Novel pyrano[2,3-c]pyrazolopyrimidines as promising anticancer agents: Design, synthesis, and cell cycle arrest of HepG2 cells at S phase

Abstract The poor selectivity, significant toxicity, high cost, and emergence of resistance of conventional chemotherapies are driving motive for the ongoing search for novel anticancer agents. New pyrano[2,3-c]pyrazolopyrimidines were synthesized and examined as antiproliferative agents, and the possible molecular mechanism(s) of action were explored. The mass and elemental analyses, alongside the IR,1H, and 13C NMR spectra, confirmed the proposed structures of the obtained compounds. Derivatives 4 and 7 demonstrated the best antiproliferative profile against HepG2 cancer cells at 4 µM, with a high selectivity index of ∼7–9 folds. They increased the S phase cell population by 51% and 40% and caused a 5- and 11-fold increase in the p21 protein. Compound 7 was superior in inhibiting HepG2 cell migration and delayed wound healing, reducing migration rates by 55% and 90%, respectively. Future studies on the pharmacokinetics, pharmacodynamics, antimetastatic, and antitumor activities in animal models would be a robust advance. Graphical Abstract

As a part of our dedication to synthesize new moieties with potential anticancer activity, [16][17][18][19][20][21][22][23][24][25] new pyrano [2,3-c]pyrazoles were synthesized and evaluated as antiproliferative agents.The effect of these compounds on the cell cycle and p21 expression was investigated, in addition to their effects on wound healing in human hepatic cancer cells (HepG2).p21, a known inhibitor of cyclin-dependent kinase (CDK), can arrest the cell cycle at different phases.The progression of cell cycle depends on the phosphorylation of retinoblastoma (Rb) by CDK-Cyclin.p21 disrupts this latter interaction and inhibits the cell cycle [26] and enhances DNA repair. [27]Although p53 is the main inducer of p21, the latter could be induced independent of p53 pathway. [28]
Compound 2 reacted with several aromatic amines including 3-aminopyridine, methyl 4-amino benzoate, and anthranilic acid to produce formamidine derivatives 3a, 3b, and 3c, respectively.When compound 2 was treated with antipyrine, it underwent a nucleophilic addition followed by cyclization, resulting in three fused rings; pyrazolopyranopyrimidine derivative 4 (Scheme 2).The structures of compounds 3a-c were proved by studying their spectral data.For instance, their IR spectra revealed C≡N absorption at ν 2208-2198 cm −1 indicating their open structures, as well as C = O absorptions for the ester group (ν 1710 cm −1 ) and carboxylic acid group (ν 1678 cm −1 ) in compounds 3b and 3c, respectively.The lower value for carbonyl absorption of 3c shows that it exists in a chelated form, as illustrated in Scheme 2.
The 1 H NMR spectra of compounds 3a and 3b revealed two singlet signals for the NH group at δ around 10.50 and 11.00 ppm (exchangeable with D 2 O).This provides strong support for their existence as a mixture of Syn-and Anti-stereoisomers.While compound 3c showed one signal for the NH group at δ 11.15 ppm.Due to carboxyl group steric hindrance, this indicates the existence of one isomer, which might be an anti-isomer.The 13 C-NMR spectra confirm the presence of a methyl ester and carboxylic acid groups in compounds 3b and 3c, respectively.The IR and 13 C NMR spectra of 4 provided an amidic carbonyl group of the pyrazolone ring at ν 1676 cm −1 and δ 162.45 ppm, respectively.The 1 HNMR spectrum displayed an exchangeable singlet signal for = NH at δ 7.63 ppm, a signal for = CH (pyrimidine ring) at δ 8.12 ppm, and two methyl signals of the pyrazolone ring.Refluxing or heating 2 with o-phenylenediamine did not provide any of the expected products, the formamidine derivative 5 or the pyrazolopyranopyrimidine derivative 6, but it afforded a compound that was identified as the enaminonitrile derivative 1 (Scheme 3).Another product was found in the mother liquor from the letter reaction, which was later identified as benzimidazole. [36]This was confirmed by testing a reliable sample obtained from refluxing o-phenylenediamine with triethyl orthoformate as shown in Scheme 3. The proposed mechanism for converting compound 2 into 1 and benzimidazole through the formation of the open structure 5 was described in Scheme 4.
Reacting compound 2 with semicarbazide hydrochloride in the presence of fused sodium acetate yielded the fused system; pyrazolopyranotriazolopyrimidine derivative 7, while the reaction of compound 2 with p-toluenesulphonyl hydrazide afforded N-sulphonamide derivative 8.In addition, refluxing compound 2 with cyanoacetohydrazide resulted in four fused rings; pyrazolopyranotriazolopyrimidine derivative 9 Scheme 3. Reactions of compound 2 with some binucleophiles.which was verified to have a methylene group by reacting it with p-chlorobenzaldehyde producing the condensation product 10 (Scheme 5).
The IR and 13 C NMR spectra of 7 confirmed the triazolone ring's C = O amidic group.Its 1 H NMR spectrum exhibited two exchangeable singlet signals for NH (triazole ring) and OH groups at δ 7.85 and 8.41 ppm, respectively, owing to the existence of 7 as a mixture of lactam-lactim tautomers.The 1 H NMR spectrum of 8 showed a broad singlet signal exchangeable by D 2 O at δ 7.90 ppm for = NH and NH (sulphonamide) and a signal at δ 2.24 ppm for the methyl protons in the toluyl moiety.
IR spectrum of 9 provided the appearance of CN group.Its 1 H NMR spectrum exhibited signals for CH 2 at δ 4.30 ppm and = CH pyrimidine at δ 8.40 ppm, indicating the cyclized form, triazolopyrimidine with a derived methylene group.The 1 H NMR spectrum of 10 revealed a singlet signal at δ 9.58 ppm for = CH olefinic that confirms the condensation on the methylene group.The 13 C NMR spectra of 8-10 were perfectly compatible with their assigned structures.The mechanistic pathway for the formation of compounds 7 and 9 was depicted in Scheme 6.

