Translate this page into:
Ultrasound-assisted synthesis and anticancer evaluation of new pyrazole derivatives as cell cycle inhibitors
⁎Corresponding author. cbleotu@yahoo.com (Coralia Bleotu)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
We designed new pyrazole derivatives as inhibitors of the cell cycle kinases and developed a simple environmentally sustainable synthesis process. We synthesized the pyrazolyl thiourea derivatives using rapid ultrasound mediated methods and confirmed their structures by NMR and IR spectra. The apoptosis and necrosis inducing effects of the new compounds were investigated. Cell cycle analysis and expression of genes involved in apoptosis, cell cycle and xenobiotic metabolism were studied. The compounds presented modest apoptotic effects in human cancer cells. The N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]carbamothioyl]-4-chloro-benzamide compound (4e) induced a significant increase of cells in G2/M phases in conjunction with an increased expression of cyclin A and cyclin B, emerging as a promising anticancer drug, to be further developed in animal models of cancer.
Keywords
Aminopyrazole
Thiourea
G2/M arrest
Apoptosis
1 Introduction
In malignant cells the initiation, progression, and completion of the cell cycle are frequently altered inducing an abnormal growth (Shapiro and Harper, 1999). A broad range of compounds are designed to interrupt cell cycle progression in order to control the growth of cancer cells. The compounds capable to arrest the cell cycle rely on various mechanisms of action. Some of the key mechanisms responsible for cell cycle progression and cell division, include the cyclin-dependent kinases (CDKs), checkpoint kinases (CHKs) and Aurora kinases (AURs), all being important targets in anticancer drug discovery (Pitts et al., 2014).
The pyrazole ring emerged as a powerful scaffold used extensively in the design of compounds targeted to block the cell cycle progression in cancer cells (Keter and Darkwa, 2012). Pharmacologic inhibitors of cyclin-dependent kinases (CDKs) have been shown to block cell cycle progression in a large variety of cell types. The use of the pyrazole ring in the design of CDKs inhibitors is demonstrated by the development of AT7519, a 4-[(2,6-dichlorobenzoyl)amino-1H-pyrazole-3-carboxamide derivative, with anti-proliferative effects in leukemia, colon and breast cancer (Sánchez-Martínez et al., 2015). A series of 4-arylazo-3,5-diamino-1H-pyrazole derivatives demonstrated anti-CDK kinase activities and anti-proliferative properties (Jorda et al., 2015).
Aurora kinases are important targets for cancer chemotherapy because of their role in the cell division, especially during mitosis and many inhibitors are currently under development (Kollareddy et al., 2012). AT9283 is a pyrazol-4-yl urea derivative and a potent AUR A and B inhibitor, capable of inhibiting growth and survival of multiple solid tumor and leukemia cell (Kimura, 2010). The importance of the pyrazole template in the design of AURs inhibitors had been demonstrated (Nitulescu et al., 2014) and can be observed in the development of both isoform non-selective inhibitors, such as SCH 1473759 (Yu et al., 2010) and ABT-348 (Curtin et al., 2012), and subtype selective inhibitors. Tozasertib and its derivative ENMD-2076 are based on the 3-aminopyrazole scaffold and are potent inhibitors of AUR A (Nitulescu et al., 2013), while AZD1152 and GSK1070916 are pyrazole derivatives designed as AUR B selective inhibitors (Zhang et al., 2010).
CHKs are key regulators of cell cycle progression serving to maintain the genomic integrity of cells and there is a significant effort to design selective inhibitors (Pitts et al., 2014). MK-8776 is a 1-methyl-1H-pyrazol-4-yl-pyrazolo[1,5-a]pyrimidine derivative selective CHKs inhibitor that interacts synergistically with DNA antimetabolite agents inducing cell death in tumor cells (Guzi et al., 2011). PF-00477736 is also a 1-methyl-1H-pyrazol-4-yl CHK1 inhibitor which was demonstrated to enhance the cytotoxicity of clinically important chemotherapeutic agents and radiation (Blasina et al., 2008).
In our previous research, we designed and developed several compounds with anti-proliferative effects by coupling the pyrazole scaffold and the thiourea moiety (Nitulescu et al., 2013). This strategy of combining the pyrazole ring and the thiourea moiety proved to be successful in the development of various anti-proliferative agents (Çalışkan et al., 2013; Karipcin et al., 2013) and apoptosis inducing agents (Nitulescu et al., 2015). Several acyl thiourea derivatives containing pyrazole ring were evaluated and demonstrated very good anticancer properties on human leukemia, colon, and liver cancer cell lines (Koca et al., 2013) by inhibiting human AURs (Ozgur et al., 2015). The thiourea and pyrazole ring were used for the design of several cell cycle inhibitors targeting CDK 2, 4 and 6 (Sun et al., 2013).
