
9
9
9
9
a (X = H, R = Et)
b (X = H, R = Pr)
c (X = H, R = Bu)
d (X = 4-Cl, R = Et)
e (X = 4-Cl, R = Pr)
f (X = 4-Cl, R = Bu)
g (X = 2,5-diCl, R = Et)
h (X = 2,5-diCl, R = Pr)
49.30 ± 0.82
25.14 ± 1.01
78.1 ± 0.71
84.67 ± 0.65
32.47 ± 6.22
35.78 ± 0.43
75.5 ± 1.11
24.03 ± 8.03
52.73 ± 0.87
14.66 ± 0.32
32.33 ± 1.1
8.33 ± 0.2
26.66 ± 1.16
30.33 ± 0.99
42.66 ± 0.68
22.66 ± 0.37

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i (X = 2,5-diCl, R = Bu)
79.75 ± 2.61
36.53 ± 1
11.66 ± 0.57
34 ± 1.04
j (X = 3,5-diCl, R = Et)
k (X = 3,5-diCl, R = Pr)
l (X = 4-OMe, R = Et)
m (X = 4-OMe, R = Pr)
n (X = 4-OMe, R = Bu)
4a (R = Et)
23.96 ± 1.3
44.79 ± 7.04
15.62 ± 2.55
31.38 ± 5.06
69.97 ± 0.91
26.61 ± 8.76
37.06 ± 0.33
78.89 ± 3.81
14.03 ± 0.41
20.66 ± 4.98
13.03 ± 0.21
27.66 ± 0.14
26.49 ± 0.1
13.33 ± 0.1
27.19 ± 0.19
13.22 ± 2.66
4b (R = Pr)
4c (R = Bu)
BBG (c)
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(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of propidium
stimulated by 1mM ATP only. Data values are expressed as means ± SDs. All experiments were repeated at least 3 and
5 times.
(b) % LDH release values at 10 µM were expressed as a percentage, relative to maximum LDH release caused
by 0.05% Triton X- 100 only. Data values are expressed as means ± SDs. All experiments were repeated at least 3 and 4
times.
(c) BBG concentration of 750 nM.
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HEK 293 cells transfected with hP2X7R demonstrated an inhibitory profile similar to MPM
using FLIPR methodology. Basically, 5e, 6a, 8h, 9d, 9i, 11, and 12 also inhibited ethidium iodide
uptake in higher antagonistic activity when compared with other molecules (Tables 4-6).
As observed for MPM, 8b, 8h, 8j, 13a, and 13b inhibited hP2X7R. Not all other ester
triazoles inhibited ATP-induced ethidium uptake with percentage higher than 75% (Table 4).
However, among ester triazoles with inhibitory action against hP2X7R, 8h was unique no
cytotoxicity. 8b, 8j, 13a and 13b promoted LDH release higher than 20% after 60 minutes of
exposition (data not shown). Thus, we selected 8h to proceed in IC determination.
5
0
Table 4. Effects of ester triazoles in HEK 293 transfected hP2X7R antagonistic activity.
Compound
% Inhibition (a)
8
8
8
8
8
8
8
8
8
8
8
8
8
8
a (X = H, R = Ph)
b (X = H, R = Me)
c (X = H, R = pentyl)
d (X = H, R = nonyl)
e (X = 4-Cl, R = Ph)
16,1 ± 1,33
81,02 ± 4,02
18,2 ± 1,32
23,19 ± 2,02
26,77 ± 1,99
9,89 ± 2,97
2,2 ± 0,97
f (X = 4-Cl, R = Me)
g (X = 4-Cl, R = pentyl)
h (X = 4-Cl, R = nonyl)
i (X = 2,5-diCl, R = Ph)
j (X = 2,5-diCl, R = Me)
k (X = 2,5-diCl, R = pentyl)
l (X = 2,5-diCl, R = nonyl)
m (X = 3,5-diCl, R = Ph)
n (X = 3,5-diCl, R = Me)
78 ± 3,2
3 ± 1,08
71,2 ± 5,41
7,28 ± 2,05
1,17 ± 0,32
2,96 ± 0,92
29,44 ± 0,9

