Synthesis, in vitro and in vivo anticancer activity of novel 1-(4-imino-1-substituted-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)urea derivatives

Abstract

A series of pyrazolo[3,4-d]pyrimidine and urea hybrids have been designed, synthesized and evaluated for their anticancer activity in vitro and in vivo cancer models. Among them, compounds 28, 30, 33, 36 and 37 showed promising cytotoxicity against tested cancer cell lines. Compound 37 (CBS-1) appeared as the most active derivative and it exhibited better cytotoxicity against all tested cell lines as compared to doxorubicin. CBS-1 successfully inhibited cell cycle progression and displayed good apoptosis in A549 cells. CBS-1 significantly induced caspase-3 activation and suppressed NF-κB and IL-6 activation in immunocytochemistry, qPCR and western blot analysis. Additionally, CBS-1 prominently displayed tumoricidal effects in lung adenocarcinoma in vivo xenograft nude mice model.

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Cancer is a major cause of death worldwide and the second biggest reason for death in developing countries.¹ The World Health Organization (WHO) accredited 8.2 million deaths to cancer in 2012, which represented 13% of all deaths.² Cancer is a multi-factorial disease that can arise from all cell types and organs. Among the twenty seven types of characterized cancers stomach, liver, bone, lung, colon and breast cancer are mainly responsible for cancer mortality.^3,4

At present, treatment of cancer is based on chemotherapy, hormone therapy, radiotherapy, surgical removal and immunotherapy.⁵ From past decades, extensive efforts have been made to manage malignancies and numerous anticancer agents have been developed from natural sources as well as synthetic approaches.⁶ Unfortunately, presently available anticancer drugs exert severe side effects.⁷ Therefore, discovery of potent anticancer agents which kill cancerous cells with minimum or no side effects are required.

Pyrazolo[3,4-d]pyrimidine is widely used heterocyclic system in drug discovery and development process. It is considered as an isostere to the purine nucleus and its derivatives shows numerous biological activities such as antiviral,⁸ antimicrobial,⁹ antileishmanial,¹⁰ anti-inflammatory,¹¹ neuroprotective¹² and cardiovascular activities.¹³ Besides these activities pyrazolo[3,4-d]pyrimidine derivatives also displayed excellent anticancer activities in various in vitro and in vivo models (Fig. 1). For example, pyrazolo[3,4-d]pyrimidine derivative 1 showed good anticancer activity against K562, MEG-01 and KU-812 cell lines. Moreover, this compound also displayed anticancer activity in xenograft mice model.¹⁴ 4-[2-(4-fluorobenzylidene)hydrazinyl]-3-(methylsulphanyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine] 2 also exhibited excellent cytotoxicity against human breast adenocarcinoma (MCF-7) cell line with an IC₅₀ value of 7.5 nM.¹⁵ Another pyrazolo[3,4-d]pyrimidine derivative 3 appeared as potent anticancer agent and produced satisfactory cytotoxicity against 57 cancer cell lines with IC₅₀ values 0.326–4.31 μM.¹⁶ Additionally, 3-(2-amino-5-benzoxazolyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (INK128, compound 4) is a potent mTOR kinase inhibitor which displayed excellent anticancer activity in numerous in vitro and in vivo models and is presently being tested in clinical trials.¹⁷

Fig. 1 Potent anticancer agents in preclinical or clinical trial studies and designed pyrazolo[3,4-d]pyrimidine and urea hybrids.

On the other hand, the urea functional group also plays an important role to generate anticancer activity and various urea containing compounds exhibited admirable anticancer activity. For instance, BAY 43-90064 (compound 5) is a novel RAF kinase and VEGFR inhibitor which showed antitumor activity in colon, breast and lung cancer xenograft models.¹⁸ Furthermore, insertion of urea pharmacophore at the C20′ position of well-known anticancer drug vinblastine (compound 6) significantly increased their potency and also displayed activity against vinblastine-resistant tumor cell line.¹⁹

Continuing our anticancer drug development program^20,21 and inspired by the excellent anticancer properties of pyrazolo[3,4-d]pyrimidine as well as urea moiety, we aimed to develop novel hybrids of pyrazolo[3,4-d]pyrimidine and urea to assess their anticancer activity. This hybridization strategy might produce potent anticancer agent because this moiety has been well studied for its ability to generate anticancer activity. Additionally, molecular hybridization is considered as effective tool to design highly potent bioactive molecules and this approach may reduce unwanted side effects.