Based on the biopharmaceutical profiling of our previously synthesized pyrazole derivatives (Anuta et al., 2014), in this work we developed new compounds by joining the pyrazole and thiourea substructures, in order to target the cell cycle kinases.
2 Methods
2.1 Chemistry
All reagents and solvents were obtained from common commercial suppliers and used without purification. The acetonitrile was dried over 3 Å molecular sieves and distillated. All reactions were followed by thin-layer chromatography analysis on silica gel 60F254 aluminum sheets, mobile phase toluene:ethyl acetate:ethanol 3:1:1, and were visualized under UV lamp at 254 nm.
Melting points (mp) were measured in open glass capillaries on an IA9000 Series melting point apparatus (Electrothermal, UK) and are uncorrected. The IR spectra were recorded on a JASCO FT/IR-4200 spectrometer (JASCO, Japan) with an ATR PRO450-S accessory. The elemental analyses were performed on a Series II 2400 CHNS/O Analyzer (Perkin Elmer, USA). The synthesis were performed using an Elmasonic S15H ultrasonic equipment operating at 37 kHz (Elma Hans Schmidbauer GmbH & Co., Singen, Germany).
The NMR spectra were performed in DMSO-d6 on a Gemini 300BB instrument (Varian, USA) operating at 300 MHz for 1H and 75 MHz for 13C. The chemical shifts were recorded as δ values in ppm units downfield to tetramethylsilane, used as internal standard. The coupling constants values (J) are reported in hertz (Hz) and the splitting patterns are abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
2.1.1 General synthesis procedure for compounds 4a–h
A solution of 4-substituted benzoic acid (0.1 mol) in anhydrous 1,2-dichlorethane is refluxed with thionyl chloride (14.5 mL, 0.2 mol) until evolution of gas is completed. The solvent and the excess thionyl chloride are removed by distillation at reduced pressure. The obtained 4-substituted benzoyl chloride (10 mmol) is dissolved in acetonitrile (10 mL) and ammonium thiocyanate (10 mmol) is added. The mixture was sonicated for 15 min at room temperature and 37 kHz. The resulting ammonium chloride was removed by filtration and the suitable aminopyrazole (10 mmol) was added to the raw benzoyl isothiocyanate solution. The mixture was subjected to ultrasonic irradiation for 15–30 min and then poured into 50 mL of 0.1 M hydrochloric solution. The compounds (4a–h) were recrystallized from isopropanol.
2.1.1.1 4-chloro-N-[[3-(4-fluorophenyl)-1H-pyrazol-5-yl]carbamothioyl]benzamide (4a)
Yield 77%, mp 234–235 °C. IR (cm−1): 3166 (N—H), 3029 (N—H), 1667 (C⚌O), 1537 (C—N). 1H NMR (DMSO-d6, ppm): 13.25 (s, 1H, NH), 12.75 (s, 1H, —CS—NH—), 11.75 (s, 1H, —CO—NH—), 7.92 (d, J = 8.6 Hz, 2H, ArH), 7.73 (dd, J = 8.9 Hz, J = 5.2 Hz, 2H, ArH), 7.54 (d, J = 8.6 Hz, 2H, ArH), 7.38 (s, 1H, Pyrz), 7.34 (t, J = 8.9 Hz, 2H, ArH). 13C NMR (DMSO-d6, ppm): 177.01 (—CS—), 167.74 (—CO—), 163.66 (1JC–F = 263.9 Hz), 147.70 (Pyrz), 141.06 (Pyrz), 138.11, 130.92, 129.11, 128.91, 127.32 (3JC–F = 8.0 Hz), 125.65, 116.08 (2JC–F = 21.5 Hz), 95.75 (Pyrz). Calcd. for C17H12ClFN4OS: C, 54.48; H, 3.23; N, 14.95; S, 8.5; Found: C, 54.55; H, 3.27; N, 15.00; S, 8.61%.
