2-(Dichloromethyl)pyrazolo[1,5-a][1,3,5]triazines: synthesis and anticancer activity

© 2020 Ye. S. Velihina et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 547.874.8:615.277.3


Introduction
Organic synthesis plays a vital role in drug discovery, and modern synthetic methods focus on increasing the efficiency of preparing small drug-like molecules which include new drugs and drug candidates and reagents used to explore biological processes [1]. Pyrazolo [1,5-a] [1,3,5]triazines were reported to behave as purine bioisosteres of various cycline-depen-dent kinases inhibitors and as inducers of cell death in a wide variety of human tumor cell lines [2], as hCRF1 receptor antagonists and effective anxiolytic drugs [3], as inhibitors of protein kinase CK2 [4,5], as antimicrobial agents [6], as active inhibition agents of bronchial construction with very low chronotropic effects [7], as anti-proliferative agents for Ye colorectal cancer cell lines [8]. 1H-Pyrazol-5amines (or 5-aminopyrazoles) comprise a class of flexible nitrogen-containing aromatic he tero cycles used as privileged organic tools for the construction of diverse fused heterocyclic scaffolds with versatile functionalities [9]. N-(2,2-Dichloro-1-cyanoethenyl)carboxamides are versatile highly reactive electrophilic reagents that are increasingly used in the organic synthesis, in particular in the synthesis of new types of heterocyclic compounds. Pioneering work on the development of cyclocondensation reactions of N-(2,2-Dichloro-1cyanoethenyl)carboxamides with N-nucleophiles originated in the late 1970's by two research groups of Matsumura and Drach; it was found that these cyclocondensations with various N-nucleophiles constitute a facile method for the synthesis of novel 5-amino-4-cyanooxazoles [10][11][12][13][14], imidazole [13,16]  NMR spectra of obtained products were recorded at Varian Unityplus 400 spectrometer in DMSO-d 6 solution with TMS as the internal standard. IR spectra were recorded on a Vertex 70 spectrometer from KBr pellets. Melting points were measured on a Fisher-Johns instrument.

Chemistry
Chromatomass spectra were recorded on an Agilent 1100 Series high performance liquid chromatograph equipped with a diode matrix with an Agilent LC/MS mass selective detector allowing a fast switching of the positive/negative ionization modes (chemical ionization).
Elemental analyses were performed at the Analytical Laboratory of the V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, their results were found to be in good agreement (±0.4 %) with the calculated values.
2-Dichloromethyl-4,7-dimethylpyra zolo [1,5-a] [1,3,5]  2-Dichloromethyl-4-methyl-7-(p-tolyl) pyrazolo [1,5-a] [1,3,5] were assigned with the NCI codes (see Table 1), respectively Primary in vitro one dose anticancer screening was initiated, in which the full NCI 60 panel lines were inoculated onto a series of standard 96-well microtiter plates on day 0 at 5000-40,000 cells/well in RPMI 1640 medium containing 5 % fetal bovine serum and 2 mM L-glutamine, and then preincubated in the absence of drug at 37 °C, and 5 % CO 2 for 24 h. Test compounds were then added in the same concentration of 10 −5 M in all 60 cell lines (drug solution preparing see in [24]), and incubated for a further 48 h under the same incubation conditions. Following this, the media were removed, the cells were fixed in situ, washed, and dried. The sulforhodamine B assay was used for cell density determination, based on the measurement of cellular protein content. After an incubation period, cell monolayers were fixed with 10 % (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye was removed by washing repeatedly with 1 % (vol/ vol) acetic acid. The bound stain was resolubilized in 10 mM Tris base solution and measured spectrophotometric ally on automated microplate readers for OD determination at 510 nm.

Five Doses Full NCI 60 Cell Panel Assay.
All the 60 cell lines, representing nine cancer subpanels (Fig. 1), were incubated at five different concentrations (0.01, 0.1, 1, 10 and 100 µM; drug solution preparing see in [23]) of the tested compounds. The outcomes were used to create log 10 concentration versus percentage growth inhibition curves and three response parameters (GI 50 , total growth inhibition (TGI) and LC 50 ) were calculated for each cell line. The GI 50 value (growth inhibitory activity) corresponds to the concentration of the compound causing 50 % decrease in net cell growth. The TGI value (cytostatic acti vity) is the concentration of the compound resulting in total growth inhibition. The LC 50 value (cytotoxic activity) is the concentration of the compound causing net 50 % loss of initial cells at the end of the incubation period of 48 h. Data calculations were made according to the method described by the NCI Development Therapeutics Program.
COMPARE correlations were performed as described in [24]. Vectors of Lg GI 50 concentrations for compound 3fa (NSC 811821) were correlated with the set of corresponding average GI 50 vectors from the standard agents database or all public NCI-60 vectors that contained at least 40 overlapping cell lines and had SD > 0.2.

