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Synthesis, Antiproliferative and Anti-inflammatory Activities of Novel Simplified Imatinib Analogues

Thiago Stevanatto Sampaio1,2, Lídia Moreira Lima1,2*, Renata da Silva Zardo1,2, Cláudia Pessoa3, Bruno Coêlho Cavalcanti3, Rosane de Paula Castro4, José Ricardo Sabino4, Patrícia Dias Fernandes1,2 and Eliezer J Barreiro1,2*

1Instituto Nacional de Ciência e Tecnologia de Fármacos e Medicamentos, Laboratório de Avaliação e Síntese de Substâncias Bioativas, Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, Rio de Janeiro-RJ, Brazil

2Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, Rio de Janeiro-RJ, Brazil

3Laboratório Nacional de Oncologia Experimental, Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza-CE, Brazil

4Instituto de Física, Universidade Federal de Goiás, Goiânia-GO, Brazil

*Corresponding Authors:
LM Lima
Laboratório de Avaliação
e Síntese de Substâncias Bioativas
Universidade Federal do Rio de Janeiro
P.O. Box 68024, Zip code: 21944-971
Rio de Janeiro, RJ, Brazil
Tel:+55(21)39386503
E-mail:[email protected]

EJ Barreiro
Laboratório de Avaliação
e Síntese de Substâncias Bioativas
Universidade Federal do Rio de Janeiro
P.O. Box 68024, Zip code: 21944-971
Rio de Janeiro, RJ, Brazil
Tel: +55(21)39386644
Fax: +55(21)39386478
E-mail: [email protected]

Received date: November 05, 2014; Accepted date: November 21, 2014; Published date: November 23, 2014

Citation: Sampaio TS, Lima LM, da Silva Zardo R, Pessoa C, Cavalcanti BC, et al. (2014) Synthesis, Antiproliferative and Anti-inflammatory Activities of Novel Simplified Imatinib Analogues. Med chem 4:756-762. doi:10.4172/2161-0444.1000226

Copyright: © 2014 Sampaio TS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

 

We described the synthesis of carboxamide derivatives designed as novel simplified imatinib analogues and their antiproliferative and anti-inflammatory activities. Compound 2c showed a unique conformation determined by X-ray diffraction and by NMR 1H and displayed antiproliferative potency in the same magnitude order than the standard imatinib. Compounds 6-10 were prepared by structural modification in carboxamide 2c, and with exception of derivative 10, they were inactive as antiproliferative agent. However, these compounds showed same anti-inflammatory potency than imatinib, standing out carboxamide 9 that was six time more potent as TNF-α inhibitor production.

Keywords

Anti-inflammatory, Antiproliferative, Carboxamide, Imatinib analogues, TNF-α inhibitor

Introduction

Imatinib, the first tyrosine-kinase inhibitor to be approved for the treatment of cancer, mainly chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GISTs) [1], is considered a milestone in small-molecule drug discovery and molecular targeted therapies. However, unlike the previous report, imatinib is not a specific inhibitor of ABL, but also inhibits PDGFRs, KIT, ARG (abl-related gene) and potentially other enzymes, which have not yet been tested [2].

Besides its antineoplastic effects, anti-inflammatory and antifibrotic activity has recently been assign to imatinib, revealing its multitarget profile [3-7]. However, its exact anti-inflammatory mechanism of action remains elusive. Nonetheless, its ability to downregulate the expression of inflammatory cytokines, like TNF-α, IL-6 and IL-1β, has been considered a possible mechanism [4,6].

In this paper we describe the synthesis, antiproliferative and anti-inflammatory activities of novel carboxamide derivatives (2a-c) designed as simplified imatinib analogues (Figure 1) in order to obtain new anti-inflammatory drug candidates.

medicinal-chemistry-carboxamide-derivatives

Figure 1: Design conception of carboxamide derivatives (2a-c) through molecular simplification from imatinib.

Results and Discussion

The carboxamide derivatives (2a-c) were planned by molecular simplification [8] in the structure of the prototype imatinib (1), resulting in the elimination of 4-(pyridin-3-yl)pyrimidin-2-amine moiety (Subunit A, Figure 1), followed by the classic ring isosterism and retroisosterism [9] of subunits B and C, respectively (Figure 1).

The regioisomers (2a-c) were synthesized in three linear steps depicted in Scheme 1. The synthetic route began with reductive amination step, involving the reaction of 2- or 3- or 4-nitrobenzaldehyde (3a-c) with 1-methylpiperazine in the presence of zinc chloride in methanol and sodium cyanoborohydride to obtain the nitrobenzyl- N-methyl-piperazines 4a-c in moderate yield [10]. Interconversion of functional group was carried out based on the reduction of nitro group in respective amine derivatives (5a-c), using metallic iron in the presence of slightly acid solution of ammonium chloride in water and ethanol (1:1) [11]. The condensation of the amines 5a-c with 2-quinaxaloyl chloride, at room temperature using dichloromethane as solvent [12], obtained the desired carboxamides 2a-c in good yields (Scheme 1). The regioisomers 2a-c were fully characterized by 1H NMR, 13C NMR and IR spectroscopy and their purity was determined by elementar analysis or HPLC. The possible conformation differences, arising from the isomerism in the benzyl-N-methylpiperazine unit, were assigned based on the X-ray crystallographic studies performed with carboxamides 2a-c (Figure 2 and Table 1).

Compound D—H...A D—H H...A D...A D—H...A
Intermolecular
2a C10—H10…O17iii 0.93 2.33 3.259(2) 124
2b N15—H15…N22iv 0.93 2.33 3.259(2) 124
2c C3—H3b...O17v 0.97 2.52 3.270(2) 135
2c C7—H7a...Cg1vii 0.97 2.45 3.367(2) 157
Intramolecular
     2c N15-H15...N1 0.86 2.24 2.915(2) 136

Table 1: Hydrogen-bonds and intermolecular interactions geometry (Â,°).

medicinal-chemistry-Synthesis

Scheme 1: Synthesis of compounds 2a-c.