2.1.1.2 N-[[3-(4-fluorophenyl)-1H-pyrazol-5-yl]carbamothioyl]-4-methyl-benzamide (4b)
Yield 69%, mp 243–245 °C. IR (cm−1): 3166 (N—H), 3016 (N—H), 1664 (C⚌O), 1534 (C—N). 1H NMR (DMSO-d6, ppm): 13.30 (s, 1H, NH), 13.25 (s, 1H, —CS—NH—), 11.60 (s, 1H, —CO—NH—), 7.92 (d, J = 8.3 Hz, 2H, ArH), 7.73 (dd, J = 8.9 Hz, J = 5.2 Hz, 2H, ArH), 7.46 (s, 1H, Pyrz), 7.34 (t, J = 8.9 Hz, 2H, ArH), 7.30 (d, J = 8.3 Hz, 2H, ArH), 2.27 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 177.21 (—CS—), 168.53 (—CO—), 162.07 (1JC–F = 254.4 Hz), 147.78 (Pyrz), 144.41, 143.84 (Pyrz), 129.11, 129.00, 128.91, 127.39 (3JC–F = 8.0 Hz), 125.71, 116.12 (2JC–F = 22.0 Hz), 95.80 (Pyrz), 21.21 (CH3). Calcd. for C18H15FN4OS: C, 61.00; H, 4.27; N, 15.81; S, 9.05; Found: C, 61.06; H, 4.31; N, 16.02; S, 8.88%.
2.1.1.3 4-chloro-N-[[3-(4-chlorophenyl)-1H-pyrazol-5-yl]carbamothioyl]benzamide (4c)
Yield 71%, mp 234–235 °C. IR (cm−1): 3155 (N—H), 3098 (N—H), 1668 (C⚌O), 1558 (C—N). 1H NMR (DMSO-d6, ppm): 13.24 (s, 1H, NH), 13.06 (s, 1H, —CS—NH—), 11.80 (s, 1H, —CO—NH—), 8.00 (d, J = 8.5 Hz, 2H, ArH), 7.78 (d, J = 8.2 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.53 (d, J = 8.2 Hz, 2H, ArH), 7.50 (s, 1H, Pyrz). 13C NMR (DMSO-d6, ppm): 177.07 (C⚌S), 167.74 (C⚌O), 147.75 (Pyrz), 138.12 (Pyrz), 132.95, 130.92, 130.76, 129.16, 128.55, 127.85, 126.90, 96.07 (Pyrz). Calcd. for C17H12Cl2N4OS: C, 52.18; H, 3.09; N, 14.32; S, 8.19; Found: C, 52.19; H, 3.20; N, 14.47; S, 8.02%.
2.1.1.4 N-[[3-(4-chlorophenyl)-1H-pyrazol-5-yl]carbamothioyl]-4-methyl-benzamide (4d)
Yield 67%, mp 243–245 °C. IR (cm−1): 3159 (N—H), 3097 (N—H), 1662 (C⚌O), 1528 (C—N). 1H NMR (DMSO-d6, ppm): 13.37 (s, 1H, NH), 13.20 (s, 1H, —CS—NH—), 11.59 (s, 1H, —CO—NH—), 7.92 (d, J = 8.4 Hz, 2H, ArH), 7.79 (d, J = 8.6 Hz, 2H, ArH), 7.54 (d, J = 8.6 Hz, 2H, ArH), 7.51 (s, 1H, Pyrz), 7.35 (d, J = 8.4 Hz, 2H, ArH), 2.39 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 177.24 (C⚌S), 168.53 (C⚌O), 143.76 (Pyrz), 140.74 (Pyrz), 132.87, 129.10, 129.02, 128.95, 128.81, 127.84, 126.83, 96.02 (Pyrz), 21.14 (CH3). Calcd. for C18H15ClN4OS: C, 58.30; H, 4.08; N, 15.11; S, 8.65; Found: C, 58.08; H, 4.02; N, 15.19; S, 8.60%.
2.1.1.5 N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]carbamothioyl]-4-chloro-benzamide (4e)
Yield 70%, mp 234–235 °C. IR (cm−1): 3165 (N—H), 3027 (N—H), 1667 (C⚌O), 1539 (C—N). 1H NMR (DMSO-d6, ppm): 13.30 (s, 1H, NH), 12.86 (s, 1H, —CS—NH—), 11.80 (s, 1H, —CO—NH—), 8.00 (d, J = 8.5 Hz, 2H, ArH), 7.72 (d, J = 8.5 Hz, 2H, ArH), 7.67 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 1H, Pyrz). 13C NMR (DMSO-d6, ppm): 177.16 (C⚌S), 167.79 (C⚌O), 147.79 (Pyrz), 138.13 (Pyrz), 134.71, 132.08, 130.78, 130.22, 130.19, 128.56, 127.17, 121.57, 96.05 (Pyrz). Calcd. for C17H12BrClN4OS: C, 46.86; H, 2.78; N, 12.86; S, 7.36; Found: C, 46.79; H, 2.83; N, 12.92; S, 7.33%.