Synthesized derivatives (28–40) have been evaluated for their antiproliferative activity against four human cancer cell lines and their structure activity relationships (SAR) were also established. The most active derivative compound 37 (CBS-1) was selected for further advance studies regarding cell cycle inhibition profile and cell death by flow cytometry in A549 (human lung adenocarcinoma) cell line. Moreover, the effect of CBS-1 on apoptosis associated proteins such as caspase-3, NF-κB and IL-6 were also studied by immunocytochemistry, qPCR and western blot analysis to understand the underlying mechanism and pathway involved. Finally, in vivo efficacy of CBS-1 has been also assessed in xenograft nude mice model of human lung adenocarcinoma. Additionally, IRDye800CW 2-DG based in vivo fluorescence imaging has been performed to visualized tumors and metastases in this model.

Designed hybrids of pyrazolo[3,4-d]pyrimidine and urea (28–40) were synthesized in four steps according to Scheme 1. Commercially available triethylorthoformate (TEOF) and malononitrile were refluxed in acetic anhydride to furnish intermediate 1. Then after, intermediate 1 was reacted with substituted phenyl hydrazines in anhydrous ethanol to yield amino cyano pyrazole derivatives (2–14). The amino group of the carbonitrile substrates (2–14) was extended with TEOF in acetic anhydride under refluxed condition to afford (E)-ethyl-N-(4-cyano-1-phenyl-1H-pyrazol-5-yl)formimidates (15–27). Finally, key intermediates 15–27 were reacted with semicarbazide hydrochloride and catalytic amount of triethyl amine (TEA) in anhydrous ethanol at RT to provide pyrazolo[3,4-d]pyrimidine-urea derivatives (28–40). All the compounds were purified and characterized with ¹H NMR, ¹³C NMR, MS and elemental analysis (ESI†).

Scheme 1 Reagents & conditions: (A) TEOF, acetic anhydride, 130 °C, 5 h; (B) substituted phenyl hydrazine, anhydrous ethanol, 100 °C, 8–12 h; (C) TEOF, acetic anhydride, 130 °C, 6 h (D) semicarbazide HCl, TEA, anhydrous ethanol, 25–30 °C, 12 h.

In vitro cytotoxicity studies of synthesized compounds 28–40 were evaluated against four types of human cancer cell lines; HepG-2 (liver), A549 (lung), MCF-7 (breast) and Hela (cervical) by MTT assay. The results were reported in term of IC₅₀ value as shown in Table 1. SAR has been elucidated on the basis of anticancer activity observed in MTT assay.

Table 1 Efficacy and anticancer activity of novel synthesized compounds (28–40)^a

Compound	IC50^b (μM)
	Hela	A549	MCF-7	HepG-2
a The data represented the mean of three experiments in triplicate and were expressed as mean.b The IC50 value was represented as the concentration at which 50% survival of cells was observed.c Used as positive control.
28	13.57	12.17	14.87	11.35
29	17.28	19.36	32.64	17.84
30	25.36	10.92	14.73	9.78
31	56.39	17.26	55.35	13.84
32	20.46	39.52	13.42	21.85
33	23.76	11.35	16.59	16.456
34	34.19	28.41	18.97	14.62
35	27.64	31.28	42.67	44.71
36	9.21	8.67	13.54	18.61
37	14.34	5.28	7.93	8.472
38	35.87	15.68	24.88	15.17
39	51.63	33.52	84.45	42.75
40	43.26	48.56	27.51	57.85
Doxorubicin^c	14.523	8.27	21.90	17.31