Single crystals of compounds 2a-c were obtained and subjected to X-ray diffraction; the ORTEP [13] view is shown in Figure 2a-2c. The different positioning of the piperazine moiety leads to changes in the carboxamide fragment conformation in solid state. These changes occur in the angles between the planes passing through the atoms of the planar subunits quinoxaline C18/N19/C20/C24/C25/ C26/C27/ C21/N22/C23 (plane α), the acetamide bridge N15/C16/C18/O17 (plane β) and the phenyl ring C8/C9/C10/C11/C12/C13 (plane γ). The piperazine conformation relative to the phenyl ring is also subject to torsional strain about the σ-bonding between N1 atom and the methylene carbon C7, where the methylene´s hydrogen is eclipsed in 2a and 2c and staggered in 2b. Bond lengths are in good agreement between the three compounds. It is worth to notice that the density increases from compound 2a to 2c, showing that in compound 2c, the intramolecular N15-H15…N1 H-bond leads to a more compact molecular conformation when compared to 2a and 2b.

medicinal-chemistry-displacement-ellipsoids

Figure 2: ORTEP view of compounds 2a-c with the atom displacement ellipsoids drawn at 30% of probability level. A) compound 2a; B) compound 2b; C) compound 2c.

In order to establish the better regioisomer template to be elected as a new lead compound, with an original structural pattern, the antiproliferative activity of carboxamides 2a-c was investigated against HL-60 (human leukemia), SF-295 (human glioblastoma) and HCT- 8 (human colorectal carcinoma) tumor cell lines [14]. In this assay, doxorubicin was used as positive control and imatinib as standard drug. As demonstrated in Table 2, the regioisomer para (2a) and meta (2b) were inactive, while the regioisomer ortho (2c) displayed antiproliferative potency similar to the prototype imatinib, against human colorectal carcinoma and human leukemia cells. In addition, compound 2c was not cytotoxic against healthy cells (i.e. human lymphocyte).

Compound Cell Lines – IC50 mM ± S.E.M Primary culture
  HL-60 SF-295 HCT-8 Human
Lymphocytes
2a
(LASSBio-1599)
> 300 208.67 ±7.28 173.41 ±5.91 194.3 ±2.17
2b
(LASSBio-1598)
> 300 287.53 ±3.15 > 300 > 300
2c
(LASSBio-1597)
74.40 ±9.80 107.64 ±5.10 39.61 ±7.05 > 300
6
(LASSBio-1724)
> 300 > 300 > 300 > 300
7
(LASSBio-1725)
> 300 > 300 > 300 > 300
8
(LASSBio-1726)
> 300 > 300 > 300 > 300
9
(LASSBio-1723)
> 300 > 300 > 300 > 300
10
(LASSBio-1727)
35.75 ±2.51 > 300 > 300 > 300
Imatinib 22.81 ±3.11 41.53 ±5.73 34.03 ±4.11 > 300
Doxorubicin 0.04 ±0.01 0.48 ±0.02 0.02 ±0.01 0.37 ±0.01

Table 2: Antiproliferative activity of carboxamides 2a-c, imatinib and doxorubicin (positive control) against HL-60, SF-295, HCT-8 tumor cell lines and their cytotoxic activity against primary culture of human lymphocytes.

The differences in the antiproliferative activity of carboxamides 2ac, especially the ortho regioisomer (2c), can be attributed to its unique conformation due to the presence of intramolecular hydrogen bond (N1 and H15) observed in the solid state, by X-ray diffraction (Figure 2C and Table 1), and confirmed in solution by 1H NMR, through the differences in the chemical shift of the amide hydrogen (CONH) of compounds 2a-c. The NH proton signal for compounds 2a and 2b is seen at 9.75 and 9.79 ppm, while for compound 2c signal moves downfield to δ 11.7 ppm, due a deshielding effect promoted by the intramolecular hydrogen bond between H15 (XONH) and N1 (piperazine), defining a six-membered ring (Figure 2C).

Once defined the ortho scaffold as the better template to achieve new imatinib analogues, the carboxamide 2c was modified varying the nature of the N-substituted piperazine (8-10) and by bioisosteric replacement [9] of the quinoxaline ring (6-7) (Figure 3). These analogues were synthesized applying the same methodology previously described for carboxamides 2a-c, exploring steps of reductive amination, functional group interconversion and condensation of amines with acyl chlorides derivatives (Scheme 2).

medicinal-chemistry-compounds

Scheme 2: Synthesis of compounds 6-10

medicinal-chemistry-Design-conception

Figure 3: Design conception of carboxamide derivatives (6-10) from the prototype 2c.

The analogues 6-10 were also evaluated as antiproliferative leadcandidates (Table 2). As show in Table 2 the attempt to replace the quinoxaline ring (2c) by the pyrazine system (6) or by the retroisoster 7 resulted in loss of activity. Also, the substitution of methyl group linked in the piperazine subunit by an aromatic group (phenyl or pyrimidine) resulted in failure of antiproliferative activity. However, compound 10, having a 1-(2-hydroxyethyl) piperazine unit, instead of the N-methylpiperazine presented in the lead 2c, seems to present a more selective cytotoxic profile, being active against human leukemia (HL- 60) and inactive against the solid tumor cells lines SF-295 and HCT-8.

Considering the recent anti-inflammatory profile described for the prototype imatinib, the carboxamides 2c and 6-10 were studied in carrageenan-induced acute inflammation in the mouse air pouch synovial model [15]. In this model, the number of leukocytes recruited into the pouch increased progressively after carrageenan, and the ability of carboxamides derivatives and the prototype imatinib to inhibit the cell migration and to reduce the cytokine TNF-α production were investigated. As shown in Table 3, imatinib (1) and carboxamides 2c and 6-10, when administered per os, were able to inhibit the leukocyte migration revealing an important anti-inflammatory profile with ED50 (in μmol/kg) values similar to imatinib. In order to verify the anti-TNFactivity of these derivatives, we quantified the TNF-α produced in the SAP exsudate. As shown in Table 3, compound 9 standing out, showing ED50 value of 0.07 μmol/kg, being more potent than imatinib (ED50 value of 0.44 μmol/kg).