2.1.1.6 N-[[3-(4-bromophenyl)-1H-pyrazol-5-yl]carbamothioyl]-4-methyl-benzamide (4f)
Yield 75%, mp 243–245 °C. IR (cm−1): 3155 (N—H), 3094 (N—H), 1667 (C⚌O), 1528 (C—N). 1H NMR (DMSO-d6, ppm): 13.37 (s, 1H, NH), 12.97 (s, 1H, —CS—NH—), 11.57 (s, 1H, —CO—NH—), 7.91 (d, J = 8.3 Hz, 2H, ArH), 7.71 (d, J = 8.6 Hz, 2H, ArH), 7.67 (d, J = 8.6 Hz, 2H, ArH), 7.48 (s, 1H, Pyrz), 7.35 (d, J = 8.3 Hz, 2H, ArH), 2.39 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 177.42 (C⚌S), 168.67 (C⚌O), 143.94 (Pyrz), 139.03 (Pyrz), 132.17, 132.14, 132.08, 129.21, 128.93, 128.80, 127.22, 121.58, 96.15 (Pyrz), 21.26 (CH3). Calcd. for C18H15BrN4OS: C, 52.06; H, 3.64; N, 13.49; S, 7.72; Found: C, 51.91; H, 3.62; N, 13.63; S, 7.85%.
2.1.1.7 4-chloro-N-[(1,5-dimethyl-3-oxo-2-phenyl-pyrazol-4-yl)carbamothioyl]benzamide (4g)
Yield 83%, mp 190–192 °C. IR (cm−1): 3132 (N—H), 3082 (N—H), 1670 (C⚌O), 1640 (C⚌O), 1530 (C—N). 1H NMR (DMSO-d6, ppm): 11.82 (s, NH), 11.51 (s, NH), 7.99 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.52 (t, J = 7.8 Hz, 2H), 7.38–7.32 (m, 3H, ArH), 3.12 (s, 3H, CH3), 2.21 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 181.95 (C⚌S), 167.30 (C⚌O), 161.10 (C⚌O), 153.12 (Pyrz), 138.05, 135.03, 131.02, 130.74, 129.18, 128.56, 126.56, 123.91, 108.28 (Pyrz), 35.71 (CH3), 11.53 (CH3). Calcd. for C19H17ClN4O2S: C, 56.93; H, 4.27; N, 13.98; S, 8.00; Found: C, 57.11; H, 4.19; N, 14.14; S, 7.97%.
2.1.1.8 N-[(1,5-dimethyl-3-oxo-2-phenyl-pyrazol-4-yl)carbamothioyl]-4-methyl-benzamide (4h)
Yield 76%, mp 203–204 °C. IR (cm−1): 3165 (N—H), 3013 (N—H), 1671 (C⚌O), 1645 (C⚌O), 1524 (C—N). 1H NMR (DMSO-d6, ppm): 11.83 (s, NH), 11.63 (s, NH), 7.89 (d, J = 8.3 Hz, 2H), 7.52 (dd, J = 8.5 Hz, J = 1.6 Hz, 2H), 7.38–7.32 (m, 5H, ArH), 3.11 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.20 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 182.28 (C⚌S), 168.22 (C⚌O), 161.15 (C⚌O), 153.11 (Pyrz), 143.77, 135.00, 129.20, 128.86, 129.24, 129.14, 126.63, 123.96, 108.32 (Pyrz), 35.71 (CH3), 21.21 (CH3), 11.55 (CH3). Calcd. for C20H20N4O2S: C, 63.14; H, 5.30; N, 14.73; S, 8.43; Found: C, 63.30; H, 5.21; N, 14.87; S, 8.33%.
2.2 Evaluation of biological activity
Human colorectal adenocarcinoma HT-29 (ATCC HTB-38) and acute monocytic leukemia THP-1 (ATCC TIB-202) cell lines were used. The adherent cell cultures were maintained in Dulbecco’s Modified Essential Medium (DMEM) (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, USA) at 37 °C, 5% CO2, in a humid atmosphere. The monocytic cell culture, THP-1, was maintained in RPMI-1640 medium (Sigma, USA) and 10% fetal bovine serum.
2.2.1 Cell viability assay
The HT-29 cells were seeded into 96-well plates at 5 × 103 cells/well and after 24 h, binary dilutions of each compound were added and the cells were maintained for other 24 h at 37 °C, 5% CO2, in a humid atmosphere. 5 × 103 THP-1 cells were added to binary dilutions of each compound and were maintained 24 h at 37 °C, 5% CO2, in a humid atmosphere. The cell viability was evaluated using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) measuring the absorbance at 490 nm in an ELISA reader.