Biology
Using the MTT assay, [37] the antiproliferative effect of the pyrano[2,3-c]pyrazole derivatives against the growth of the human liver HepG2 was investigated.The cytotoxic activity of these compounds was also examined against the normal human fibroblasts WI-38.Derivatives 3c and 10 were highly toxic to the normal fibroblasts without any significant antiproliferative activity against the cancer cells with selectivity index (SI) of Scheme 5. Reactions of compound 2 with some hydrazide derivatives.0.4-0.8µM (Table 1).The presence of substituted benzene moiety in pyranopyrazoles 3c and 10 could be responsible for their cytotoxicity against the normal fibroblasts.According to the National Cancer Institute's (NCI) criteria, substances are only deemed hits if they exhibit antiproliferative activity at a concentration of less than 10 µM.Derivatives 4 and 7 showed the best antiproliferative profile at very low concentrations; ~4 µM and very safe to the normal cells with high SI at ~7-9 folds indicating high selectivity against liver cancer cells.The incorporation of pyrazolone nucleus within a tricyclic system in compound 4 as well as the presence of triazolo ring bridgehead with pyrazolopyranopyrimidine motif in compound 7 enhanced the antiproliferative activity against the cancer cells.Furthermore, the presence of two NH groups may also be responsible for the significant antiproliferative activity in compound 4, and NH 2 group in compound 5, which can form hydrogen bonds with DNA's nucleic bases.These  derivatives had an antiproliferative activity comparable to that of doxorubicin in the efficacy but much safer to the normal cells.These active compounds (4 and 7) could be carcinostatic rather than merely cytotoxic.Derivatives similar to compounds 4 and 7, showed significant antiproliferative activities against the growth of HepG2 through various mechanisms.Pyrazolo[4′,3′:5,6]pyrano [2,3-d]pyrimidine and pyrano[2,3-c]pyrazole derivatives showed a strong antiproliferative activity against the growth of HepG2 with IC 50 at 0.31 to 0.71 μM. [38]However, they did not investigate the mechanism behind this spectacular antiproliferative effect.In another study, pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffolds had an IC 50 against HepG-2 with range of 48-90 nM.This was attributed to cell cycle arrest through inhibition of CDK2/cyclin A2. [39] Therefore, we decided to examine their effects on the cell cycle using the flow cytometry.
The IC 50 was calculated from a dose-response curve.Doxorubicin (Dox) was used as a positive control.The data are expressed as mean ± SEM from three independent experiments.a Significant antiproliferative activity as compared to the untreated cells.b SI: selectivity index = WI-38 IC 50 /IC 50 against cancer cells .
Compounds 4 and 7 were selected for further studies to explore the possible mechanism(s) of action.As shown in Fig. 2, the analysis of cell cycle has shown a significant increase in the proportion of cells in the S phase after treating the cells with compounds 4 (Fig. 2b, ~51%), and 7 (Fig. 2c, ~40%) compared to the control cells treated with DMSO (Fig. 2a, ~16%).Any damage of DNA would arrest the cell cycle giving time for repair and/or induce apoptosis.Arresting the cells at G1/S and G2/M boundaries are well-known mechanisms for many anticancer agents. [40,41]Although arresting the cycle at the S phase is less common, it has been reported as an efficient target for anticancer agents. [42]The effect of these compounds on the activity and gene expression of caspase 3 was examined (data not shown).They had very little negative effects on the expression of caspase 3 in HepG2 and almost no effect on the activity of the cleaved active enzyme.Caspase 3 is the main executioner enzyme in the apoptosis. [43]Therefore, it is evident that the cells evaded apoptosis and that the cell cycle arrest is the main mechanism of action of compounds 4 and 7. Cyclins E1 and E2 induce the activity of CDK2 which leads to G1/S transition.The transition from S phase to mitosis depends on the degradation of cyclin E and replacement by cyclin A. p21 binds and inhibits cyclin A/CDK1, 2 arresting the cells at the S phase. [44]Therefore, we proposed that compounds 4 and 7 arrested the cell cycle at the S phase by inducing p21 since this pathway does not induce apoptosis. [27]The effect of compounds 4 and 7 on the protein level of p21 was investigated by Western blotting after incubation with the HepG2 cells for 24 hours.
As shown in Fig. 3, both compounds 4 and 7 caused ~ 5-and 11-fold increase in the p21 protein, respectively.p21 inhibits the activity of many CDKs such as CDK2, CDK3, CDK4, and CDK6 and arrests the cells at the S phase. [45,46]p21 could be regulated at multiple levels by p53-dependent and -independent mechanisms. [26]ince HepG2 cells express functional p53, [47] we may attribute the elevation of p21 to a p53-dependent pathway; a speculation that has not been verified in the current study.
p21 is not only a CDK inhibitor, but it has intricate pathways that affect many cellular and molecular aspects.p21 affects the cytoskeleton, cell motility, proliferation, and migration of cells. [27]Overexpression of p21 is associated with halting the metastasis of cancer. [48]To assess the role of p21 on the metastasis, we have examined the effect of compounds 4 and 7 on the wound healing of HepG2.Both compounds 4 and 7 inhibited the migration of HepG2 cells and delayed the wound healing but compound 7 was superior to compound 4 after 48 hours of incubation (Fig. 4).The migration rate ((initial wound width -final wound width)/time) was calculated and found to be reduced by 55% and 90% after treatment with compounds 4 and 7, respectively.This magnificent percentage of inhibition of migration could be attributed to the overexpression of p21. [49]In the present study, the elevations (5-and 11-fold) of p21 protein caused by compounds 4 and 7 were directly related to the inhibition of wound healing or migration rate (55 and 90%).