Compound ED50 (µmol/kg)
  Leukocyte migration TNF-α production
ASA 128.0* N.R.
imatinib 5.0* 0.44*
2c (LASSBio-1597) 5.1* 4.4*
6 (LASSBio-1724) 5.35* 9.26*
7 (LASSBio-1725) 6.1* 8.8*
8 (LASSBio-1726) 3.2* 8.6*
9 (LASSBio-1723) 4.45* 0.07*
10 (LASSBio-1727) 5.4* 0.9*

Table 3: Effects of carboxamides 2c, 6-10, imatinib and acetylsalicylic acid (ASA) in leukocyte migration and TNF-α production induced by carrageenan injection into the subcutaneous air pouch.

Conclusion

In summary, similar to imatinib (1) and the initial prototype 2c, all carboxamide derivatives described herein (6-10) showed an important anti-inflammatory and anti-TNF-α activity in vivo. Compound 9, having a pyrimidine group linked to piperazine ring was six times more potent as TNF-α production inhibitor than imatinib (1). It is noteworthy that modifications introduced in quinoxaline ring or piperazine unit were able to dissociate the anti-inflammatory effect in the detriment of the antiproliferative activity, as exemplified by compounds 6-9. From this series of simplified imatinib analogues, only compounds 2c and 10 showed both cytotoxic and anti-inflammatory activities.

Experimental Section

Chemistry

All commercially available reagents and solvents were used without further purification. Reactions were routinely monitored by thinlayer chromatography (TLC) in silica gel (F245 Merck plates) and the products visualized with ultraviolet lamp (254 and 365 nm). 1H and 13C nuclear magnetic resonance (NMR) spectra were determined in CDCl3 solution using a Bruker AC-200. The chemical shifts are given in parts per million (δ) from solvent residual peaks and the coupling constant values (J) are given in Hz. Signal multiplicities are represented by: s, singlet; d, doublet; m, multiplet; Is, large signal. Infrared (IR) spectra were obtained with ABB spectrophotometer, FTLA 2000-100 model, by using potassium bromide plates. Elemental analysis results were obtained with a Thermo CE scientific apparatus, EA 1112 Series model. Melting points were determined using a Quimis Melting Point Q-340S13 and are uncorrected. Flash chromatography was performed using a LaFlash system (VWR) with Merck silica gel (25-40 μm).

The TLC was performed on 2.0 × 6.0 cm aluminium sheets covered with silica gel (Sorbent, 200 μm thickness) under ultraviolet light (254- 365 nm).

The high performance liquid chromatography was performed using Shimadzu – LC20AD with Kromasil column 100-5C18 (4,6 mm × 250 mm), SPD-M20A detector (Diode array) at a wavelength of 254 nm. The mobile phases used: 30% acetonitrile and 70% aqueous trifluoroacetic acid 0,01% or 50% acetonitrile and 50% aqueous trifluoroacetic acid 0,01%

Analyses by differential scanning calorimetry were obtained with DSC-60 Shimadzu. The samples were heated at a rate of 20ºC/min, with a temperature range of 30-300°C under a nitrogen flow.

General procedure for the preparation of compounds 4a-c, 11-13 [10]: To a 100 mL flask 6.6 mmol of nitrobenzaldehyde was dissolved in methanol (15 mL). Then, 26.4 mmol of 1-methylpiperazine were added. After 30 minutes, 6.6 moles of sodium cyanoborohydride and 3.3 mol of zinc chloride, previously dissolved in 10 mL of methanol, were added. The mixture was stirred for 2 hours. The end of this step was observed by TLC and desired product was extracted using 20 mL of NaOH 0.1N and 3 × 30 mL of ethyl acetate. Afterwards, organic phases was put together, dried and solvent was evaporated under reduced. The products were purified by column chromatography on silica gel (eluent n-hexane:Ethyl Acetate 50:50)

1-(4-nitrobenzyl)-4-methylpiperazine (4a): Yield: orange oil (41%); 1H NMR (200 MHz, CDCl3) δ 2.26 (s, 3H, CH3), 2.45 (ls, 8H, Ha and Hb), 3.56 (s, 2H, CH2), 7.47 (d, 2H, J = 8.5 Hz, H3’ and H5’), 8.12 (d, 2H, J = 8.5 Hz, H4’ and H6’); 13C NMR (50 MHz, CDCl3) δ 46.1 (Cc), 53.2 (Ca and Ca’), 55.1 (Cb and Cb’), 62.2 (Cd), 123.6 (C-2 and C-2’), 129.6 (C-3 and C-3’), 146.5 (C-4), 147.3 (C-1); IR (KBr) 3050 cm-1 (υ C-H aromatics), 2937 and 2793 cm-1 (υ CH2), 1514 cm-1 a NO2), 1342 cm-1 (υ s NO2), 855 cm-1out of the plane C-H of aromatic ring para-substituted).

1-(3-nitrobenzyl)-4-methylpiperazine (4b): Yield: orange oil (45%); 1H NMR (200 MHz, CDCl3) δ 2.30 (s, 3H, CH3), 2.48 (ls, 8H, Ha and Hb), 3.60 (s, 2H, CH2), 7.48 (t, 1H, J=7.8 Hz, H5’), 7.68 (d, 1H, J = 7.5 Hz, H4’), 8.10 (d, 1H, J = 8.1 Hz, H6’), 8.21 (s, 1H, H2’); 13C NMR (50 MHz, CDCl3) δ 46.0 (Cc), 53.1 (Ca and Ca’), 55.1 (Cb and Cb’), 62.0 (Cd), 122.2 (C-6), 123.7 (C-2), 129.1 (C-5), 135.0 (C-4), 140.9 (C-3), 148.5 (C-1); IR (KBr) 3045 cm-1 (υ C-H aromatic), 1527 cm-1a NO2), 1318 cm-1s NO2), 751 and 689 cm-1out of the plane C-H of aromatic rings meta-substituted).