2.2.2 Flow cytometry analysis of cellular apoptosis
Apoptosis detection was made using Annexin V-FITC Apoptosis Detection Kit I (BD Bioscience Pharmingen, USA) according to manufacturer protocol. For this purpose, 3 × 105 HT-29 cells were seeded in 3.5 cm diameter wells and treated with 50 μg/mL of the tested compounds for 24 h. Both adherent and detached cells were re-suspended in 100 μL of binding buffer and stained with 5 μL Annexin V-FITC and 5 μL propidium iodide for 10 min in dark. At least 10,000 events from each sample were acquired using a Beckman Coulter EPICS XL flow cytometer (Fullerton, CA, USA). The percentage of treatment-affected cells was determined by subtracting the percentage of apoptotic/necrotic cells in the untreated population from percentage of apoptotic cells in the population. Early apoptosis was defined as Annexin V positive and PI negative, and late apoptosis, as Annexin V and PI positive.
2.2.3 Flow cytometry analysis of cell cycle
After treatment with 50 μg/mL of tested compounds for 24 h, the cells were harvested, washed in cold solution of PBS (pH 7.5), and then fixed in cold 70% ethanol and stored at −20 °C overnight. Samples were centrifuged, washed with PBS and then re-suspended in 100 μl PBS, treated with RNase A (1 mg/mL) and stained with propidium iodide (100 μg/mL), at 37 °C for 1 h. The DNA content of cells was quantified on a Beckman Coulter EPICS XL flow cytometer (Fullerton, CA, USA) and analyzed using FlowJo 8.8.6 software (Ashland, Oregon, USA).
2.2.4 Quantification of the expression of genes involved in apoptosis, cell cycle and xenobiotic metabolism
Total RNA was extracted with Trizol Reagent (Invitrogen, USA) according to the manufacturer’s protocol from HT-29 cells treated for 24 h with tested compounds (10 μg/mL, 50 μg/mL). The concentration and purity of the RNA samples were determined using Nanodrop equipment. For each sample, 2 μg of total RNA was used for reverse transcription with High Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystem), and 50 ng cDNA from each sample was used in real-time PCR reaction. Real-Time PCR was performed on an ABI 7300 Real-Time PCR System using pre-validated Taqman Gene Expression Assays kits (Applied Biosystems). Human beta-actin was used as endogenous control. Each experiment was performed three times. Results were analyzed with RQ study software (Applied Biosystems). The ΔΔCT method was used to compare the relative gene expression levels.
3 Results and discussion
3.1 Chemistry
Numerous investigations now routinely use non-traditional synthetic methodologies such as ultrasound mediated synthesis (Rao et al., 2015). In order to develop environmentally sustainable processes we optimized the synthesis of the acylthiurea derivatives and reduced the energy consumption by the use of ultrasounds.
The 1H-pyrazol-5-ylthiourea and (1,5-dimethyl-3-oxo-2-phenyl-pyrazol-4-yl)thiourea derivatives were synthesized using the method presented in Scheme 1 starting from 4-substituted benzoic acids (1) that were treated with thionyl chloride to yield the corresponding 4-substituted benzoyl chlorides (2). The ultrasound-assisted reaction of 4-R-benzoyl chlorides with ammonium isothiocyanate in acetonitrile afforded the corresponding 4-R-benzoyl isothiocyanates (3). The sonication of 3 with suitable aminopyrazoles afforded the target compounds (4a–h).
Reagents: (a) SOCl2, C2H4Cl2, reflux; (b) NH4SCN, CH3CN, ultrasonic irradiation; (c) R2–C6H4–C3H2N2–NH2, CH3CN, ultrasonic irradiation; (d) C11H11N2–NH2, CH3CN, ultrasonic irradiation.
The structures of the synthesized compounds were confirmed by NMR and IR spectral data. In the infrared spectra of compounds 4a–h, can be observed the absorptions between 3166 and 3013 cm−1 relating to the thiourea NHs stretch and the absorptions in the 1645–1670 cm−1 range produced by the carbonyl bond stretching. The C—N bond of the acyl thiourea moiety has an intense band in the range 1566–1521 cm−1. The band has unexpectedly high intensity compared with amides, explaining the involvement of the carbonyl group in an intra-molecular hydrogen bond.