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o (X = 4-OMe, R = Ph)
10,55 ± 1,01
9,12 ± 1,03
8,15 ± 2,01
88,21 ± 5,78
80,1 ± 6,02
10,2 ± 2,99
19,16 ± 3,11
90,08 ± 9,94
p (X = 4-OMe, R = Me)
q (X = 4-OMe, R = pentyl)
3a (R = Ph)
3b (R = Me)
3c (R = pentyl)
3d (R = nonyl)
BBG (b)
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6
6
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(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of ethidium
bromide stimulated by 1mM ATP only. Data values are expressed as means ± SDs. All experiments were repeated at
least 3 and 5 times.
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Regarding to alcohol (5e, 12), aldehyde (6a), and oxime (11) triazoles, they inhibited
hP2X7R, in a similar manner to mP2X7R (Table 5). Not all other alcohol, aldehyde and oximes
tested inhibited hP2X7R as observed for mP2X7R.
Table 5. Effects of alcohol, aldehyde and oxime triazoles in HEK 293 transfected hP2X7R
antagonistic activity.
Compound
% Inhibition (a)
BBG (b)
90,08 ± 9,94
Alcohol
5
5
5
5
5
1
a (X = H)
30,13 ± 1,04
26,13 ± 2,09
38,11 ± 3,04
41,09 ± 3,79
94,12 ± 2,05
90,22 ± 2,77
b (X = 4-Cl)
c (X = 2,5-diCl)
d (X = 3,5-diCl)
e (X = 4-OMe)
2
Aldehyde
Oxime
6a (X = H)
6b (X = 2,5-diCl)
6c (X = 4-OMe)
10
95,3 ± 2,87
44,02 ± 5,71
50,28 ± 4,06
42,12 ± 6,08
7
7
11
a (X = H)
b (X = 3,5-diCl)
64,3 ± 1,92
62,11 ± 5,02
94,3 ± 1,03
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650
651
652
653
(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of ethidium
stimulated by 1mM ATP only, Data values are expressed as means ± SDs. All experiments were repeated at least 3 and
5 times.
(b) BBG concentration of 750 nM.

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Ether triazoles also showed inhibitory profile similar to mP2X7R, because 9c, 9d, and 14a
reduced ATP-induced dye uptake above 75% in comparison to ATP alone. Other ether triazoles did
not inhibit or they acted partially (Table 6).
Table 6. Effects of ether triazoles in HEK 293 transfected hP2X7R antagonistic activity.
Compound
% Inhibition (a)
9
9
9
9
9
9
9
9
9
9
9
9
9
9
1
1
1
a (X = H, R = Et)
b (X = H, R = Pr)
c (X = H, R = Bu)
56,73 ± 5,35
20,08 ± 3,6
67,52 ± 2,84
96,87 ± 0,22
62,37 ± 4,41
15,08 ± 2,02
45,3 ± 6,02
46,61 ± 3,13
90,07 ± 0,99
51,22 ± 4,7
33,66 ± 3,9
45,02 ± 3,13
35,77 ± 3,82
51,29 ± 1,79
79,08 ± 3,44
41,19 ± 7,03
16,11 ± 1,09
90,08 ± 9,94
d (X = 4-Cl, R = Et)
e (X = 4-Cl, R = Pr)
f (X = 4-Cl, R = Bu)
g (X = 2,5-diCl, R = Et)
h (X = 2,5-diCl, R = Pr)
i (X = 2,5-diCl, R = Bu)
j (X = 3,5-diCl, R = Et)
k (X = 3,5-diCl, R = Pr)
l (X = 4-OMe, R = Et)
m (X = 4-OMe, R = Pr)
n (X = 4-OMe, R = Bu)
4a (R = Et)
4b (R = Pr)
4c (R = Bu)
BBG (b)
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660
661
662
(a) % Inhibition values at 10 µM were expressed as a percentage, relative to maximum uptake of ethidium
stimulated by 1mM ATP only, Data values are expressed as means ± SDs. All experiments were repeated at least 3 and
5 times.
(b) BBG concentration of 750 nM.
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670
671
672
673
After the screening phase, we selected triazoles 5e, 6a, 8h, 9d, 9i, 11, and 12 to investigate
their potential as P2X7R antagonists in more detail. As indicated in Fig. 3, we performed dye
uptake experiments using flow cytometry to obtain inhibition curves for the selected analogs. ATP
treatment increased dye uptake 4 times in comparison with negative control (compare Fig.s 3A1
with 3A2). ATP-induced dye uptake was inhibited after pretreating with BBG (Fig. 3A3), and
fluorescence was restored to basal levels. All 1,2,3-triazole analogs (Fig.s 3A4 - 3A10) reduced the
ATP effect in a dose-dependent manner (Fig. 3B). The IC50 values obtained through the dose-
response experiment were 488.4 nM for analog 5e, 167.4 nM for analog 6a, 106.8 nM for analog
8h, 83.40 nM for analog 9d, 316.9 nM for analog 9i, 349.2 nM for analog 11 and 95.96 nM for
analog 12 (Table 7). In comparison with BBG (which has an IC value of 110.3 nM), analogs 6a,
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9d, 8h and 12 were more potent and effective at inhibiting ATP-induced dye uptake. Analogues 5e,
9i and 11 displayed potency similar or lower than BBG (Table 7).
6
6
77
78
Fig. 3. Dose-concentration inhibition curves for selected 1,2,3-triazole derivatives on pore
formation activity. (A) The dot plots are related to ATP-induced dye uptake alone or in the presence
of the selected 1,2,3-triazole derivatives. (B) The dose response curves of selected 1,2,3-triazole
derivatives with 1 mM ATP for 25 min. as analyzed by flow cytometry to detect the PI uptake.
Values represent the mean ± SEM. The profiles are representative of 3-6 independent experiments.
679
680
681
682
683
684
Table 7. Antagonistic effect of triazoles in mice P2X7R.
P2X7R antagonists and Triazoles *IC (µM)
5
0
BBG
0.343
0.488
0.167
0.107
0.083
0.317
5e
6a
8
9
h
d
9i