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SAR studies revealed that phenyl derivative (compound 28) showed good cytotoxicity against Hela, MCF-7 and HepG-2 cells with an IC₅₀ value of 13.57 μM, 14.87 μM and 11.35 μM respectively, while m-fluoro phenyl derivative (compound 29) only displayed satisfactory cytotoxicity against HepG-2 cells with an IC₅₀ value of 17.84 μM. p-Fluoro phenyl containing compound 30 exhibited cytotoxicity toward A549, MCF-7 and HepG-2 cells with an IC₅₀ value of 10.92 μM, 14.73 μM, 9.78 μM, respectively. However, chloro substitution on phenyl ring did not produced remarkable cytotoxicity and m-chloro phenyl derivative (compound 31) only produced cytotoxicity against HepG-2 cells (IC₅₀ = 13.84 μM) while p-chloro phenyl derivative (compound 32) was effective towards MCF-7 cells (IC₅₀ = 13.42 μM). Further, bromo substitution enhanced cytotoxic effect and m-bromo phenyl bearing derivative (compound 33) showed remarkable cytotoxicity against A549 (IC₅₀ = 11.35 μM), MCF-7 (IC₅₀ = 16.59 μM) and HepG-2 (IC₅₀ = 16.45 μM) cells although p-bromo phenyl derivative (compound 34) displayed acceptable cytotoxicity for MCF-7 (IC₅₀ = 18.97 μM) as well as HepG-2 (IC₅₀ = 14.62 μM) cells. Cyano substitution on phenyl ring led to complete abolition of cytotoxicity against all tested cell lines as compared to doxorubicin. However, substitution with the strong electron withdrawing nitro group substitution on phenyl ring remarkably improved cytotoxicity potential of compounds. m-Nitro phenyl derivative (compound 36) displayed adequate cytotoxicity against all tested cell lines and this compound showed an IC₅₀ value 9.21 μM, 8.67 μM, 13.54 μM, and 18.61 μM against Hela, A549, MCF-7 and HepG-2 cells, respectively. Moreover, p-nitro phenyl derivative (compound 37) demonstrated excellent cytotoxicity against all tested cell lines and its cytotoxic effect was better than doxorubicin. This compound displayed an IC₅₀ value 14.34 μM, 5.28 μM, 7.93 μM and 8.47 μM for Hela, A549, MCF-7 and HepG-2 cells, respectively. Electron donating group substitution on phenyl ring did not produce effective derivative and m,p-methyl phenyl derivative 39 and p-methoxy phenyl derivative 40 was unable to produced significant cytotoxicity against all tested cell line as compare to doxorubicin. Though, p-methyl phenyl derivative 38 showed noteworthy cytotoxicity against HepG-2 cells.

Overall, SAR studies clearly indicate that strong electron withdrawing group substitution is beneficial to produced excellent anticancer effect. Although, phenyl, m-fluoro phenyl and m-bromo phenyl substitution was also generated reasonable anticancer effects (Fig. 2).

Fig. 2 Essential SAR features of synthesized pyrazolo[3,4-d]pyrimidine and urea hybrids.

In vitro cytotoxicity studies of synthesized derivatives (28–40) highlighted that most of the derivatives showed promising anticancer effect against tested cell lines. However, compound 37 (CBS-1) appeared as a most potent derivative among the entire series and it displayed excellent cytotoxicity against A549 with an IC₅₀ value 5.28 μM as compared to doxorubicin which possess an IC₅₀ value 8.27 μM towards this cell line. Additionally, CBS-1 significantly reduced cell viability up to 48 h in time course studies against A549 cells (Fig. 3). Therefore, CBS-1 has been chosen for further experiments to prove its potential as potent anticancer agent in vitro as well as in vivo.

Fig. 3 CBS-1 inhibits proliferation of A549 cells in dose and time dependent manner compared to doxorubicin (A) phase contrast micrographs showed cytotoxic effect exerted by CBS-1 (5 μM and 10 μM) and doxorubicin (5 μM and 10 μM) against A549 cells after 24 h; (B) A549 cells were treated with varying concentrations of CBS-1, doxorubicin and the percentage viability and IC₅₀ were determined by MTT assay at 24 h. (C) Time course studies of CBS-1 against A549 up to 48 h.

It is well studied that anticancer agents slow down growth and proliferation of cancerous cells by averting cell division at various checkpoints of cell cycle.²² Various potent anticancer agents distinguish cells in different phases of the cell cycle.²³ To assess whether CBS-1 alters the cell cycle in A549 cells, a flow cytometric analysis was carried out. In this study A549 cells were treated with CBS-1 for 24 h at 5 μM and 10 μM concentration. Result of this study indicate that CBS-1 successfully arrest the cell cycle at the G2/M phase as compared to untreated cells (Fig. 4 and Table 2). CBS-1 significantly increased population of A549 cells in G2/M phase as compared to untreated cells. After treatment of CBS-1, the percentage of cells in G2/M phase was 17.66% at 5 μM and 26.74% at 10 μM concentration while untreated cells shows 7.29% cells in G2/M phase. This study reveals that CBS-1 at 10 μM notably arrests cell cycle at G2/M phase as compared to doxorubicin.