1-(2-nitrobenzyl)-4-methylpiperazine (4c): Yield: yellow solid (44%); MP: 170-171 ºC; 1H NMR (200 MHz, CDCl3) δ 2.25 (s, 3H, CH3), 2.44 (ls, 8H, Ha and Hb), 3.53 (s, 2H, CH2), 7.41 (t, 1H, J = 7.5 Hz, H4’), 7.60 (d, 1H, J = 7.6 Hz, H3’), 8.03 (m, 1H, H5’), 8.14 (ls, 1H, H6’); 13C NMR (50 MHz, CDCl3) δ 46.0 (Cc), 53.0 (Ca and Ca’), 55.1 (Cb and Cb’), 62.0 (Cd), 122.3 (C-2), 123.8 (C-6), 129.3 (C-5), 135.1 (C-4), 140.9 (C-3), 148.5 (C-1); IR (KBr) 2938 and 2797 cm-1 (υ CH2), 1528 cm-1a NO2), 1356 cm-1s NO2).

1-(2-nitrobenzyl)-4-phenylpiperazine (11): Yield: yellow solid (71%); MP: 108-109 ºC; 1H NMR (200 MHz, CDCl3) δ 2.52 (t, 4H, J = 4.9 Hz, Ha), 3.08 (t, 4H, J = 4.9 Hz, Hb), 3.78 (s, 2H, CH3), 6.77 (m, 3H, H3’’, H4’’ and H5’’), 7.16 (m, 2H, H2’’ and H6’’), 7.31 (m, 1H, , H5’), 7.46 (m, 1H, H4’), 7.57 (d, 1H, J = 6.9 Hz, H3’), 7.74 (d, 1H, J = 7.9 Hz, H6’); 13C NMR (50 MHz, CDCl3) δ 49.2 (Cb and Cb’), 53.2 (Ca and Ca’), 59.0 (Cc), 116.2 (C-2’ and C-6’), 119.8 (C-2), 124. 5 (C-4’), 128.1 (C-6), 129.1 (C-3’ and C-5’), 131.1 (C-5), 132.5 (C-4), 133.5 (C-3), 150.0 (C- 1), 151.3 (C-1’); IR (KBr) 2952 and 2817 cm-1 (υ CH2), 1596, 1492 and 1443 cm-1 (υ C=C aromatic), 1525 cm-1a NO2), 1348 cm-1s NO2).

1-(2-nitrobenzyl)-4-(2-pyrimidyl)piperazine (12): Yield: white solid (71%); MP: 110-112 °C; 1H NMR (200 MHz, CDCl3) δ 2.37 (t, 4H, J = 4.6 Hz, Ha), 3.65 (t, 4H, J = 4.5 Hz, Hb), 3.75 (s, 2H, CH2), 6.60 (t, 1H, J = 4.6 Hz, H4’’), 7.48 – 7.56 (m, 1H, H3’ and H5’), 7.65 (m, 2H, H4’), 7.87 (d, 1H, J = 7.8 Hz, H6’), 8.32 (m, 2H, H3’’ and H5’’); 13C NMR (50 MHz, CDCl3) δ 43.8 (Cb and Cb’), 53.0 (Ca and Ca’), 58.7 (Cc), 110.7 – 161.6 (C-aromatics); IV (KBr) 2936 and 2821 cm-1 (υ CH2), 1587, 1498 and 1446 cm-1 (υ C=C aromatic), 1541 cm-1a NO2), 1355 cm-1s NO2).

1-(2-nitrobenzyl)-4-(2-hydroxyethyl)piperazine (13): Yield: yellow oil (47%); 1H NMR (200 MHz, CDCl3) δ 2.43 – 2.50 (m, 8H, Ha and Hb), 2.74 (ls, 2H, Hd), 3.54 (t, 2H, J = 5.3 Hz, He), 3.73 (s, 2H, CH2), 7.28 – 7.36 (m, 1H, H4’), 7.42 – 7.52 (m, 2H, H3’ and H5’), 7.73 (d, 1H, J = 7.7 Hz, H6’); 13C NMR (50 MHz, CDCl3) δ 53.0 (Ca and Ca’), 53.1 (Cb and Cb’), 57.8 (Cc), 59.1 (Cd), 59.4 (Ce), 124.5 (C-2), 128.1 (C-6), 131.1 (C-5), 132.4 (C-4), 133.8 (C-3), 150.0 (C-1); IV (KBr) 3390 cm-1a OH), 2940 and 2816 cm-1 (υ CH2), 1529 cm-1a NO2), 1356 cm-1s NO2).

General procedure for preparation of intermediates 5a-c, 14-16 [11]: To a 125 mL flask containing 1.3 mmol of nitro-compound and 90 mL of ethanol:water (2:1) were added 7.3 mmol of iron powder and 2.1 mmol of ammonium chloride. The mixture was refluxed for 1 hour. The hot mixture was filtered through Celite and concentrated in vacuo. The residue was diluted with H2O and extracted with AcOEt. Afterwards, organic phases was dried over anhydrous Na2SO4 and concentrated to give the amine intermediates.

4-[(4-methylpiperazin-1-yl)methyl]benzenamine (5a): Yield: yellow solid (83%); MP: 68-70 ºC; 1H NMR (200 MHz, CDCl3) δ 2.27 (s, 3H, CH3), 2.44 (ls, 8H, Ha and Hb), 3.39 (s, 2H, CH2), 3.51 (s, 2H, NH2), 6.62 (d, 2H, J = 7.9 Hz, H3’ and H4’), 7.08 (d, 2H, J = 7.8 Hz, H2’ and H6’); 13C NMR (50 MHz, CDCl3) δ 46.1 (Cc), 53.0 (Ca and Ca’), 55.2 (Cb and Cb’), 62.7 (Cd), 114.9 (C-2 and C-2’), 129.0 (C-4), 130.4 (C-3 and C-3’), 145.5 (C-1); IV (KBr) 3444 cm-1a NH2), 3361 cm-1s NH2), 2933 and 2807 cm-1 (υ CH2), 1621, 1518 and 1450 cm-1 (υ C=C aromatic), 826 cm-1out of the plane C-H of aromatic rings parasubstituted).