In the 1H NMR spectra of the compounds 4a–f the hydrogen of the pyrazole ring presents one singlet at 13.37–13.24 ppm, the thiourea hydrogen atoms give two broad singlets at 13.25–12.75 ppm (NH—C⚌S) and 11.80–11.59 ppm (NH—C⚌O), and the hydrogen on the two phenyl groups can be observed in the range of 8.00–7.30 ppm. The methyl substituted compounds present a singlet in range of 2.39–2.27 ppm. The structure of the compounds 4g–h is confirmed by the broad singlets of the thiourea moiety at 11.82–11.63 ppm (NH—C⚌S) and 11.63–11.51 ppm (NH—C⚌O), the signals of the hydrogen on the benzene rings observed in the range of 7.99–7.32 ppm, and the two methyl groups at 3.12–3.11 ppm and 2.21–2.20 ppm.
In the 13C NMR spectra of the compounds 4a–f are observed a signal in the range of 177.42–177.01 ppm is related to the thiourea carbon (C⚌S), a signal at 168.67–167.74 ppm assignable to the carbonyl (C⚌O), and the pyrazole ring signals at 147.83–143.40 ppm (C-3), 96.15–95.75 ppm (C-4) and 143.84–138.13 ppm (C-5). The methyl group presented a signal in the range of 21.26–21.14 ppm. In the 13C NMR spectra of the compounds 4g–h we can observe the C⚌S carbon signal at 182.28–181.95 ppm, the carbonyl carbon at 168.22–167.30 ppm and the signals of the methyl groups at 35.71 ppm and 11.55–11.54 ppm.
The compound’s purity was certified by elemental analyses and the results were within ± 0.4 of the theoretical values
3.2 Evaluation of biological activity
3.2.1 Cell viability assay
Anticancer drugs are designed to kill selectively tumor cells. At the primary screening stage, in vitro toxicity assay was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Kit (Promega, USA). The pyrazole antitumor activity was tested using adherent HT29 cells and suspension monocyte line THP1. The THP1 line was more susceptible to the action of synthesized compounds. The viability of THP1 cells, grown in the presence of 50 μg/ml pyrazole, decreased in all cases, and the toxic effects were manifested to a concentration of 25 μg/ml in the case of 4e (Fig. 1). IC50 of the compound 4e was 40.34, comparing with other compounds whose IC 50 was included in the range 42.97–48.96.
Results of THP1 cells viability (%) after 24 h exposure to 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, and 50 μg/mL of the new compounds using the MTS assay.
3.2.2 Analysis of cellular cytotoxicity by flow cytometry
The apoptosis- and necrosis-inducing effects of the compounds were tested in HT-29 cells using propidium iodide (PI) in conjunction with Annexin-V-FITC. The tested compounds produced only modest effects on apoptosis/necrosis induction. The compound 4e has the highest necrosis-inducing effect, while compound 4g has the highest apoptosis-inducing effect (Table 1).
Sample
Necrosis
Early apoptosis
Late apoptosis
Viable cells
Control
1.02
2.26
0.11
96.6
4a
1.66
1.64
0.15
96.5
4b
1.62
1.56
0.04
96.8
4c
0.87
3.01
0.22
95.9
4d
1.82
2.13
0.27
95.8
4e
8.43
5.69
1.16
84.7
4f
2.66
2.67
0.84
93.8
4g
1.12
6.95
0.54
91.4
4h
1.02
4.66
0.14
96.8
Flow cytometry diagrams of apoptosis/necrosis effects in HT-29 cells treated for 24 h with 4a–h compounds are presented (Fig. 2). Dot plots are representation of logarithmic Annexin V fluorescence versus PI fluorescence. The cells in region Q4 represent living cells, Q3 early apoptotic cells, Q2 late apoptotic cells and Q1 those in necrosis.
Flow cytometry diagram of double-staining with Annexin V-FITC/PI after treatment with 50 μg/mL of the new compounds.
3.2.3 Quantitation of genes expression involved in apoptosis
In order to identify the mechanism of apoptosis, we performed real-time PCR analysis for expression of genes implicated in apoptosis. It was observed that treatment of HT-29 cells with 50 μg/ml of 4 g and 4 h induced inhibition in Bax and MCL1 pro-apoptotic genes expression. Furthermore, inhibition of expression of caspases 8 was observed in the presence of 50 μg/ml of 4 h suggesting extrinsic apoptotic pathway inhibition and increases in expression level of caspase 9 and caspase 3 (slightly) was associated with activation of intrinsic pathway. After 24 h, all other compounds induced an increase in the pro-apoptotic gene expression of extrinsic /intrinsic pathways (Fig. 3).
The effects exerted by the new compounds (50 μg/mL) on the expression of apoptotic genes. Results are expressed as log10-relative quantitation levels.