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2
6
1
1
0.349
0.096
12
6
6
85
86
*IC50 values were obtained from flow cytometry in propidium uptake assay. Data values are expressed as
means ± SDs. All experiments were repeated at least 3 times.
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
Whole cell patch clamp used to study mP2X7R pore macroscopic ionic current evoked after
treating with ATP (1 mM ATP) for 5 minutes (black bar), with or without BBG or 1,2,3-triazole
analogs (gray bar) in both cases (Fig. 4). ATP-induced ionic currents is represented for Fig. 4A1.
BBG reduced ATP-induced current as exhibited at Fig. 4A2. All 1,2,3-triazole analogs diminished
ATP-induced macroscopic currents (Fig.s 4A3-4A9). In comparison with BBG, analogs 11, 5e and
9i demonstrated inferior inhibition profile (Fig. 4A7-A9). By contrast, analogs 6a, 8h, 9d and 12
showed an inhibition superior to that of BBG (Fig. 4A3-6). Dose-response curves for selected
triazoles and BBG confirms this inhibitory profile (Fig. 4B). As demonstrated in the Table 8, 6a,
8h, 9d and 12 were more potent than BBG to inhibit P2X7R expressed in MPM. Analogs 6a and 8h
obtained a performance similar to BBG. In the other hand, BBG exhibited a higher potency than
05e, 9i and 11 (Table 8).
Our results indicate that these four compounds could be promising P2X7R antagonists. All
of them inhibited P2X7R at IC values that were lower than classical P2X7R antagonists, namely
5
0
suramin, PPADS, KN-62 and oxidized ATP [75,76], and similar to novel antagonists such as
A740003, A438079, A804598, AZ10606120, and AZ11645373 [77].

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7
7
7
05
06
707
708
709
710
711
712
713
714
715
716
Fig. 4. Macroscopic current induced by ATP is inhibited by selected 1,2,3-triazole derivatives. (A)
Whole cell recordings of the cationic P2X7R activated by 1 mM ATP for 5 min. on peritoneal
macrophages from 30-37 ºC. Adding BBG (100 nM) or the selected 1,2,3-triazole derivatives when
incubating for 10 min. of the total. The initial 5 min. during which the antagonists were added alone
and the last 5 min. in conjunction with ATP. (B) The plot represents the quantification of the data
observed in A; the % relative current recorded as a function of the ratio between the amplitude of
the ionic current and the cell capacitance. The values represent the mean ± SEM of the total % ATP
effect. The profiles are representative of 3-6 independent experiments for whole cell recordings.
Table 8. Antagonistic effect of triazoles in mice P2X7R.
P2X7R antagonists and Triazoles
*IC (µM)
50
BBG
0.109
0.341
0.103
0.103
0.069
0
0
5e
6a
0
0
8h
9d

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2
8
0
1
1
9i
1
2
0.316
0.457
0.081
7
7
17
18
*IC values were obtained from whole cell configuration. Data values are expressed as means ± SDs. All
50
experiments were repeated at least 3 times.
719
720
721
722
723
724
725
726
727
Cytotoxicity of the selected triazoles. Cytotoxicity in MPM was measured in crescent
selected triazole analogue concentrations as shown in Fig. 5 after continuous exposition for 24
hours. Triazoles 5e, 9i, and 11 caused cell toxicity at 362 µM, 505 µM and 250.2 µM, respectively.
By contrast, analogs 6a, 8h, 9d and 12 reached their CC at 3.73 mM, 2.84 mM, 4.04 mM and 2.35
50
mM, respectively (Fig. 5 and Table 9). Triazoles 6a, 8h, 9d and 12 exhibit cytotoxicity effect in
concentrations at least 1,000 times lower than IC values. Therefore, they are good candidates to
5
0
continue the studies.
n= 4
1
50
1
0
1
0
0
0
0
1
8h
2
100
6a
9d
5e
9i
5
0
0
-
10
-8
-6
-4
-2
0
[
Analogs]M
7
28
29
7
Fig. 5. Toxicity of selected 1,2,3-triazole derivatives on mouse peritoneal macrophages. The dose
response curve of selected 1,2,3-triazole derivatives to peritoneal macrophages at concentrations
ranging from 1 nM - 10 mM for 24 h. The profiles are representative of 3-6 independent
experiments
7
7
7
7
7
30
31
32
33
34
Table 9. Cytotoxicity of selected triazoles in mice P2X7R.
P2X7R antagonists and Triazoles
*CC (µM)
50
0
0
0
5e
6a
8h
0.363
3.739
2.841