Fig. 4 Effect of CBS-1 and doxorubicin on cell cycle progression in A549 cells incubated for 24 h at 5 μM and 10 μM concentrations.

Table 2 Effects of CBS-1 and doxorubicin on cell cycle progression in A549 cells^a

Group	Conc. (μM)	% of cells at indicated checkpoints of cell cycle
		M1 = G0	M2 = G1	M3 = G1/S	M4 = G2/M
a The percentage of distributed cells in different cell cycle checkpoints are presented as mean (n = 3).b p < 0.05 versus the percentage of apoptotic cells of the control.c p < 0.01 versus the percentage of apoptotic cells of the control.
Control	0	8.88	69.91	13.92	7.29
Doxorubicin	10	3.88	55.99	19.41	21.07^c
CBS-1	5	6.98	54.05	22.27	17.66^b
CBS-1	10	11.25	37.23	24.78	26.74^c

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Apoptosis is a most imperative approach by which most of the anticancer drugs kill tumor cells.²⁴ The stimulation of apoptosis has been considered as a standard and best strategy in anticancer therapy.²⁵ Therefore, apoptotic effect of CBS-1 has been evaluated by Hoechst 33342 staining and biparametric cytofluorimetric analysis, using propidium iodide (PI) and Annexin-V FITC double staining method in A549 cells. Morphological changes in the apoptotic cells were visualized by the Hoechst 33342 staining as shown in Fig. 5. In the untreated A549 cell, the nuclei were stained feeble homogeneous blue while the cells treated with CBS-1 (10 μM), bright chromatin condensation and nuclear fragmentation were observed prominently as compared to cell treated with doxorubicin (10 μM). Moreover, cell treated with CBS-1 at 10 μM produced significant apoptosis associated morphological changes as compared to CBS-1 at 5 μM and doxorubicin at 10 μM concentration.

Fig. 5 Effect of CBS-1 on A549 cells morphological changes: fluorescent images of Hoechst staining displaying CBS-1 and doxorubicin induced cell death after 24 h treatment at 5 μM and 10 μM concentration. The yellow arrows indicate the morphological changes (the untreated cells are flat with oval-shaped nuclei uniformly blue; the nuclei of early apoptotic cells lobular or fragmented blue bodies as karyorrhexis) after treatment of CBS-1 and doxorubicin.

To study apoptosis by flow cytometer, A549 cells were treated with CBS-1 (5 μM and 10 μM) and doxorubicin (10 μM) for 24 h. In result, CBS-1 at 10 μM concentration showed significant (p < 0.05) apoptosis against A549 cells and it was found better than doxorubicin (10 μM) as shown in Fig. 6 and Table 3. Result points out that CBS-1 (10 μM) showed 50.98% apoptosis and while doxorubicin (10 μM) displayed 22.74% apoptosis in 24 h. Thus, Hoechst 33342 staining and flow cytometry studies evidently indicate that CBS-1 (10 μM) showed better apoptosis against A549 cells as compared to doxorubicin.

Fig. 6 Flow cytometric quadrant dot plot of apoptotic A549 cells after 24 h treatment with CBS-1 and doxorubicin. Apotosis of A549 cancer cells as detected by Annexin-V-FITC/PI dual staining method.

Table 3 Quantitative apoptosis assay of A549 using Annexin-V/PI dual staining method by FACS^a

Group	Conc. (μM)	Viable cells (Q1%)	Early apoptosis cells (Q2%)	Late apoptosis cells (Q3%)	Necrotic cells (Q4%)	Apoptotic cells (Q2 + Q3)
a The percentage of viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells are presented as mean (n = 3).b p < 0.05 versus the percentage of apoptotic cells of the control.c p < 0.01 versus the percentage of apoptotic cells of the control.
Control	0	94.90	0.17	0.01	4.92	3.42
Doxorubicin	10	68.03	5.38	17.09	9.50	22.47^c
CBS-1	5	91.96	2.55	3.71	1.78	6.26^b
CBS-1	10	35.23	29.94	21.04	13.79	50.98^c