3.2.4 Flow cytometry analysis of cell cycle
Analyses of the cell cycle distribution of HT-29 cells after exposure for 24 h to 50 μg/mL of each newly synthesized pyrazole derivatives showed different modification patterns depending on the chemical structure (Fig. 4, Table 2). The 5-aminopyrazole derivatives (4a–f) increased the G2/M, accompanied by a decrease in the number of cells in the G0/G1 indicating mitotic inhibition, while the 4-aminopyrazolone derivatives (4g–h) produced a slight decrease in G2/M. The compound 4e induced a drastic increase in G2/M cell populations, accompanied by the reduction of cells in G0/G1 phase.
HT-29 cells exposed for 24 h to 50 μg/mL of the new compounds. Evaluation of the cell cycle by flow cytometry and the FlowJo 8.8.6 software.
Sample
G0/G1
S
G2/M
Control
65.81
22.49
10.03
4a
61.66
26.75
11.31
4b
62.35
19.78
21.41
4c
63.64
24.20
10.52
4d
64.11
26.47
10.75
4e
4.17
11.32
80.28
4f
54.02
25.32
22.57
4g
73.40
18.06
5.96
4h
66.67
21.65
8.35
3.2.5 Expression of genes involved in cell cycle
We analyzed the effect of the synthesized substances on the gene expression of cyclin A, cyclin B, CDK1 and CDC20 at the level of mRNA expression in HT-29 cells after a 24 h exposure. It was observed an increased expression of cyclin A, cyclin B, CDK1 and CDC20 after treatment of cells with 50 μg/mL of 4e, which correlated with the sharp increase in G2/M and thus associated with a blockage at this level (Fig. 5). Also, in case of 4b and 4f the increased expression of cyclin A was associated with a mild G2/M arrest.
The influence of the tested compounds on the expression of genes involved in the cell cycle of HT-29 cells.
3.2.6 Expression of genes involved in xenobiotics metabolism
It is very important to evaluate the involvement of different enzymes in the biotransformation reactions for drug detoxification and/or bio-activation, taking into account that the proper drug metabolism and elimination will avoid or diminish the adverse effects.
NAT catalyzes the biotransformation of aromatic and heterocyclic amines. These enzymes participate in both detoxification and activation reactions, because NAT can facilitate the detoxification of carcinogenic arylamines to harmless metabolites by N-acetylation or promote their metabolic activation by electrophilic binding to DNA via O-acetylation. NAT1 and NAT2 expression was inhibited in the presence of the 4b and 4h, and increased by 4f, 4a and 4c indicating that these last compounds might be metabolized via NAT pathway (Fig. 6).
The influence of the tested compounds on the expression of genes involved in the xenobiotic metabolism.
In the human genome, 57 genes encoding for cytochrome P450 enzymes have been identified, out of which in xenobiotic metabolism they are involved mainly those belonging to CYP1, CYP2 and CYP3A families. Of these CYP1A2, CYP2A6, CYP2C, CYP2D6, CYP2E1 and CYP3A are involved in the metabolism of 90% of the drugs.
CYP3A4 is responsible for the metabolism of 50% of the drugs, followed by 2D6, 2C9 and 1A2, which metabolize 25%, 15% and 5% respectively. CYP1A, 1B, 2A, 2B and 2E catalyze the conversion of a large number of proto-toxins and carcinogenic substances in terminally reactive metabolites. CYP1A2 is the predominant form of the enzyme CYP1A, its substratum being represented by planar aromatic molecules. All new pyrazole compounds showed increased expression of CYP1A1. CYP2C19 oxidizes especially unionized acid molecules and is inhibited in the presence of 4a–h compounds. CYP3A4 is the major P450 isoform of 3A family, the typical substratum of the CYP3A4 being represented by the highly hydrophobic compounds. CYP3A4 expression is increased by the treatment with the 4g and 4h (Fig. 6).
4 Conclusion
By combining the pyrazole ring and the thiourea moiety we designed new compounds targeting the cell cycle kinases. We have described a simple, rapid and accessible ultrasound-assisted method to obtain these derivatives and confirmed their structures by IR and NMR spectroscopic analysis and elemental analytical data. The compounds 4e and 4g present a modest apoptosis-inducing effect. Exposure of THP-1 cells to the compounds at 50 μg/mL resulted in cell viability decrease, but produced a small effect at concentrations under 25 μg/mL.
The compound 4e induced a drastic increase in G2/M cell population, accompanied by the reduction of cells in G0/G1 phase in HT-29 cells after exposure to 50 μg/mL. The cell blockade in G2/M was correlated with an increased expression of cyclin A, cyclin B, CDK1 and CDC20 genes. The compound 4e displayed the best anti-proliferative effect and emerged as the lead molecule for new pyrazole/thiourea based derivatives.
