Biological Validation of Novel Polysubstituted Pyrazole Candidates with in Vitro Anticancer Activities

With the aim of developing novel antitumor scaffolds, a novel series of polysubstituted pyrazole derivatives linked to different nitrogenous heterocyclic ring systems at the C-4 position were synthesized through different chemical reactions and characterized by means of spectral and elemental analyses and their antiproliferative activity against 60 different human tumor cell lines was validated by the U.S. National Cancer Institute using a two stage process. The in vitro anticancer evaluation revealed that compound 9 showed increased potency toward most human tumor cell lines with GI50MG-MID = 3.59 µM, as compared to the standard drug sorafenib (GI50 MG-MID = 1.90 µM). At the same time, compounds 6a and 7 were selective against the HOP-92 cell line of non-small cell lung cancer with GI50 1.65 and 1.61 µM, respectively.


Introduction
The development of new antitumor agents is an important field of scientific activity, due to the toxic side effect problems of recent drugs. Many of the obtainable anticancer agents display unwanted side effects such as reduced bioavailability, toxicity and drug-resistance [1][2][3][4][5]. Incorporation of a pyrazole ring into different heteroaryl ring systems results in significant anticancer activities [6][7][8][9]. Different substituted pyrazole compounds have also been examined for their antiproliferative activities in vitro and antitumor activity in vivo, resulting in promising target products [10][11][12]. On the other hand, compounds containing pyrazole derivatives represent an advantageous choice for the synthesis of compounds with a broad spectrum of pharmacological activities, including anti-inflammatory [13], antibacterial, antifungal [14], inhibition of cyclooxygenase-2 [15], antiangiogenic [16], antipyretic [17], antihypertensive [18], antiplatelet [19], nitric oxide synthase (NOS) inhibitors [20] and anticancer activities [21]. Based on these observations and in continuation of our research on biologically active heterocycles [22][23][24][25], it was of interest to incorporate the 1,2,4-polysubstituted pyrazole ring system into different heteroaryl ring systems in one molecule in an attempt to obtain a new target anticancer agents.

In Vitro Anticancer Screening
The target compounds were selected by the U.S. National Cancer Institute (NCI), for anticancer activity screening. The screening is a two-stage process, beginning with the evaluation at a single dose (10 µM) and the compounds which display significant growth inhibition are then evaluated at five concentration levels. The first screening, where the selected compounds are evaluated at a single dose (10 µM) and the culture is incubated for 48 h, utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of lung, colon, central nervous system (CNS), ovary, kidney, prostate as well as breast. Finally, α,β-unsaturated ketone 2 was reacted with hydroxylamine hydrochloride in refluxing ethanol in the presence of sodium hydroxide as alkaline medium to afford the corresponding isoxazoline 7. Treatment of 2 with guanidine sulfate in ethanolic sodium hydroxide gave 2-aminopyrimidine derivative 8, which was reacted with thiourea in the presence of sodium hydroxide to give the corresponding pyrimidine-2-thione derivative 9 (Scheme 2).

