Convenient Synthesis , Characterization , Cytotoxicity and Toxicity of Pyrazole Derivatives

3-Methyl-1H-pyrazol-5(4H)-one (1) was used as a template to develop new anticancer compounds and investigate their SAR. The ring modification of compound 1 occurred through its reaction with aromatic aldehydes and various reagents to afford the corresponding 6-oxopyrano[2,3-c]pyrazoles 4a–c and their amino analogues 6-aminopyrano[2,3-c]pyrazoles 6a–c,8; the pyrazolopyrano[2,3-b]pyridines 10a–c and the chromenopyrano[2,3-c]pyrazolones 13,14. The reaction of 1 with thiourea and appropriate aromatic aldehydes afforded the pyrazolo[3,4-d]pyrimidine derivatives 17a–c. On the other hand, the pyrazolo[3,4-d]thiazole derivatives 22a–d were obtained via the reaction of 1 with sulfur and aryl isothiocyanates in the presence of triethylamine. The reaction of 1 with phenylisothiocyanate followed by treatment with the α-halocarbonyl compounds 24a–c afforded the thiazole derivatives 25a–c. The synthesized products were evaluated for their cytotoxicity against cancer and normal cell lines. Most compounds showed significant anticancer activity without affecting the normal fibroblast cells. The toxicity of the most pontent cytotoxic compounds was measured using Brine-Shrimp Lethality Assay.


Introduction
Cancer is a major public health problem in the world.Chemotherapy is still one of the primary modalities for the treatment of cancer.However, the use of this method is limited mainly due to the small number of the available chemotherapeutic agents to choose among them and also because the use of these agents is often accompanied by undesirable side effects.This clearly underlies the urgent need for developing novel chemotherapeutic agents with more potent antitumor activities and reduced side effects.8][9] Celecoxib, sulfaphenazole, CDPPB, linazolac, mepiprazole, and rimonabant are some of the pyrazole-based drugs available today in the market (Figure 1). 10 Moreover, the chemistry of fused pyrazole derivatives has received great attention due to their pharmacological importance. 11,12It has been found that pyranopyrazoles are an important class of biologically active heterocycles.They are reported to possess a multiplicity of pharma-cological properties including anticancer, 13 antimicrobial, 14 anti-inflammatory, 15 insecticidal and molluscicidal activities. 16,17They are also potential inhibitors of human Chk1 kinase. 18On the other hand, pyrazolopyrimidines which are the fused heterocyclic ring systems that structurally resemble purines, prompted biological investigations to assess their potential therapeutic significance.They are known to play a crucial role in numerous disease conditions.3][24] The synthesis of some new pyrazole-based 1,3-thiazoles as anticancer agents was reported. 25Most recently, excellent anticancer effectiveness of pyrazolylthiazole derivatives was also reported, via EGFR TK inhibition that plays an important role in cell growth regulation. 26However, according to the literature and to our knowledge, the discovery of the potential anticancer activity of pyrazolothiazoles is still essentially in the development stage.In view of the aforementioned facts, our efforts were directed towards the uses of 3-methyl-1H-pyra-zol-5(4H)-one to prepare heterocyclic and fused derivatives together with evaluation of their activity towards cancer and normal cell lines.