Acknowledgments
This work received financial support through the project entitled “CERO – Career profile: Romanian Researcher”, Grant Number POSDRU/159/1.5/S/135760, co-financed by the European Social Fund for Sectoral Operational Programme Human Resources Development 2007–2013.
References
- Biopharmaceutical profiling of new antitumor pyrazole derivatives. Molecules. 2014;19:16381-16401.
- [CrossRef] [Google Scholar]
- Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1. Mol. Cancer Ther.. 2008;7:2394-2404.
- [CrossRef] [Google Scholar]
- Synthesis and evaluation of analgesic, anti-inflammatory, and anticancer activities of new pyrazole-3(5)-carboxylic acid derivatives. Med. Chem. Res.. 2013;22:782-793.
- [CrossRef] [Google Scholar]
- Thienopyridine ureas as dual inhibitors of the VEGF and Aurora kinase families. Bioorgan. Med. Chem. Lett.. 2012;22:3208-3212.
- [CrossRef] [Google Scholar]
- Targeting the replication checkpoint using SCH 900776, a potent and functionally selective CHK1 inhibitor identified via high content screening. Mol. Cancer Ther.. 2011;10:591-602.
- [CrossRef] [Google Scholar]
- Novel arylazopyrazole inhibitors of cyclin-dependent kinases. Bioorg. Med. Chem.. 2015;23:1975-1981.
- [CrossRef] [Google Scholar]
- Structural, spectral, optical and antimicrobial properties of synthesized 1-benzoyl-3-furan-2-ylmethyl-thiourea. J. Mol. Struct.. 2013;1048:69-77.
- [CrossRef] [Google Scholar]
- Perspective: the potential of pyrazole-based compounds in medicine. BioMetals 2012
- [CrossRef] [Google Scholar]
- AT-9283, a small-molecule multi-targeted kinase inhibitor for the potential treatment of cancer. Curr. Opin. Invest. Drugs. 2010;11:1442-1449.
- [Google Scholar]
- Synthesis and anticancer activity of acyl thioureas bearing pyrazole moiety. Bioorg. Med. Chem.. 2013;21:3859-3865.
- [CrossRef] [Google Scholar]
- Aurora kinase inhibitors: progress towards the clinic. Invest. New Drugs. 2012;30:2411-2432.
- [CrossRef] [Google Scholar]
- New potential antitumor pyrazole derivatives: synthesis and cytotoxic evaluation. Int. J. Mol. Sci.. 2013;14:21805-21818.
- [CrossRef] [Google Scholar]
- Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines. Bioorganic Med. Chem. 2015 doi:10.1016/j.bmc.2015.07.010
- [Google Scholar]
- In the search of Aurora-A kinase inhibitors as antitumor agents. Rom. J. Biophys.. 2014;24:243-254.
- [Google Scholar]
- Acyl Thiourea Derivatives Containing Pyrazole Ring Selective Targeting of Human Aurora Kinases in Breast and Bone Cancer. Lett. Drug Des. Discov.. 2015;12:180-189.
- [Google Scholar]
- Targeting nuclear kinases in cancer: development of cell cycle kinase inhibitors. Pharmacol. Ther.. 2014;142:258-269.
- [CrossRef] [Google Scholar]
- Ultrasound mediated synthesis of 6-substituted 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinoline derivatives and their pharmacological evaluation. Arab. J. Chem. 2015
- [CrossRef] [Google Scholar]
- Cyclin dependent kinase (CDK) inhibitors as anticancer drugs. Bioorg. Med. Chem. Lett.. 2015;25:3420-3435.
- [CrossRef] [Google Scholar]
- Anticancer drug targets: cell cycle and checkpoint control. J. Clin. Invest.. 1999;104:1645-1653.
- [Google Scholar]
- Synthesis, biological evaluation and molecular docking studies of pyrazole derivatives coupling with a thiourea moiety as novel CDKs inhibitors. Eur. J. Med. Chem.. 2013;68:1-9.
- [CrossRef] [Google Scholar]
- Discovery of a potent, injectable inhibitor of Aurora Kinases based on the imidazo-[1,2- a ]-pyrazine core. ACS Med. Chem. Lett.. 2010;1:214-218.
- [CrossRef] [Google Scholar]
- 3D-QSAR and molecular docking studies on derivatives of MK-0457, GSK1070916 and SNS-314 as inhibitors against aurora B kinase. Int. J. Mol. Sci.. 2010;11:4326-4347.
- [CrossRef] [Google Scholar]