Synthesis of New 1,3,5-Triazine-Based 2-Pyrazolines as Potential Anticancer Agents

A new series of 1,3,5-triazine-containing 2-pyrazoline derivatives (8–11)a–g was synthesized by cyclocondensation reactions of [(4,6-bis((2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amine]chalcones 7a–g with hydrazine hydrate and derivatives. Chalcones 7a–g were obtained by Claisen-Schmidt condensation between aromatic aldehydes and triazinic derivative 5, which was synthesized in high yield by a microwave-assisted reaction. Seventeen of the synthesized compounds were selected and tested by the US National Cancer Institute (NCI) for their anticancer activity against 58 different human tumor cell lines. Compounds 7g and 10d,e,g showed important GI50 values ranging from 0.569 to 16.6 µM and LC50 values ranging from 5.15 to >100 µM.


Introduction
Cancer continue being one of the main causes of death worldwide [1]. Chemotherapy is the most common procedure used for its treatment, despite of its known side-effects. For that reason, many researchers are focusing on the design of new more selective anticancer agents, able to combat the resistance of cancer cells and hence, able to reduce their secondary clinical drawbacks. One strategy to obtain new anticancer therapeutic agents is the combination of two or more biologically active molecules/pharmacophores in only one structure (i.e., molecular hybridization), which by synergistic effects between them, could afford biologically and pharmacologically improved therapeutic entities [2,3]. For example, triazine-pyrazoline-based hybrids have been synthesized and reported to possess important biological activities, particularly, anticancer activity [4,5].
Recently, we have reported the synthesis and anticancer activity of some novel pyrazolines and their chalcone-precursors with outstanding GI 50 values in the range of 0.13 to 0.99 µM [32], 0.28 to 11.7 µM [33] and 0.04 to 11.4 µM [34], when they were subjected to in vitro assays. Thus, as a continuation of our current studies on the synthetic utility of chalcones and derivatives [32][33][34], and based on the proven anticancer properties of triazinic and pyrazolinic derivatives, we report here the synthesis from chalcone derivatives of 1,3,5-triazine-2-pyrazoline hybrids and their subsequent evaluation (along with their triazino-ketone and chalcone precursors), by the US National Cancer Institute (NCI) against 58 different human tumor cell lines.
The structural elucidation of compounds 7a-g was performed by analysis of their spectroscopic data (FTIR, 1 H-NMR, 13 C-NMR and mass spectrometry). We discuss here the spectroscopic data of compound 7a as representative of this series. The IR spectrum of compound 7a shows absorption bands at 3362, 3287 and 1645 cm −1 corresponding to stretching vibration of N-H, O-H and C=O bonds, respectively. In the 1 H-NMR spectrum the signals of the methylene groups protons appeared at 3.33-3.39 and 3.48-3.59 ppm, respectively. Two doublets assigned to both vinylic protons of the α,β-unsaturated moiety, with a coupling constant of 3 J = 15. 6 Hz are observed at 7.70 and 7.94 ppm; this coupling constant corroborates the E-configuration of the carbon-carbon double bond. The mass spectrum shows a molecular ion peak at m/z 420, which is in agreement with the structure proposed for 7a. The synthesis of target 2-pyrazolines 8(a-g)-11(a-g) was performed through 1,2-dinucleophilic cyclocondensation reactions under different experimental conditions (Scheme 2). Thus, the N-acetyl pyrazolines 8a-g and N-formyl pyrazolines 9a-g were obtained by reaction of chalcones 7a-g with hydrazine monohydrate and their subsequent functionalization with acetic anhydride and formic acid, respectively, under stirring in ethanol at room temperature for 2.5-3.5 h. On the other hand, the treatment of chalcones 7a-g with 3,5-dichlorophenyl hydrazine and 4-chlorophenyl hydrazine under reflux in ethanol for 2 h afforded the N-3,5-dichlorophenylpyrazolines 10a-g and the N-4-chlorophenylpyrazolines 11a-g, respectively, Scheme 2.
The obtained pyrazolinic compounds 8(a-g)-11(a-g) showed wide FT-IR absorption bands in the range of 3228-3309 cm −1 assigned to O-H groups. The IR spectra also showed absorption bands at 1514-1604 and 1562-1651 cm −1 assigned to C=C and C=N functionalities, respectively. In the 1 H-NMR spectrum of N-3,5-dichlorophenylpyrazoline 10e, for example, the protons on the diastereotopic center C-4, of the pyrazoline ring appears as two double doublets at δ 3.17 ppm (with 2 J AM = 17.6 Hz and 3 J AX = 4. 8 Hz), and at δ 3.93 ppm (with 2 J AM = 17.6 Hz, and 3 J MX = 12.0 Hz), while the H-5 proton is observed as a double doublet at δ 5.63 ppm (with 3 J MX = 12.0 Hz and 3 J AX = 4.8 Hz), confirming the existence of an AMX coupling system in the pyrazoline ring. The mass spectrum showed molecular ion peak at m/z 612 (in agreement with the expected mass), and a 100:81: 32 6] + ), isotopic profile, respectively, characteristic of a compound bearing three chlorine atoms in its structure.