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The caspase-3 belongs to a member of the cysteine–aspartic acid protease family and gets activated in both extrinsic as well as intrinsic pathways of cell apoptosis.²⁶ Caspase-3 plays a crucial role in the execution phase of apoptosis activated by various stimuli.²⁷ Among all caspases, caspase-3 is the best implicated in several apoptotic pathways.²⁸ To study the impact of CBS-1 on caspase-3 activation, A549 cells were treated with CBS-1 at 10 μM concentration for 24 h and the results were verified with immunocytochemistry, qPCR as well as western blot analysis. Immunocytochemistry, qPCR and western blot analysis clearly revealed that CBS-1 significantly activate caspase-3 level in A549 cells as compared to control as well as doxorubicin (Fig. 7 and 8). qPCR study indicates that CBS-1 increased caspase-3 mRNA levels almost 5 fold as compared to untreated cell and it was significantly (p < 0.001) higher than doxorubicin (Fig. 8A). Similarly, in western blot analysis almost 4 fold induction of caspase-3 protein level by CBS-1 was observed in A549 cells as compared to untreated cells (Fig. 8B and C). These result visibly indicate that CBS-1 have immense capability to induce cell death through caspase dependent apoptosis pathway.

Fig. 7 Immunofluorescent staining of A549 cells showing effect of CBS-1 on caspase-3 and NF-kB expression. A549 cells were treated with CBS-1 and doxorubicin for 24 h then stained with mouse monoclonal caspase-3 and NF-kB antibodies and followed by incubation with FITC (green) and Alexa flour 546 (red) conjugated goat anti-mouse IgG. The images were visualized under a fluorescence microscope.

Fig. 8 Effect on caspase-3 activation after treatment of CBS-1 and doxorubicin; (A) effect on mRNA level (B) caspase-3 activation observed by western blot analysis, DMSO: only DMSO treated cell, control: doxorubicin treated cell, test: CBS-1 treated cells, (C) relative band intensity of caspase-3 by ImageJ software.

Several studies confirmed that NF-κB is activated by various carcinogens and tumor promoters and it is important regulator of tumor development.²⁹ Studies have also shown that NF-κB activation promotes cell proliferation and its suppression led to stop proliferation. Therefore, suppression of NF-κB activity is needed for stimulation of apoptotic response and it is an important criterion in the selection of drugs for therapy of cancer.³⁰ To elucidate NF-κB activity suppression by CBS-1, immunofluorescence staining, qPCR and immunoblot analysis were performed. For these studies A549 cells were treated with CBS-1 at 10 μM concentration for 24 h. Results showed that CBS-1 appreciably suppress NF-κB activity as compared to untreated cells and doxorubicin (10 μM) (Fig. 7 and 9). Suppression of NF-κB activity was visibly observed by immunofluorescent studies. Moreover, CBS-1 showed 7.5 fold suppression of NF-κB mRNA levels in qPCR assay and 4 fold suppression in western blot analysis (Fig. 9A–C). Significant suppression of NF-κB activity by CBS-1 is indicating its participation in NF-κB pathway for induction of apoptosis as well as suppression of tumor.

Fig. 9 Down regulation of NF-κB after treatment of CBS-1 and doxorubicin; (A) CBS-1 down regulates NF-κB relative mRNA level compared to doxorubicin (B) oncogenic NF-κB protein suppression observed by western blot analysis, DMSO: only DMSO treated cells, control: doxorubicin treated cells, test: CBS-1 treated cells (C) relative band intensity of NF-κB calculated using ImageJ software, DMSO: only DMSO treated cells, Dox: doxorubicin treated cells, test: CBS-1 treated cells.

Interleukin-6 (IL-6) is attracting therapeutic target for cancer and its inhibition may improve cancer therapy by inducing apoptosis in cancers cells. Studies have illustrated that cytokines including IL-6 activation suppress induction of apoptosis leading to enhance cell survival.³¹ Moreover, IL-6 is regulated by transcription factor NF-κB and plays an important part in linking inflammation initiation and progression of oncogenesis.³² Therefore, role of CBS-1 on suppression of IL-6 activation was studied in A549 cells. Results of this study revealed that CBS-1 evidently suppressed IL-6 activation in A549 cells as compared to untreated cells and doxorubicin (Fig. 10). In qPCR 3 fold IL-6 mRNA levels has been suppressed by CBS-1 and in immunoblot analysis CBS-1 suppressed almost 5 fold IL-6 activation as compared to untreated cells (Fig. 10A–C). Present studies clearly evoke that CBS-1 successfully suppressed IL-6 activation mediated by NF-κB and further support their efficacy in inducing apoptosis in A549 cells by adopting NF-κB–IL-6 pathway.