1. Chemistry
The present investigation mainly on the synthesis of molecules derived from pyrazole-5-one and evaluation of their cytotoxicity against cancer and normal cell lines.The synthetic strategies adopted for the synthesis of the intermediate and target compounds are depicted in Schemes 1-4.One pot multicomponent reactions (MCR) were utilized to prepare the target compounds.The reaction of the 3-methyl-1H-pyrazol-5(4H)-one (1) with each of benzaldehyde (2a), 4-methoxybenzaldehyde (2b) or 4-chlorobenzaldehyde (2c) and ethyl cyanoacetate (3) afforded the 6-oxopyranopyrazole derivatives 4a-c.The structure of the latter products was confirmed on the basis of their respective analytical and spectral data.Thus, 1 H NMR spectrum of 4a revealed the presence of a singlet at δ 2.49 ppm indicating the presence of the CH 3 group, a multiplet at δ 7.59-8.41ppm equivalent to the C 6 H 5 group and a singlet at δ 10.40 ppm corresponding to the NH group.Moreover the 13 C NMR spectrum demonstrated a signal at δ 14.1 equivalent to the CH 3 group, δ 116.0 corresponding to the CN group, signals at δ 128.6, 129.5, 129.7, 129.8,  131.3, 131.8, 133.0, 133.9 corresponding to the phenyl, pyran and pyrazole carbons and a signal at δ 155.7 corres-ponding to C=O.Meanwhile, the reaction of 1 with either of 2a, 2b or 2c and malononitrile (5) in ethanol containing triethylamine gave the 6-amino-3-methyl-4-aryl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile derivatives 6a-c, respectively.The analytical and spectral data of 6a-c were in consistence with their respective structures.The latter compounds were previously reported to be prepared via a one pot, four component reaction between aldehydes, hydrazine hydrate, malononitrile and ethyl acetoacetate in the presence of different catalysts. 27On the other hand, the reaction of compound 1 with pyridine-3-aldehyde (7) and malononitrile (5) afforded the 6-amino-3methyl-4-(pyridin-3-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (8).The structure of the latter product was based on its respective analytical and spectral data.Thus, the 1 H NMR spectrum showed the presence of a singlet at δ 1.79 ppm indicating the CH 3 group, a singlet at δ 4.69 ppm equivalent to the pyran H-4, a singlet at δ 6.95 ppm for the NH 2 group and a multiplet at δ 7.32-8.46ppm corresponding to the pyridine protons.
Moreover, the reaction of 1 with the aromatic aldehydes 2a-c and 2-aminoprop-1-ene-1,1,3-tricarbonitrile (9) in ethanol containing a catalytic amount of triethylamine afforded the pyrazolopyrano[2,3-b]pyridine-6-carbonitrile derivatives 10a-c. 1 H NMR of 10a (as an example) showed the presence of a singlet at δ 2.49 ppm corresponding to the CH 3 group, a singlet at δ 4.58 ppm for the pyran H-4, two singlets at δ 7.10 and 8.02 ppm indicating the presence of the two NH 2 group.Moreover, 13   148.4, 150.6, 154.3, 154.0 equivalent to the phenyl, pyrazole, pyran and pyridine carbons.On the other hand, the reaction of the compound 6b with phenylisothiocyanate (11) in 1,4-dioxane afforded the corresponding thiourea derivative 12, the structure of which was based on analytical and spectral data.
Reaction of compound 1 with triethylorthoformate (18) in an oil bath at 120 o C afforded the 4-(ethoxymethylene)-3-methyl-1H-pyrazol-5(4H)-one (19).The structure of 19 was established on the basis of analytical and spectral data.Thus, the 1 H NMR spectrum showed a triplet and quartet at δ 1.29 and 4.15 ppm corresponding to the ethyl group and a singlet at δ 7.38 ppm indicating CH=C group.Meanwhile, the reaction of 1 with malononitrile and triethylorthoformate in ethanol afforded 20.The presence of the two CN groups was indicated by the presence of two absorption bands in the IR spectrum at ν 2204, 2179 cm -1 , respectively. 1 H NMR spectrum showed a sin-glet at δ 8.66 ppm corresponding to the CH=C group.Further confirmation of the structure of compound 20 was obtained through its synthesis via another reaction route.Thus, the reaction of malononitrile (5) with 19 gave the same product 20 (m.p. and mixed m.p. and finger print IR).Moreover, the reaction of compound 1 with elemental sulfur and either phenylisothiocyanate (11), 4-methoxyphenylisothiocyanate (21a), 4-chlorophenylisothiocyanate (21b), or 4-bromophenylisothiocyanate (21c) in 1,4-dioxane containing triethylamine gave the pyrazolo [3,4d]thiazole derivatives 22a-d.The structure of the latter products was based on the analytical and spectral data.Thus, the 1 H NMR spectrum of 22a (as an example) showed the presence of a singlet at δ 2.49 ppm corresponding to CH 3 group, a mutiplet at δ 7.09-7.50ppm corresponding to the phenyl protons and a singlet at δ 9.75 equivalent to the NH group.Moreover, the 13 C NMR spectrum showed the presence of the CH 3 group at δ 12.27, the phenyl and pyrazole carbons at δ 124.5, 128.5,128.9,129.4, 130.4, 137.8, 139 and the C=S group at δ 180.1.
The methylene group present in the pyrazole 1 was reported to show high reactivity towards thiazole formation via its reaction with phenylisothiocyanate in basic DMF solution followed by heterocyclization with α-halocarbonyl compounds. 28,29Thus, 1 was reacted with phenylisothiocyanate in DMF/KOH solution to give the intermediate potassium sulfide salt 23.The reaction of the latter intermediate with either 2-bromo-1-phenylethanone (24a), 2-bromo-1-(4-chlorophenyl)ethanone (24b) or ethyl chloroacetate (24c) gave the thiazole derivatives 25a-c.The structures was of the latter products were established on the basis of their respective analytical and spectral data.