Anticancer Activity
The two-stage screening process started with the selection of seventeen of the obtained compounds (i.e., 5, 7f,g, 8c,e,f,g, 9e,f,g, 10a,d,e,g, 11b,c,e) by the Drug Evaluation Branch of National Cancer Institute (NCI-USA) through the COMPARE program [36].
The selected compounds were subjected to a primary in vitro evaluation against 58 cell lines at a single dose of 10 µM during an incubation time of 48 h, (it is referred to as one-dose assay). The 58 cell panel is derived from nine different cancer strains: leukemia, lung, melanoma, colon, CNS, ovary, renal, breast and prostate cancers. The output from the one-dose screening was reported as a mean graph of the growth percent (GP) of the treated cells supplied by the NCI for analysis; the values are illustrated in Table 1. The mean graph is drawn with the GP data corresponding to the growth relative to the no-drug control, and relative to the time zero number of cells. This graph allows detection of both growth inhibition (GI) (i.e., GP values between 0 and 100) and lethality (i.e., negative values). For example, a GP value of 100 means no growth inhibition. A GP value of 30 would mean 70% growth inhibition. A GP value of 0 means no net growth over the course of the experiment. A GP value of −30 would mean 30% lethality and a GP value of −100 means all cells are dead. Table 1. Percentages of mean growth and growth inhibition of NCI human cancer cell lines treated with selected compounds (5, 7f,g, 8c,e-g, 9e-g, 10d,e,g, 11b,c,e) at one-dose of 10 µM.

Compound
Mean Growth (%) Most Sensitive Cell Line As shown in Table 1, compounds 7g and 10d,e,g displayed an outstanding anticancer behavior against diverse cell lines, at lethality level, because of their larger number of negative growth inhibition values. Compounds 10a and 11b,e also showed remarkable anticancer behavior against a single cell line with lethality values of −36.95% (LOX IMVI, melanoma), −12.62% (RXF 393, renal) and −26.15% (RXF 393, renal), respectively. Compounds 7f and 11c displayed moderate GI values against HCT-116 of renal cancer cell line (i.e., 86.27%) and RXF 393 also of renal cancer cell line (i.e., 64.68%), respectively. While the remaining compounds showed less activity with GI values in the range of 14.43-34.82. This primary assay showed that compounds 7g and 10d,e,g satisfied the predetermined threshold inhibition criteria, therefore they were considered active. For that, these compounds were subjected to a second in vitro screening in order to determine their cytostatic activity (GI 50 and LC 50 ), against the full 58 cells panel at five concentrations of 10-fold dilution (i.e., 100, 10, 1.0, 0.1 and 0.01 µM) (it is referred to as five-dose assay). The test consisted of a 48 h continuous drug exposure protocol by using sulforhodamine B (SRB) protein assay to estimate cell growth. More details of this evaluation method and the complementary information which is encoded by the activity pattern over all cell lines have been published elsewhere [37][38][39]. The GI 50 value (growth inhibitory activity) corresponds to the molar concentration of the compounds required to inhibit 50% of the growth of cell lines (relative to untreated cells), and LC 50 value (cytotoxic activity) is the molar concentration needed to kill 50% of the cells at the end of the incubation period of 48 h [40].
As shown in Table 2, compounds 7g and 10d,e,g showed outstanding values of GI 50 against several cell lines, some of them lower than 1.00 µM. Chalcone 7g showed GI 50 values in the range of 1.54-15. respectively. Compound 10e showed the best cytotoxicity value against SF-539 (CNS, LC 50 = 5.15 µM), while compound 10g showed the best cytotoxicity value against ACHN (renal, LC 50 = 5.16 µM). Table 2. In vitro testing expressed as growth inhibition GI 50 and lethal concentration LC 50 of cancer cell lines for compounds 7g and 10e,d,g a .