Fig. 10 Effect on IL-6 suppression after treatment of CBS-1 and doxorubicin; (A) effect on relative mRNA level exert by CBS-1 and doxorubicin (B) inflammatory protein IL-6 suppression observed by western blot analysis, DMSO: only DMSO treated cells, control: doxorubicin treated cells, test: CBS-1 treated cells (C) relative band intensity of IL-6 measured by ImageJ software, DMSO: only DMSO treated cells, Dox: doxorubicin treated cells, test: CBS-1 treated cells.

The in vivo anticancer activity of CBS-1 was assessed in lung cancer in vivo xenograft animal model using SPF/VAF immunodeficient nude mouse. 5-Week-old SPF/VAF immunodeficient nude mice were purchased from KS HI-TEC, Inc Korea. They were housed under suitable environmental and nutritional conditions. The research proposal and the relevant experimental procedures were approved by the institutional review board of the Department of Animal Biotechnology, Jeju National University, Jeju Special Self-Governing Province, Korea. Thus, all experiments were performed in compliance with the relevant laws and institutional guidelines, and the institutional committee has approved the experiments. In this experiment developed model was treated with CBS-1 at the dose 5 mg kg⁻¹ bwt and 10 mg kg⁻¹ bwt for 45 days. Doxorubicin at 10 mg kg⁻¹ bwt also treated for 45 days to compare efficacy of CBS-1. Infra-red (IR) dye-800CW-2-deoxy-D-glucose (2DG) wavelength-based whole body images were taken to visualize the reduction of tumor size and metastases in CBS-1 treated animal as compared to control and doxorubicin.

2DG is widely used as a tracer for identification of tumors and their metastases because the rate of glycolysis is enhanced in highly proliferative tumors and metastasized body organs. 2DG guided fluorescence imaging studies displayed that CBS-1 at the dose 10 mg kg⁻¹ reduced size of tumor and metastases as compared to control and doxorubicin treated animals (Fig. 12B and C). CBS-1 at the dose 10 mg kg⁻¹ significantly enhanced the survival rate of A549 tumor bearing mice 32–47% as compared to doxorubicin (32–42%). To support our studies, tumor of animals in all groups were removed and weighed. It was observed that CBS-1 at the dose 10 mg kg⁻¹ significantly (p < 0.01) reduced tumor weight as compared to control (Fig. 11 and 12) and reduction of tumor weight in CBS-1 (10 mg kg⁻¹) treated animals was higher than doxorubicin (10 mg kg⁻¹) treated animals (Fig. 12D). Furthermore, reduction in tumor volume was also measured and results showed that CBS-1 (10 mg kg⁻¹) significantly reduced tumor volume over time as compared to control and doxorubicin (Fig. 11D and 12A). These results noticeably support efficacy of CBS-1 to slow down lung adenocarcinoma progression in vivo model. This result also supports in vitro anticancer potential of CBS-1 where it showed fabulous apoptosis in A549 cells.

Fig. 11 Suppression of A549 induced lung adenocarcinoma tumorigenesis and metastasis by CBS-1 inhibition in SPF/VAF immunodeficient nude mice. (A) Kaplan–Meier analysis of percent survival of subcutenous tumor development, mice that had been treated with CBS-1 as indicated (n = 5/group). For control mice, for CBS-1 administered mice group versus non-treated, P = 0.0285 (significant) by the log rank test. Percentage survival untreated animals and CBS-1 (10 mg kg⁻¹) treated animals. (B) Doxorubicin versus non-treated P = 0.2765 by the log rank test (C) representation of tumor reduction after treatment of CBS-1 and doxorubicin (Dox) as compared to untreated animals. (D) Extraction of tumor after 45 days, control: represent untreated tumor, Dox: represent doxorubicin (10 mg kg⁻¹) treated tumor and CBS-1: represent CBS-1 (10 mg kg⁻¹) treated tumor for 45 days.