2. 1. Effect on the Growth of Human Cancer Cell Lines
The heterocyclic compounds prepared in this study were evaluated according to standard protocols for their in vitro cytotoxicity against six human cancer cell lines inclu-ding cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), human breast cancer (MCF) and normal fibroblast cells (WI38).For comparison, CHS 828 was used as the standard anticancer drug.All of IC 50 values in (nM) are listed in Table 1 and the results are presented graphically in Figures 2-4.Many of the synthesized heterocyclic compounds were observed with significant cytotoxicity against most of the cancer cell lines tested (IC 50 <1000 nM).Normal fibroblasts cells (WI38) were affected to a much lesser extent (IC 50 >10,000 nM).Among the tested compounds the 3-methyl-6-phenyl-1H-pyrazolo [3,4-d]

Structure Activity Relationship
In the present study, a series of heterocyclic derivatives incorporating a pyrazole moiety were synthesized and evaluated for their cytotoxicity aiming at investigating their SAR.Thus, 6-oxopyranopyrazoles 4a-c and their amino analogs 6a-c and 8 were prepared.Refering to the IC 50 values listed in Table 1, 4a bearing a phenyl substituent exhibited significant broad spectrum cytotoxic activity in the range of IC 50 120-527 nM.Meanwhile, 4b bearing a 4-OCH 3 C 6 H 4 group showed selective activity against liver cancer HEPG2 (IC 50 428 nM) and breast cancer MCF (IC 50 580 nM).The 4-ClC 6 H 4 substituted derivative 4c demonstrated better activity compared to 4a and 4b especially against gastric cancer NUGC (IC 50 60 nM).Among the 6-amino-4-substituted pyranopyrazole derivatives 6a-c and 8, derivative 6a carrying a phenyl group was found to have selective activity against the human liver cancer cell line HEPG2 (IC 50 399 nM) and colon cancer cell line DLDI (IC 50 890 nM).However, 6b bearing 4-OCH 3 C 6 H 4 group was completely devoid of cytotoxic activity.On the other hand, 6c bearing the 4-ClC 6 H 4 moiety showed high activity against all cancer cell lines except breast cell line MCF in the range of IC 50 120-359 nM.The presence of pyridine ring in 8 is most probably res-ponsible for its high potency against human liver cancer cell line HA22T (IC 50 58 nM) and nasopharyngeal cancer cell line HONE1 (IC 50 nM).The previous result suggests that the replacement of the 6-amino group in compounds 6a-c by a 6-oxo group in compounds 4a-c in the latter pyranopyrazole derivatives leads to compounds with enhanced cytotoxic effect which might be attributed to the presence of the electronegative oxygen moiety.Meanwhile, replacement of the 2-amino group in 6b by a phenylthiourea moiety afforded 12 which demonstrated a dramatic increase in the cytotoxic activity with the highest activity exhibited against NUGC (IC 50 36 nM).
The investigation of the cytotoxicity of the pyrazolo [4',3':5,6]pyrano [2,3-b]pyridine derivatives 10a-c revealed that 10a bearing a phenyl group exhibited selective activity against MCF (IC 112 nM).On the other hand, 10b bearing the 4-OCH 3 C H 4 group was found to be active against most cancer cell lines with the highest activity against NUGC (IC 50 122 nM) and DLDI (IC 50 90nM).The 4-ClC 6 H 4 substituted derivative 10c showed high cytotoxic activity against four cancer cell lines with potent activity against NUGC (IC 50 40 nM) and DLDI (IC 50 60 nM).Meanwhile, the tetracyclic chromenopyranopyrazoles 13 and 14 were found to be almost devoid of cytotoxic acti- vity which might be attributed to the existence of the annelated ring system.Compound 14 showed only moderate selective activity against HEPG2 (IC 50 410 nM).

3 Toxicity Testing
Bioactive compounds are often toxic to shrimp larvae.Thus, in order to monitor these chemicals' in vivo lethality to shrimp larvae (Artemia salina), Brine-Shrimp Lethality Assay as described by Choudhary et al. in 2001 was used. 30Results were analysed with LC 50 program to determine LC 50 values and 95% confidence intervals. 31esults are given in Table 2 for the compounds which exhibited optimal cytotoxic effect against cancer cell lines; these are the following fourteen compounds 4a, 4c, 6c, 10b, 10c, 12, 17b, 19, 20, 22a, 22b, 22c, 22d and 25c.The shrimp lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials, natural and synthetic organic compounds.It has also been shown that A. salina toxicity test results have a correlation with rodent and human acute oral toxicity data.Generally, a good correlation was obtained between A. salina toxicity test and the rodent data.Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in humans, including A. salina toxicity test, was slightly better than the rat test for test compounds. 32n order to prevent the toxicity results from possible false effects originating from solubility of compounds and DMSO's possible toxicity effect, compounds were prepared by dissolving in DMSO in the suggested DMSO volume ranges.It is clear from Table 2 that compounds 4a, 6c, 17b, 22a and 22b were found to be nontoxic against the tested organisms.It is of great value to mention that compound 22a which is of optimum cytotoxicity was also found to be nontoxic.