3-[(4-methylpiperazin-1-yl)methyl]benzenamine (5b): Yield: yellow solid (78%); MP: 70-71 ºC; 1H NMR (200 MHz, CDCl3) δ 2.20 (s, 3H, CH3), 2.38 (ls, 8H, Ha and Hb), 3.33 (s, 2H, CH2), 3.53 (m, 2H, NH2), 6.47 (m, 1H, H2’), 6.61 (d, 2H, J = 7.0 Hz, H4’ and H6’), 7.00 (t, 1H, J = 7.7 Hz, H5’); 13C NMR (50 MHz, CDCl3) 46.1 (Cc), 53.2 (Ca and Ca’), 55.2 (Cb and Cb’), 63.1 (Cd), 113.9 (C-6), 115.8 (C-2), 119.5 (C- 4), 129.1 (C-5), 139.5 (C-3), 146.5 (C-1); IV (KBr) 3408 cm-1a NH2), 3307 cm-1s NH2), 2946 and 2794 cm-1 (υ CH2), 1606, 1494 and 1454 cm-1 (υ C=C aromatic), 783 and 692 cm-1out of the plane C-H of aromatic rings meta-substituted).

2-[(4-methylpiperazin-1-yl)methyl]benzenamine (5c): Yield: yellow solid (74%); MP: 85-87 ºC; 1H NMR (200 MHz, CDCl3) δ 2.21 (s, 3H, CH3), 2.37 (ls, 8H, Ha and Hb), 3.44 (s, 2H, CH2), 4.51 (s, 2H, NH2), 6.58 (m, 2H, J = 8.0 Hz, H4’ and H5’), 6.89 – 7.05 (m, 2H, H3’ and H6’); 13C NMR (50 MHz, CDCl3) δ 46.1 (Cc), 52.7 (Ca and Ca’), 55.4 (Cb and Cb’), 62.0 (Cd), 115.6 (C-6), 117.7 (C-4), 122.4 (C-2), 128.5 (C-3), 130.6 (C-5), 147.1 (C-1); IV (KBr) 3364 cm-1a NH2), 3303 cm-1s NH2), 2938 and 2806 cm-1 (υ CH2), 750 cm-1out of the plane C-H of aromatic rings ortho-substituted).

2-[(4-phenylpiperazin-1-yl)methyl]benzenamine (14): Yield: white solid (84%); MP: 178-179 ºC; 1H NMR (200 MHz, CDCl3) δ 2.51 (t, 4H, J = 4.9 Hz, Ha), 3.09 (t, 4H, J = 4.9 Hz, Hb), 3.50 (s, 2H, CH2), 4.51 (ls, NH2), 6.54 – 7.21 (9H, H-aromatics); 13C NMR (50 MHz, CDCl3) δ 49.4 (Cb and Cb’), 52.9 (Ca and Ca’), 62.0 (Cc), 115.8 (C- 6), 116.2 (C-2’ and C-6’), 117.8 (C-4), 119.9 (C-4’), 121.9 (C-2), 128.8 (C-3), 129.3 (C-3’ and C-5’), 130.8 (C-5), 147.2 (C-1), 151.4 (C-1’); IV (KBr) 3443 cm-1a NH2), 3311 cm-1s NH2), 2932 and 2817 cm-1 (υ CH2), 1599, 1498 and 1451 cm-1 (υ C=C aromatic), 746 cm-1out of the plane C-H of aromatic rings ortho-substituted).

2-{[4-(pyrimidin-2-yl)piperazin-1-il]methyl}benzenamine (15): Yield: yellow solid (81%); MP: 100-102 ºC; 1H NMR (200 MHz, CDCl3) δ 2.44 (t, 4H, J = 5.0 Hz, Ha), 3.50 (s, 2H, CH2), 3.75 (t, 4H, J = 4.9 Hz, Hb), 6.41 (t, 1H, J = 4.7 Hz, H4’’), 6.61 (t, 2H, J = 6.9 Hz, H3’ and H5’), 6.92 (d, 1H, J = 6.6 Hz, H6’), 7.05 (t, 1H, J = 8.2 Hz, H4’), 8.22 (d, 2H, J = 4.7 Hz, H3’’ and H5’’); 13C NMR (50 MHz, CDCl3) δ 43.9 (Cb and Cb’), 52.8 (Ca and Ca’), 62.1 (Cc), 110.1 (C-5’), 115.9 (C-6), 117.9 (C-4), 121.7 (C-2), 128.9 (C-3), 130.9 (C-5), 147.2 (C-1), 157.9 (C-4’ and C-6’), 161.8 (C-2’); IV (KBr) 3421 cm-1a NH2), 3302 cm-1s NH2), 2929 and 2814 cm-1 (υ CH2), 1586, 1495 and 1448 cm-1 (υ C=C aromatic), 751 cm-1out of the plane C-H of aromatic rings ortho-substituted).

2-[(4-hydroxyethyl-piperazin-1-yl)methyl]benzenamine (16): Yield: yellow solid (76%); MP: 63-65 ºC; 1H NMR (200 MHz, CDCl3) δ 2.43 – 2.50 (m, 10H, Ha, Hb and Hd), 3.44 (s, 2H, CH2), 3.55 (t, 2H, J = 5.6 Hz, He), 4.03 (ls, 2H, NH2), 6.58 (t, 2H, J = 8.2 Hz, H4’ and H5’), 6.93 (d, 1H, J = 7.1 Hz, H3’), 7.01 (t, 1H, J = 7.6 Hz, H6’); 13C NMR (50 MHz, CDCl3) δ 52.7 (Ca and Ca’), 53.2 (Cb and Cb’), 57.9 (Cc), 59.5 (Cd), 62.0 (Ce), 115.6 (C-6), 117.7 (C-4), 122.2 (C-2), 128.5 (C-3), 130.6 (C-5), 147.0 (C-1); IV (KBr) 3373 cm-1a OH), 3307 cm-1a NH2), 3212 cm-1s NH2), 2942 and 2817 cm-1 (υ CH2), 1634, 1495 and 1456 cm-1 (υ C=C aromatic), 762 cm-1out of the plane C-H of aromatic rings ortho-substituted).

General procedure for preparation of the compounds 2a-c, 6-10 [12]: To a flask of the 50 mL containing 1 mmol of acyl chloride derivative in dichloromethane (20mL) treated with 2.2 mmol of triethylamine was added 1 mmol of amine-compound and the mixture stirred at room temperature for 2 hours. The mixture was diluted with additional dichloromethane (10 mL) and brine (20 mL) and washed with 0.05M HCl (10 mL) and brine (20 mL). The organic portion was dried (Na2SO4), filtered and concentrate in vacuo.