Fig. 12 In vivo tumoricidal effects of CBS-1 and doxorubicin in the A549 xenograft cancer mouse model; (A) tumor volume in untreated animals, doxorubicin (10 mg kg⁻¹) treated animals and CBS-1 (10 mg kg⁻¹) treated animals. (B) LI-COR Pearl image of untreated mice (control), doxorubicin (10 mg kg⁻¹) treated mice and CBS-1 (5 mg kg⁻¹ and 10 mg kg⁻¹) treated mice. (C) LI-COR Pearl image (2DG guided) of untreated tumor (control), doxorubicin (10 mg kg⁻¹) treated tumor and CBS-1 (5 mg kg⁻¹ and 10 mg kg⁻¹) treated tumor and (D) tumor weight in untreated animals, doxorubicin (10 mg kg⁻¹) treated animals and CBS-1 (5 mg kg⁻¹ and 10 mg kg⁻¹) treated animals.

Conclusion

In conclusion, by employing hybrid drug designing strategy a series of pyrazolo[3,4-d]pyrimidine-urea derivatives have been synthesized. p-Nitro phenyl group containing derivative CBS-1 appeared as potent anticancer agent in vitro as well as in vivo studies. CBS-1 displayed excellent cytotoxicity against all tested cell lines and particularly it showed superior cytotoxicity for A549 cells with an IC₅₀ value of 5.28 μM. FACS analysis against A549 cell revealed that CBS-1 arrest cell cycle at G2/M phase and displayed 50.98% apoptosis as compared to doxorubicin, which showed 22.47% apoptosis. Further, induction of caspase-3 by CBS-1 was indicative of their caspase dependent apoptotic ability. Moreover, CBS-1 significantly (p < 0.05) suppressed NF-κB and IL-6 activation in immunocytochemistry, qPCR as well as western blot analysis which are suggestive of apoptotic efficacy of this compound through these pathways also. Additionally, CBS-1 successfully reduced tumor volume and tumor weight in optical probe (2DG) guided A549 xenograft SPF/VAF immunodeficient nude mice model. The degree of reduction of tumor volume and tumor weight was better than standard anticancer drug doxorubicin. Over all, in vitro as well as in vivo studies clearly indicate that CBS-1 emerged as a potent anticancer agent and may develop as a potent lead in the therapy of lung adenocarcinoma in future. However, further studies are required to explain the mechanism by which CBS-1 produces such type of excellent anticancer effects.

Acknowledgements

The authors CBM is thankful to the University Grants Commission (UGC) for the award of Dr D. S. Kothari Post-doctoral fellowship, SK is thankful to UGC for financial support, MT is thankful to University of Delhi for financial assistance. RKM and DKJ are thankful to Next-Generation Bio Green 21 Program organization for providing financial support, grant number PJ01117401, Rural Development Administration, Republic of Korea. University Science Instrumentation (USIC), University of Delhi is acknowledged for providing NMR and Mass spectral characterization of the synthesized compounds.