In Vitro Anticancer Screening
The target compounds were selected by the U.S. National Cancer Institute (NCI), for anticancer activity screening. The screening is a two-stage process, beginning with the evaluation at a single dose (10 µM) and the compounds which display significant growth inhibition are then evaluated at five concentration levels. The first screening, where the selected compounds are evaluated at a single dose (10 µM) and the culture is incubated for 48 h, utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of lung, colon, central nervous system (CNS), ovary, kidney, prostate as well as breast. The percentages of growth of the tested compounds against the full 60-cell line panel are listed in Table 1. The one dose mean graphs of the selected compounds revealed that compounds 6a, 7 and 9 showed increased potency against most human cancer cell lines, so these compounds were selected for further evaluation at five dose concentration levels (0.01-100 µM).Regarding sensitivity against individual cell lines, compound 9 showed potent anticancer activity against all human cancer cell lines with GI50 1.9-5.50 µM. It had the highest selectivity against the non-small cell lung cancer cell line EKVX, with GI50 1.9 µM. At the same time, compounds 6a and 7 showed the highest activity against the cell line HOP-92 belonging to the non-small cell lung cancer class with GI50 1.7 and 1.6 µM, respectively, and against the renal cancer cell line A498 with GI50 1.47 and 1.81 µM, respectively.
Examination of all the ligand structural modifications performed at the 4-position of 1,3,4-trisubstituted pyrazole scaffold to establish the structure activity relationships with anticancer acitivity shows the following results: first, introduction of the pyrimidine-2(1H)-thione moiety in the 4-position of the pyrazole moiety in compound 9 enhanced the potency towards most cancer cell lines. It has GI50 MG-MID = 3.6 µM against all subpanel tumor cell lines, comparable to that of sorafenib (GI50 MG-MID = 1.90 µM. Concerning the pyrazolyl derivative 6a, it was observed that the activity was enhanced in 2-amino-6-oxopyrimidine-5-carbonitrile, which suggests that the polar 6-aminopyrimidine moiety has a role in enhancing anticancer activity (GI50 MG-MID = 5.70 µM), but less so than the 2-mercaptopyrimidine group. Finally, introduction of a 1-carbothioamide to a pyrazole or isoxazoline group substituted on the pyrazole greatly reduces the activity and these compounds showed the least potent activity. The results are presented in Tables 1-6. The percentages of growth of the tested compounds against the full 60-cell line panel are listed in Table 1. The one dose mean graphs of the selected compounds revealed that compounds 6a, 7 and 9 showed increased potency against most human cancer cell lines, so these compounds were selected for further evaluation at five dose concentration levels (0.01-100 µM).Regarding sensitivity against individual cell lines, compound 9 showed potent anticancer activity against all human cancer cell lines with GI 50 1.9-5.50 µM. It had the highest selectivity against the non-small cell lung cancer cell line EKVX, with GI 50 1.9 µM. At the same time, compounds 6a and 7 showed the highest activity against the cell line HOP-92 belonging to the non-small cell lung cancer class with GI 50 1.7 and 1.6 µM, respectively, and against the renal cancer cell line A498 with GI 50 1.47 and 1.81 µM, respectively.
Examination of all the ligand structural modifications performed at the 4-position of 1,3,4-trisubstituted pyrazole scaffold to establish the structure activity relationships with anticancer acitivity shows the following results: first, introduction of the pyrimidine-2(1H)-thione moiety in the 4-position of the pyrazole moiety in compound 9 enhanced the potency towards most cancer cell lines. It has GI 50 MG-MID = 3.6 µM against all subpanel tumor cell lines, comparable to that of sorafenib (GI 50 MG-MID = 1.90 µM. Concerning the pyrazolyl derivative 6a, it was observed that the activity was enhanced in 2-amino-6-oxopyrimidine-5-carbonitrile, which suggests that the polar 6-aminopyrimidine moiety has a role in enhancing anticancer activity (GI 50 MG-MID = 5.70 µM), but less so than the 2-mercaptopyrimidine group. Finally, introduction of a 1-carbothioamide to a pyrazole or isoxazoline group substituted on the pyrazole greatly reduces the activity and these compounds showed the least potent activity. The results are presented in Tables 1-6.

General Information
Melting points were measured in open capillary tubes using a Griffin apparatus and are uncorrected. Structures of compounds were confirmed by routine spectrometric analysis. Elemental analyses were carried results were within˘0.4% of the theoretical values. Infrared spectra were recorded on a 435 IR spectrophotometer (Shimadzu Bruker, Tokyo, Japan) using KBr discs. 1 H-NMR and 13 C-NMR spectra were obtained on a Gemini 500 MHz spectrophotometer (Varian, Polo Alto, Ca, USA) or on a Bruker 500 MHz spectrophotometer, and measured in δ scale using TMS as an internal standard. Mass Spectra were recorded on a 5988 spectrometer (Hewlett Packard, California, USA). Analytical thin layer chromatography (TLC) was performed using silica gel aluminum sheets, 60 F 254 (E. Merck, Darmstadt, Germany) for the progress of reactions and visualization with ultraviolet light (UV) at 365 and 254 nm.

Measurement of Anticancer Activity
The experimental method used in anticancer screening has been adopted by U.S. National Cancer Institute according to reported standard procedure [28][29][30].

Conclusions
In summary, we have synthesized a series of novel pyrazole derivatives incorporated different heteroaryl ring systems in one molecule and evaluated these compounds for their anticancer activities against different 60 human cancer cell lines representing leukemia, melanoma and cancers of lung, colon, brain, ovary, breast, prostate and kidney cancer using a two-stage process. The pyrimidine-2(1H)-thione derivative 9 showed good anticancer activity with (GI 50 MG-MID = 3.59 µM) compared to the standard drug sorafenib. The structures of the new compounds were elucidated using spectroscopic and elemental analysis.