N-{4-[(4-methylpiperazin-1-yl)methyl]phenyl}quinoxaline-2- carboxamide (2a; LASSBio-1599): Yield: yellow solid (64%). When necessary, the product was purified by flash chromatography on silica gel (elution with 1% triethylamine in ethyl acetate); MP: 156 ºC; 1H NMR (200 MHz, CDCl3) δ 2.26 (s, 3H, CH3), 2.47 (ls, 8H, Ha and Hb), 3.44 (s, 2H,CH2), 7.19 (d, 2H, J = 8.2 Hz, H3’ and H5’), 9.70 (d, 2H, J = 8.2 Hz, H2’ and H6’), 7.77 – 7.82 (m, 2H, H5 and H8), 8.07 – 8.15 (m, 2H, H6 and H7), 9.67 (s, 1H, H3), 9.75 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 46.0 (Cc), 53.0 (Cb and Cb’’), 55.2 (Ca and Ca’), δ62.6 (Cd), 119. 9 (C-2 and C-6), 129.7 (C-6’), 130.1 (C-3 and C-5), 131.2 (C- 8’), 132.0 (C-9’), 134.9 (C-7’), 136.4 (C-4), 140.2 (C-1), 143.5 (C-10’), 144.0 (C-2’), 144.2 (C-3’ and C-5’), 161.0 (CONH); IR (KBr) 3280 cm-1 (υ CONH), 2931 and 2793 cm-1 (υ CH2), 1682 cm-1 (υ CONH), 830 cm-1out of the plane C-H of aromatic rings para-substituted); Elementar Analysis C21H23N5O (361): Calculated - C, 69.44; H, 6.41; N, 19.38. Found – C, 69.78; H, 6.60; N, 18.98.

N-{3-[(4-methylpiperazin-1-yl)methyl]phenyl}quinoxaline-2- carboxamide (2b; LASSBio-1598): Yield: yellow solid (53%). When necessary, the product was purified by flash chromatography on silica gel (elution with 1% triethylamine in ethyl acetate); MP: 142 ºC; 1H NMR (200 MHz, CDCl3) δ 2.27 (s, 3H, CH3), 2.49 (ls, 8H, Ha and Hb), 3.49 (s, 2H, CH2), 7.07 (d, 1H, J = 7.4 Hz, H4’), 7.30 (t, 1H, J = 7.7 Hz, H5’), 7.65 (s, 1H, H6’), 7.74 (s, 1H, H2’), 7.79 – 7.84 (m, 2H, H6 and H7), 8.09 – 8.16 (m, 2H, H5 and H8), 9.69 (s, 1H, H3), 9.79 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 45.8 (Cc), 52.8 (Cb and Cb’’), 55.0 (Ca and Ca’), 62.8 (Cd), 118.8 – 144.1 (14C, C-aromatic), 161.0 (CONH); IV (KBr) 3257 cm-1 (υ CONH), 2945 and 2794 cm-1 (υ CH2), 1680 cm-1 (υ CONH) 1607, 1537 and 1488 cm-1 (υ C=C aromatic); Elementar Analysis C21H23N5O (361): Calculated - C, 69.44; H, 6.41; N, 19.38. Found – C, 69.32; H, 6.18; N, 19.38.

N-{2-[(4-methylpiperazin-1-yl)methyl]phenyl}quinoxaline-2- carboxamide (2c; LASSBio-1597): Yield: yellow solid (51%). When necessary, the product was purified by flash chromatography on silica gel (elution with 1% triethylamine in ethyl acetate); MP: 132 ºC; 1H NMR (200 MHz, CDCl3) δ 2.30 (s, 3H, CH3), 2.64 (ls, 8H, Ha and Hb), 3.64 (s, 2H, CH2), 6.99 – 8.43 (m, 8H, H-aromatics), 9.69 (s, 1H, H3’), 11.70 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 45.7 (Cc), 52.3 (Cb and Cb’’), 55.1 (Ca and Ca’), 61.5 (Cd), 121.9 – 145.03 (14C, C-aromatics), 162.0 (CONH); IR (KBr) 3238 cm-1 (υ CONH), 2933 and 2795 cm-1 (υ CH2), 1684 cm-1 (υ CONH) 1590, 1525 and 1450 cm-1 (υ C=C aromatic), 753 cm-1out of the plane C-H of aromatic rings ortho-substituted); Elementar Analysis C21H23N5O (361): Calculated - C, 69.78; H, 6.41; N, 19.38. Found: C, 69.44; H, 5.99; N, 19.21; HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (30:70), 254 nm): Retention time: 7.64 min. Peak Area: 98.928 %.

N-{2-[(4-methylpiperazin-1-yl)methyl]phenyl}pyrazine-2- carboxamide (6; LASSBio-1724): Yield: yellow solid (72%). When necessary, the product was purified by flash chromatography on silica gel (elution with 1% triethylamine in ethyl acetate); MP: 134 ºC; 1H NMR (200 MHz, CDCl3) δ 2.35 (s, 3H, CH3), 2.62 (ls, 8H, Ha and Hb), 3.59 (s, 2H, CH2), 6.98 – 7.13 (m, 2H, H3’ and H4’), 7.30 (t, 1H, J = 7.0 Hz, H5), 8.37 (d, 1H, J = 8.0 Hz, H6’), 8.52 (s, 1H, H6), 8.72 (d, 1H, J = 2.3 Hz, H5), 9.45 (s, 1H, H3), 12.12 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 45.7 (Cc), 52.1 (Cb and Cb’’), 54.8 (Ca and Ca’), 61.6 (Cd), 121.5 (C-6), 124.2 (C-4), 126.8 (C-5), 128.7 (C-3), 130.2 (C-2), 137.9 (C-1), 142.5 (C-3’), 145.2 (C-5’), 145.8 (C-2’), 147.2 (C-6’), 161.5 (CONH); IR (KBr) 3456 cm-1 (υ CONH), 2937 and 2795 cm-1 (υ CH2), 1677 cm-1 (υ CONH) 1590, 1534 and 1452 cm-1 (υ C=C aromatic), 756 cm-1out of the plane C-H of aromatic rings ortho-substituted); HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (30:70), 254 nm): Retention time: 3.136 min. Peak Area: 95.355 %.