1. Chemistry
All melting points were determined on a Stuart apparatus and the values given are uncorrected.IR spectra (KBr, cm -1 ) were determined on a Shimadzu IR 435 spectrophotometer (Faculty of Pharmacy, Cairo University, Egypt). 1 H and 13 C NMR spectra were recorded on Varian Gemini 300 MHz (Microanalysis Center, Cairo University, Egypt) and Bruker Ascend 400 MHz spectrophotometers (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt) using TMS as internal standard.Chemical shift values are recorded in ppm on δ scale.Mass spectra were recorded on a Hewlett Packard 5988 spectrometer (Microanalysis Center, Cairo University, Egypt).Elemental analyses were carried out at the Microanalysis Center, Cairo University, Egypt; found values were within ±0.35% of the theoretical ones.Progress of the reactions was monitored using thin layer chromatography (TLC) sheets pre-coated with UV fluorescent silica gel Merck 60F 254 and were visualized using UV lamp.Kamel: Convenient Synthesis, Characterization, ...

1. 1. General Procedure for the Synthesis of Compounds 4a-c and 6a-c
To a solution of 1 (0.98 g, 0.01 mol) and the appropriate aldehyde (0.01 mol) in ethanol (30 mL) containing triethylamine (1.0 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added.The reaction mixture, in each case, was heated under reflux for 1 h, left to cool and the formed solid product, in each case, was collected by filtration and crystallized from ethanol.

1. 8. General Procedure for Synthesis of Compounds 22a-d
To a solution of compound 1 (0.98 g, 0.01 mol) in 1,4-dioxane (30 mL) containing triethylamine (1.0 mL) each of elemental sulfur (0.32 g, 0.01 mol) and the appropriate arylisothiocyanate (0.01 mol) was added.The whole reaction mixture, in each case was heated under reflux for 3 h, left to cool then poured onto ice/water mixture containing few drops of hydrochloric acid.The formed solid product was collected by filtration and crystallized from ethanol.[3,4-  [3,4-
Cell cultures: were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt).They grow as monolayer and were routinely maintained in RPMI-1640 medium supplemented with 5% heat-inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37 °C in a humidified atmosphere containing 5% CO 2 .Exponentially growing cells were obtained by plating 1.5 × 10 5 cells / mL for the six human cancer cell lines followed by 24 h of incubation.The effect of the vehicle solvent (DM-SO) on the growth of these cell lines was evaluated in all experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.

Brine Shrimp Lethality Bioassay
The brine shrimp lethality bioassay was used to predict the toxicity of the synthesized compounds.For the experiment 4 mg of each compound was dissolved in dimethylsulfoxide (DMSO) and solutions of varying concentrations (10, 100, 1000 mg/mL) were obtained by the serial dilution technique using simulated seawater.The solutions were then added to the pre-marked vials containing 10 live brine shrimp nauplii in 5 mL simulated seawater.After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted.The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 s of observation.From this data, the percent of lethality LC 50 of the brine shrimp nauplii for each concentration and control was calculated.

Conclusions
The present research reports the successful synthesis, characterization and evaluation of anticancer activity Kamel: Convenient Synthesis, Characterization, ... of pyrazolone, pyranopyrazolone, pyrazolopyrimidine and pyrazolothiazole derivatives.Several compounds showed potent cytotoxic effect with IC 50 <100 nM.Among these derivatives the pyrazolothiazoles exhibited significant cytotoxic activity.Compound 22a showed the maximum cytotoxicity among the tested compounds.Moreover, it was found to be nontoxic against shrimp larvae (Artemia salina).Normal fibroblast cells (WI38) were affected to a much lesser extent (IC 50 >10,000 nM).The obtained results suggest that these compounds may serve as lead chemical entities for further modification in the search of new classes of potential anticancer agents.It could be also concluded that while some of the compounds were not the most potent, their specific activity against particular cell lines makes that of interest for further development as anticancer drugs.
Cytotoxicity of the synthesized compounds against a variety of cancer cell lines a [IC 50 b (nM)].

Table 2 .
Toxicity of the most optimal cytotoxic compounds against shrimp larvae a Ten organisms (A.salina) tested for each concentration.