Conclusion
Two pyrano[2,3-c]pyrazolopyrimidines (4 and 7) exhibited a significant antiproliferative activity against the growth of human liver cancer cells (HepG2) with IC 50 at ~4 µM.These compounds were highly selective against the cancer cells.Compounds 4 and 7 arrested the cell cycle at the S phase (51% and 40%, respectively) by elevating the protein level of p21 (5-and 11-fold, respectively).Compound 7 in particular resulted in a significant halting in the proliferation and migration of HepG2 cells.Taken together, the promising antiproliferative activity, cell cycle arrest, and halting of the wound healing and cell migration caused by compounds 4 and 7 merit further investigations of these compounds on solid tumors in animal models with the exploration of the pharmacodynamics and pharmacokinetics properties of these compounds, possible mechanisms of action in vivo, and the potential side effects to overcome the shortcomings of the vitro study.

Chemistry
Open capillary tubes on electrothermal melting point apparatus were used to measure the melting points which were uncorrected.The infrared spectra were recorded using the KBr wafer technique on Fourier Transform Infrared Thermo Electron Nicolet iS10 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) at the Chemistry Department Laboratory, Faculty of Science, Ain Shams University.The 1 H and 13 C NMR spectra were measured on BRUKER 300; 400 and 75 MHz Spectrometer, with chemical shift (δ) expressed in ppm downfield with tetramethyl silane (TMS) as an internal standard, in DMSO-d 6 at the Microanalytical Center (Faculty of Science, Cairo University).The mass spectra were recorded on a direct probe controller inlet part to single quadrupole mass analyzer (Thermo Scientific GCMS MODEL (ISQ LT)) using the Thermo X-CALIBUR software at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.Elemental analyses were carried out at Ain Shams University's Faculty of Science using a PerkinElmer 2400 CHN elemental analyzer (PerkinElmer, Waltham, MA).Thin-layer chromatography (TLC) was run using TLC aluminum sheets silica gel F254 (Merck, Whitehouse Station, NJ).