N-{2-[(4-methylpiperazin-1-yl)methyl]phenyl}quinoxaline-6- carboxamide (7; LASSBio-1725): Yield: yellow solid (64%). When necessary, the product was purified by flash chromatography on silica gel (elution with 1% triethylamine in ethyl acetate); MP: 128 ºC; 1H NMR (200 MHz, CDCl3) δ 2.31 (s, 3H, CH3), 2.59 (sl, 8H, Ha and Hb), 3.66 (s, 2H, CH2), 6.97 – 7.13 (m, 2H, H3’ and H4’), 7.31 (t, 1H, J = 8.3 Hz, H5’), 8.16 (d, 1H, J = 8.7 Hz, H6’), 8.31 – 8.42 (m, 2H, H4 and H5), 8.62 (s, 1H, H7), 8.86 (s, 2H, H9 and H10), 11.61 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 45.6 (Cc), 52.5 (Cb e Cb’), 54.7 (Ca e Ca’), 62.4 (Cd), 121.0 – 146.4 (14C, C-aromáticos), 164.2 (CONH); IR (KBr) 3467 cm-1 (υ CONH), 2937 e 2809 cm-1 (υ CH2), 1672 cm-1 (υ CONH) 1591, 1529 e 1448 cm-1 (υ C=C aromático), 749 cm-1out of the plane C-H aromatic rings ortho-substituted); HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (30:70), 254 nm): Retention time: 3.688 min. Peak Area: 95.913 %.

N-{2-[(4-phenylpiperazin-1-yl)methyl]phenyl}quinoxaline-2- carboxamide (8; LASSBio-1726): Yield: yellow solid (59%). When necessary, the product was purified by flash chromatography on silica gel (elution with 20% ethyl acetate in n-hexane); MP: 179 ºC; 1H NMR (200 MHz, CDCl3) δ 2.69 (t, 4H, J = 4.5 Hz, Ha), 3.29 (t, 4H, J = 4.7 Hz, Hb), 3.67 (s, 2H, CH2), 6.74 – 8.42 (13H, H-aromatics), 9.66 (s, 1H, H3), 11.90 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ49.4 (Cb and Cb’), 53.0 (Ca and Ca’’), 62.0 (Cc), 116.3 – 151.3 (20C, C-aromatics), 162.3 (CONH); IR (KBr) 3217 cm-1 (υ CONH), 2938 and 2825 cm-1 (υ CH2), 1679 cm-1 (υ CONH) 1591, 1527 and 1452 cm-1 (υ C=C aromatic), 754 cm-1out of the plane C-H of aromatic rings ortho-substituted); HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (50:50), 254 nm): Retention time: 5.055 min. Peak Area: 97.413 %.

N-{2-[(4-(pyrimidin-2-yl)piperazin-1-yl)methyl]phenyl} quinoxaline-2-carboxamide (9; LASSBio-1723): Yield: yellow solid (58%). When necessary, the product was purified by flash chromatography on silica gel (elution with 30% ethyl acetate in n-hexane); MP: 216 ºC; 1H NMR (200 MHz, CDCl3) δ 2.60 (ls, 4H, Ha), 3.66 (s, 2H, CH2), 3.92 (ls, 4H, Hb), 6.39 – 8.47 (11H, H-aromatics), 9.72 (s, 1H, H3), 11.91 (s, 1H, CONH); 3C NMR (50 MHz, CDCl3) δ 44.0 (Cb and Cb’), 52.8 (Ca and Ca’’), 62.0 (Cc), 110.1 – 161.7 (18C, C-aromatics), 162.2 (CONH); IV (KBr) 3221 cm-1 (υ CONH), 2930 and 2817 cm-1 (υ CH2), 1681 cm-1 (υ CONH) 1585, 1539 and 1447 cm-1 (υ C=C aromatic), 753 cm-1out of the plane C-H of aromatic rings orthosubstituted); HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (50:50), 254 nm): Retention time: 3.300 min. Peak Area: 98.053 %.

N-{2-[(4-(2-hydroxyethyl)piperazin-1-yl)methyl]phenyl} quinoxaline-2-carboxamide (10; LASSBio-1727): Yield: yellow solid (62%). When necessary, the product was purified by flash chromatography on silica gel (elution with ethyl acetate); MP: 150 ºC; 1H NMR (200 MHz, CDCl3) δ 2.65 (ls, 8H, Ha and Hb), 3.11 (ls, 2H, Hc), 3.59 (t, 3H, J = 5.0 Hz, He and Hf), 3.64 (s, 2H, CH2), 7.00 – 8.44 (8H, H-aromatics), 9.70 (s, 1H, H3), 11.75 (s, 1H, CONH); 13C NMR (50 MHz, CDCl3) δ 52.7 (Ca and Ca’), 53.1 (Cb and Cb’), 57.8 (Cd), 59.4 (Cc), 61.6 (Ce), 121.9 – 145.0 (14C, C-aromatics), 162.0 (CONH); IR (KBr) 3390 cm-1a OH), 3213 cm-1 (υ CONH), 2934 and 2812 cm-1 (υ CH2), 1680 cm-1 (υ CONH) 1591, 1538 and 1452 cm-1 (υ C=C aromatic), 755 cm-1out of the plane C-H of aromatic rings ortho-substituted); HPLC (C-18, acetonitrile: aqueous trifluoroacetic acid (50:50), 254 nm): Retention time: 6.463 min. Peak Area: 98.748 %.

X-ray crystallography

Single crystals of the compounds 2a-c (Table 4), suitable for X-ray study, were obtained by slow evaporation of a solution of ethanol-dimethylformamide (3:1), ethanol-dimethyl sulfoxidedichloromethane (1:1:1) and methanol:dichloromethane (10:1), respectively, at room temperature 295(2) K. Data collection was performed using the Kappa Apex II Duo diffractometer operating with Mo-Kα radiation (λ=0.71073 Å) an room temperature. Raw data integration was performed with Saint (Bruker, 2009) and the scaling were carried out using SADABS (Bruker, 2009). Structure solution was obtained using Direct Methods implemented in SHELXS and the model refinement was performed with full matrix least squares on F2 using SHELXL. The programs ORTEP-3, [13] SHELXS/SHELXL [18] were used within WinGX [19] software package.