Panel Cell Line Compounds
A raw SAR analysis (from the preliminary one-dose and five-dose data) shows that N-acetyl and N-formyl structures 8 and 9, respectively, were the less actives of the whole evaluated series, while structures 10 were the most active ones, followed by pyrazolines 11 and chalcones 7. It is remarkable that the presence of halogen atoms (Cl and F), in most cases, increased the activity of their structures. In consequence, the halogenated structures 7g,f, 10a,d,e,g and 11b,c,e were the most active compounds of their corresponding series. Finally, it seems that the presence of the N-(3,5-dicholorophenyl) substituent in the pyrazoline ring of compounds 10 is very convenient for improving their relative activity in comparison with the N-(4-cholorophenyl) substituent in pyrazolines 11. This suggestion is supported by the fact that three pyrazolines 10 (i.e., 10d,e,g) were promoted by the NCI to five-dose assays (due to their outstanding inhibitory values at one-dose), against none pyrazoline 11 (see Tables 1 and 2). The above findings and in particular the high activity displayed by structures 7g and 10d,e,g will be used as reference for further structural-reforming experiments addressed to find the most potent as possible chalcone or pyrazoline-triazine lead hybrid for the developing of potential anticancer agents based in our synthesized compounds 7 or 10.

General Information
Reagents and solvents used were obtained from commercial sources and used without further purification. Melting points were measured using a Stuart SMP10 melting point device (Cole-Parmer Ltd., Stone, Staffordshire, UK) and are uncorrected. FTIR spectra were obtained with a IRAffinity-1 spectrophotometer (Shimadzu, Columbia, MD, USA). The 1 H-and 13 C-NMR spectra were run on a DPX 400 spectrometer (Bruker, Billerica, MA, USA) operating at 400 and 100 MHz respectively, using DMSO-d 6 as solvent and TMS as internal standard. The mass spectra were obtained on a Shimadzu-GCMS-QP2010 spectrometer (Shimadzu, Kyoto, Honshu, Japan) operating at 70 eV. The elemental analyses were obtained using an Agilent CHNS elemental analyzer (Thermo Fischer Scientific Inc., Madison, WI, USA) and the values are within ±0.4% of the theoretical values. Thin layer chromatography (TLC) were performed on 0.2 mm pre-coated aluminium plates of silica gel 60 F 254 (Merck, Darmstadt, Hesse, Germany).

General
Procedure for the Synthesis of 5-(4-((4,6-bis((2-Hydroxyethyl) amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(aryl)-4,5-dihydro-1H-pyrazol-1-carbaldehydes 9a-g N-Formylpyrazolines 9a-g were synthesized using a similar methodology for obtaining N-acetyl pyrazolines 8a-g, but formic acid was used instead of acetic anhydride. The solid obtained was filtered and washed with water. No further purification was required.   The human cancer cell lines of the cancer screening panel were grown in an RPMI-1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96-well microtiter plates. After cell inoculation, the microtiter plates were incubated at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity for 24 h prior to the addition of the tested compounds. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of sample addition (Tz). The samples were solubilized in dimethyl sulfoxide (DMSO) at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of compound addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin. An additional four 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus the control. Aliquots of 100 µL of these different sample dilutions were added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final sample concentrations [37][38][39]. After the tested compounds were added, the plates were incubated for an additional 48 h at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 • C. The supernatant was discarded, and plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. Using the seven absorbance measurements [time zero (Tz), control growth in the absence of drug, and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as: [(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti > Tz, and [(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz. Two dose-response parameters were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells and the LC 50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning), indicating a net loss of cells; calculated from [(Ti − Tz)/Tz] × 100 = −50). Values were calculated for each of these two parameters if the level of activity is reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested [37][38][39]41].

Conclusions
In summary, the trisubstituted [1,3,5]-triazine precursor 5 was obtained in good yield under microwave irradiation after a short reaction time. Then, starting from 5 and mediated by chalcone intermediates 7, four new series of 1,3,5-triazine-2-pyrazoline hybrids (8-11)a-g were successfully prepared in acceptable to excellent yields through a multistep method. Seventeen of the obtained compounds were subjected under evaluation of their in vitro growth inhibition properties at one-and five-dose levels against a panel of 58 human cancer cell lines by the U.S. NCI. The screening results showed that chalcone 7g and 3,5-dichlorophenylpyrazolines 10d,e,g exhibited the more remarkable activities against several cancer cell lines (i.e., renal, cns and breast), with GI 50 values in the range of 0.569-16.6 µM and low toxicity. Therefore, compounds 7g and 10d,e,g are promising building blocks for further structural modifications in order to improve their activities and hence develop possible new antitumor agent leaders.