Chemistry
For synthesis of pyrazolo[1,5-a][1,3,5]triazines 3 we investigated different reaction conditions (at room temperature, under reflux in different solvents, with or without a base catalyst) and the most promising results were achieved when starting reagents were heated in tetrahydrofuran in the presence of triethylamine. Thus, it has been found that the addition of one equivalent of 1H-pyrazol-5-amines 2 to a stirred solution of N-(2,2-dichloro-1cyanoethenyl)carboxamides 1 in THF containing one equivalent of triethylamine under reflux gave 2-(dichloromethyl)pyrazolo[1,5-a] [1,3,5] triazines 3 which were the major products of this one-pot reaction. Apparently, the heterocyclization proceeded in several stages, starting with the addition of an NH 2 group to the activated C=C bond to formed intermediate A, followed by the elimination of hydrogen cyanide promoted by triethylamine (and intermediate B creation) with further intramolecular condensation into the final product 3. Recrystallization of crude products easily yielded the pure target compounds. Our method is convenient due to mild reaction conditions, short time of the key reaction, and high degree of purity and good yields of the products.
Compounds 3 are tan solids, melting in the range of 110-210 °C, their structure was established with the help of IR, NMR spectroscopy, mass spectrometry, and X-Ray Analysis of compound 3db (CCDC1920913, deposit@ccdc.cam.ac.uk). 1 H NMR signal of CHCl 2 and pyrazole CH group occurs in the region 6.7-7.5 ppm. In the spectrum of 3ba, for example, there are two distinguished one proton singlets at 6.80 and 7.40 ppm. For other samples, one or both of these signals overlap with ArH multiplets.

In Vitro Screening
One Doses Assay. The initial assessment made it possible to identify the eight most promising structures from the collection of synthesized compounds for the one-dose assay: substances 3aa, 3ab, 3ba, 3bb, 3ca, 3fa, 3fb, 3gc. Their results are represented in Table 1.
So, the average value of the effect of the substance 3aa with two methyl substituents on the growth of cancer cells is close to 100 %, and the range of values is also relatively narrow, which indicates its low cytotoxicity. The anticancer properties of substance 3ab with methyl substituent in position 4 of pyrazolo [1,5a] [1,3,5]triazine system and phenyl in 7 are very low too; and some growth inhibition was observed only in the case of line EKVX of non-small cell lung cancer ( Table 1). tert-Butyl derivative 3gc also only slightly slows the growth of cancer cells.
However, the presence of aryl substituent (instead of alkyl) in position 4 leads to a swift increase in activity. Substances 3ba, 3bb, 3ca, 3fa, 3fb can effectively inhibit the growth of certain cancer cell lines. The character of the substituent in position 7 is not so important; and high cytotoxicity is inherent to the substance 3fa with a 7-methyl group and diphenyl derivative 3bb.
Five Doses Assay. According to the results of single dose tests, the most perspective substances 3ba, 3bb, 3ca, 3fa, 3fb were selected for five doses assay to establish the parameters GI 50 , TGI and LC 50 . In Table 2 the mean values of these parameters are given, as well as their values for the cell lines referred in Table 1.

NCI 60 Cell Panel COMPARE Correlations
COMPARE analysis [24] was performed to propose a mechanism of action of the investigated compounds. Only for compound 3fa (NSC 811821) the correlation, computed as the GI 50 vector, exceeded 0.5 in comparison with Fluorouracil. This antineoplastic agent produces active metabolites that incorporate into RNA and DNA and inhibit their processing, thereby inhibiting cell growth [25]. For other investigated compounds (3ba, bb, ca, fb) no analogues of anticancer mechanism were found, therefore 4-(dichloromethyl)pyrazolo[1,5-a] [1,3,5]triazine derivatives could potentially be a new class of anticancer agents.

Conclusions
By starting from N-(2,2-dichloro-1-cyanoe thenyl)carboxamides 1 and 1H-pyrazol-5-  Compounds 3ba, 3bb, 3ca, 3fa, 3fb with an aromatic substituent in position 4 can effectively inhibit the growth of certain cancer cell lines, whereas compounds with an alkyl substituent in the same position possess low cytotoxicity. Future work will be focused on the improvement of their biophysical properties to yield drug-like pre-clinical candidates for in vivo animal studies. COMPARE analysis did not reveal any known anticancer drugs with a similar action, which warrants a more detailed study of the anticancer action mechanism of the obtained 4(dichloromethyl)pyrazolo[1,5-a] [1,3,5]triazine.