Compound 2a 2b 2c
Crystal system Monoclinic Monoclinic Monoclinic
Space group P21/c P21/c P21/c
Unit cell dimensions
a(Å) 17.651(4) 6.3082(3) 5.7947(12)
b(Å) 5.8253(14) 26.4257(14) 16.576(4)
c(Å) 19.925(4) 11.5607(6) 20.465(4)
β(°) 108.344(7) 98.802(3) 106.312(11)
Volume (Å3) 1944.6(7) 1904.46(17) 1886.7(7)
Z 4 4 4
Density Calculated (Mg/m3) 1.235 1.261 1.272
Absorption coefficient (mm-1) 0.079 0.081 0.082
F(000) 768 768 768
Crystal size (mm3) 0.25×0.2×0.2 0.32×0.19×0.04 0.41×0.14×0.07
θ range (°) 2.11 to 26.23 1.54 to 25.39 1.61 to 26.47
Inde× ranges
h -21 to 21 -7 to 7 -7 to 7
k -7 to 7 -30 to 31 -19 to 20
l -24 to 15 -13 to 13 -25 to 23
Reflections collected 14313 9930 14347
Independent reflections 3881 3410 3858
R(int) 0.0459 0.0500 0.0223
Completeness to θ = 25° (%) 99.1 99.3 99.2
Data / restraints / parameters 3881 / 0 / 245 3410 / 0 / 244 3858 / 0 / 246
Goodness-of-fit on F2 1.064 1.021 1.044
Final R1 indices [I>2σ(I)] 0.0410 0.0542 0.0399
Final wR2 indices [I>2σ(I)] 0.1276 0.1325 0.1059
R indices (all data) 0.0577 0.0908 0.0544
R indices (all data) 0.1442 0.1532 0.1146
Extinction coefficient 0.0113(19)   0.0083(12)
Largest diff. peak / hole (e.Å-3) 0.214 / -0.174 0.156 / -0.167 0.168 / -0.152

Table 4: Crystal data and structure refinement details of compounds 2a-c.

Biological assays

All experiments were performed with male Swiss mice (20-25 g) obtained from Instituto Vital Brazil (Niterói, Brazil). Animals were maintained in temperature-controlled room (22 ± 2°C) with a 12 h light/dark cycles and free access to food and water. Twelve hours prior to each experiment, the animals received only water in order to avoid food interfering with the substance absorption. Animal care and research protocols (DFBCICB015-04/16) were in accordance with the principles and guidelines adopted by the National Council of Control of Animal Experimentation (CONCEA).

MTT assay [14]: The tumor cells used HL-60 (human leukemia – ATCC code: CCL-240™), HCT-8 (human colorectal carcinoma – ATCC code: CCL-244™) and SF-295 (human glioblastoma) were provided by the National Cancer Institute (USA). Human lymphocytes were isolated from heparinized blood from healthy, non-smoker donors who had not taken any medication at least 15 days prior to sampling by a standard method of density-gradient centrifugation on Histopaque-1077 (Sigma Aldrich Co. - St. Louis, MO/USA). Cancer cells and lymphocytes were incubated in RPMI 1640 supplemented with 10% (tumor cells) or 20% (lymphocytes) fetal calf serum. Lymphocytes cultures were also supplemented with 2% phytohaemagglutinin. Cells were seeded in 96 well plates at the following densities: 0.3 × 106 (HL-60), 0.7 × 105 (HCT8, SF-295), and 1 × 106 (lymphocytes) cells/mL. After 24 hour, the compounds were added at concentrations ranging from 0.1 μM – 300 μM and then incubated at 37°C and atmosphere containing 5 % CO2 for 72 hours. After that, it was added 100 μL of a solution of MTT (5 mg/ mL) in RPMI medium and incubated for 3 hour at the same conditions. After incubation time, it was added 100 μL of DMSO for solubilization of MTT-formazan crystals formed by the metabolism of MTT salt by the viable cells. Finally, the plates were read on an ELISA reader at a wavelength of 550 nm.

The experiments were analyzed according to their means and standard error media (S.E.M.) confidence intervals from nonlinear regression in GraphPad Prism. Each sample was tested in triplicate in two independent experiments.

Air pouch synovial model [15]: The method was similar to that described by Romano et al. [16] with several modifications described in Raymundo et al. [17]. Briefly, air pouches were produced by subcutaneous injection of sterile air (10 ml) into the intrascapular region of the mice. After three days, another injection of air (10 ml) was performed in order to maintain the pouches. Three days after the last injection of air, animals received an injection of sterile carrageenan suspension (1%) into the SAP. Mice were pre-treated with oral doses of substances, ASA or vehicle 1 h before carrageenan injection in the SAP. Animals were sacrificed 24 h after carrageenan injection and the cavity was washed with 1 ml of sterile PBS. The total number of cells was determined with the aid of a haemocytometer (CellPoucH, Merck, USA). The exudates were centrifuged at 170 x g for 10 min at 4 °C, and the supernatants were collected and stored at −20°C to further analysis. Supernatants from exudates collected in the SAP were used to measure tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay (ELISA), using the protocol supplied by the manufacturer (B&D, USA).

Statistical analysis

The results are presented as mean ± standard deviation (SD), mean ± error deviation or mean ± confidence interval using the GraphPad Prism vs. 5.0. Statistical significance between groups was calculated using analysis of variance (ANOVA) followed by Bonferroni or Dunnet tests. P values less than 0.05 (*p<0.05) were used as significant level.

Acknowledgments

The authors would like to thank INCT-INOFAR (573.564/2008-6 and E-26/170.020/2008), CNPq (BR), FAPERJ (BR) and CAPES (BR) for fellowship and financial support. We also thank Dr. Nailton Monteiro do Nascimento Júnior for his valuable help with the NMR spectra analysis.

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