Cell lines and reagents
HepG2 cells were purchased from Nawah Scientific Company, Egypt.Cells were grown in DMEM medium (BioWhittaker™) containing 10% bovine serum albumin (Life Science Group L, UK, Cat No: S-001B-BR) and 100 IU/mL penicillin/streptomycin (100 µg/mL) (Lonza, 17-602E).The tested pyrazole derivatives were prepared in dimethyl sulfoxide (10 mM stock) (DMSO, Cat.No. 20385.02,Serva, Heidelberg, Germany) and stored at −20 °C. [37]he MTT assay was used to measure the antiproliferative activity of pyrazole derivatives (0.5-100 µM) against the growth of human liver (HepG2) and normal fibroblasts (WI-38), and the results were compared with those of doxorubicin.The selectivity index (SI) was then calculated; SI = IC 50 against WI-38/IC 50 against cancer cells.After 48 hours of incubation with the pyrazole derivatives, MTT was applied to the cells.The cells were further incubated for four hours.After adding DMSO, the absorbance at 570 nm was measured.The data are expressed as mean ± SEM for three independent experiments.The study was approved by the Research Ethics Committee at Ain shams University.

Wound healing assay in HepG2 cells
To investigate cell migration in vitro, the wound healing experiment was run.[54] The cells were treated with compound 4, or compound 7 at their IC 50 concentration reported in Table 1.The images were taken using Optika B-159 (OPTIKA S.r.l., Italy).The size of the wound was measured using Image J 1.51 software from three independent experiments.

Cell cycle analysis by flow cytometry
HepG2 cells were seeded (1 × 10 5 ) in 6-well plate (2 mL/well) and incubated overnight at 37 °C and 5% CO 2 .Cell synchronization at G1 phase was performed by incubating the cells with serum-free medium for 24 hours.Then, cells were incubated with compound 4 or 7 at the IC 50 concentration (reported in Table 1) for 48 hours.The details could be reviewed elsewhere. [55]Propidium (PI)/RNase was obtained from BD Biosciences (BDB550825, NJ, USA).The experiment was repeated thrice for validity.

Measurement of p21 by Western blotting
HepG2 cells were seeded (1.5*10 cells/ml) in 25 cm flask (3 ml per flask) and incubated overnight.The cells were then treated either with DMSO (0.1%), compound 4, or compound 7 at their IC 50 concentrations for 24 hours at 37 °C and 5% CO 2 .The mouse anti-human p21 and β-actin were obtained from Cell signaling technology (MA, USA).The details could be reviewed elsewhere. [56]The fold of change in p21 level was calculated using GraphPad Prism 8 Software after normalization to the level of β-actin.

Statistical analysis of data
The Kolmogorov-Smirnov test was used to analyze the data distribution.The findings were presented as mean ± SEM.One Way ANOVA was used for the statistical analysis, and the Tukey-HSD test was used for multiple comparisons.When a difference was p < .05, it was deemed significant.

Scheme 6 .
Scheme 6. Proposed mechanism for the conversion of 2 into 7 and/or 9.

Figure 2 .
Figure 2. effect of a) DmSo, B) derivative 4, and c) derivative 7 on the cell cycle progression of hepG2 cells after incubation with the ic 50 concentration for 48 hours.the upper panel is representative of the flow cytometry chart, and the lower panel is the pie chart of the mean from three independent experiments.

Figure 3 .
Figure 3. effect of compounds 4 and 7 on the protein level of p21 in hepG2 cells by Western blotting after incubation for 24 hours.a) a representative photo of the Western blot of p21 as well as the house-keeping β-actin.B) Data are expressed as the mean ± Sem of two samples and from two independent experiments.* Significant at p < .05 as compared to control.For presentation and clarity, the gels were cropped.Please see supplementary file for the original gel.

Figure 4 .
Figure 4. Representative photos of wound healing or migration of hepG2 cells after treatment with a) DmSo, B) compound 4, and c) compound 7 at their ic 50 concentrations at 0, 24, and 48 h intervals.the experiment was repeated thrice.