Notes and references

1. X. Gao, Y. Lu, L. Fang, X. Fang, Y. Xing, S. Gou and T. Xi, Eur. J. Med. Chem., 2013, 69, 1.
2. S. Y. Wang, L. J. Wang, B. Jiang, N. Wu, X. Q. Li, J. Luo, B. C. Wang, R. S. Zhang, Q. Xu and D. Y. Shi, RSC Adv., 2015, 5, 91795.
3. R. Baskar, K. A. Lee, R. Yeo and K. W. Yeoh, Int. J. Med. Sci., 2012, 9, 193.
4. S. Grosse, V. Mathieu, C. Pillard, S. Massip, M. Marchivie, C. Jarry, P. Bernard, R. Kiss and G. Guillaumet, Eur. J. Med. Chem., 2014, 84, 718.
5. S. K. Baloch, L. J. Ling, H. Y. Qiu, L. Ma, H. Y. Lin, S. C. Huang, J. L. Qi, X. M. Wang, G. H. Lu and Y. H. Yang, RSC Adv., 2014, 4, 35588.
6. X. H. Wang, H. H. Su, C. S. Chen and X. F. Cao, RSC Adv., 2015, 5, 15597.
7. X. Liang, C. Chen, Y. Zhao and P. C. Wang, Methods Mol. Biol., 2010, 596, 467.
8. B. G. Ugarkar, H. B. Cottam, P. A. McKernan, R. K. Robins and G. R. Revankar, J. Med. Chem., 1984, 27, 1026.
9. M. Bakavoli, G. Bagherzadeh, M. Vaseghifar, A. Shiri, M. Pordel, M. Mashreghi, P. Pordeli and M. Araghi, Eur. J. Med. Chem., 2010, 45, 647.
10. D. L. Looker, J. J. Marr and R. L. Berens, J. Biol. Chem., 1986, 261, 9412.
11. R. A. Nugent, C. J. Dunn, N. D. Staite, M. J. Murphy, S. T. Schlachter, D. G. Aspar, S. K. Shields and L. A. Galinet, Phosphorus, Sulfur Silicon Relat. Elem., 1996, 109, 229.
12. A. Kumar, A. S. Jaggi and N. Singh, Int. J. Neurosci., 2014, 124, 777–786.
13. Y. Xia, S. Chackalamannil, M. Czarniecki, H. Tsai, H. Vaccaro, R. Cleven, J. Cook, A. Fawzi, R. Watkins and H. Zhang, J. Med. Chem., 1997, 40, 4372.
14. A. Rossi, S. Schenone, A. Angelucci, M. Cozzi, V. Caracciolo, F. Pentimalli, A. Puca, B. Pucci, R. La Montagna and M. Bologna, FASEB J., 2010, 24, 2881.
15. M. K. Abd El Hamid, M. D. Mihovilovic and H. B. El-Nassan, Eur. J. Med. Chem., 2012, 57, 323.
16. M. M. Kandeel, L. W. Mohamed, K. Mohammed, A. Hamid and A. T. Negmeldin, Sci. Pharm., 2012, 80, 531.
17. S. Sun, Cancer Lett., 2013, 340, 1.
18. S. M. Wilhelm, C. Carter, L. Tang, D. Wilkie, A. McNabola, H. Rong, C. Chen, X. Zhang, P. Vincent, M. McHugh, Y. Cao, J. Shujath, S. Gawlak, D. Eveleigh, B. Rowley, L. Liu, L. Adnane, M. Lynch, D. Auclair, I. Taylor, R. Gedrich, A. Voznesensky, B. Riedl, L. E. Post, G. Bollag and P. A. Trail, Cancer Res., 2004, 64, 7099.
19. E. K. Leggans, K. K. Duncan, T. J. Barker, K. D. Schleicher and D. L. Boger, J. Med. Chem., 2013, 56, 628.
20. R. K. Mongre, S. S. Sodhi, M. Ghosh, J. H. Kim, N. Kim, Y. H. Park, S. J. Kim, Y. J. Heo, N. Sharma and D. K. Jeong, Int. J. Oncol., 2015, 46, 2573.
21. R. K. Mongre, S. S. Sodhi, N. Sharma, M. Ghosh, J. H. Kim, N. Kim, Y. H. Park, Y. G. Shin, S. J. Kim, Z. J. Jiao, D. L. Huynh and D. K. Jeong, Int. J. Oncol., 2016, 48, 84.
22. J. Massague, Nature, 2004, 432, 298.
23. S. Senese, Y. C. Lo, D. Huang, T. A. Zangle, A. A. Gholkar, L. Robert, B. Homet, A. Ribas, M. K. Summers, M. A. Teitell, R. D. Amoiseaux and J. Z. Torres, Cell Death Dis., 2014, 5, 1462.
24. Y. C. Duan, Y. C. Zheng, X. C. Li, M. M. Wang, X. W. Ye, Y. Y. Guan, G. Z. Liu, J. X. Zheng and H. M. Liu, Eur. J. Med. Chem., 2013, 64, 99.
25. M. Hassan, H. Watari, A. AbuAlmaaty, Y. Ohba and N. Sakuragi, BioMed Res. Int., 2014, 1.
26. G. S. Salvesen, Cell Death Differ., 2002, 9, 3.
27. E. S. Alnemri, D. J. Livingston, D. W. Nicholson, G. Salvesen, N. A. Thornberry, W. W. Wong and J. Yuan, Cell, 1996, 87, 171.
28. I. N. Lavrik, A. Golks and P. H. Krammer, J. Clin. Invest., 2005, 115, 2665.
29. S. Shishodia, H. Amin, R. Lai and B. B. Aggarwal, Biochem. Pharmacol., 2005, 70, 700.
30. A. C. Bharti and B. B. Aggarwal, Biochem. Pharmacol., 2002, 64, 883.
31. J. Lotem and L. Sachs, Apoptosis, 1999, 4, 187.
32. D. Iliopoulos, H. A. Hirsch and K. Struhl, Cell, 2009, 139, 693.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra26939c

‡ These authors are equally contributed.

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