Novel Pyrazolo[3,4-d]pyrimidine Derivatives as Potential Antitumor Agents: Exploratory Synthesis, Preliminary Structure-Activity Relationships, and in Vitro Biological Evaluation

In a cell-based screen of novel anticancer agents, the hit compound 1a which bears a pyrazolo[3,4-d]pyrimidine scaffold exhibited high inhibitory activity against a panel of four different types of tumor cell lines. In particular, the IC50 for A549 cells was 2.24 µM, compared with an IC50 of 9.20 µM for doxorubicin, the positive control. Four synthetic routes of the key intermediate 3 of 1a were explored and 1a was prepared via route D on the gram scale for further research. Two analogs of 1a were synthesized and their preliminary structure-activity relationships were studied. Flow cytometric analysis revealed that compound 1a could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. These results suggest that the target compound 1a and its analogs with the pyrazolo[3,4-d]pyrimidine scaffold might potentially constitute a novel class of anticancer agents, which requires further studies.


Introduction
Cancer is a major health problem worldwide. Improvements in treatment and prevention have led to a decrease in cancer deaths, but the number of new diagnoses continues to rise [1]. Chemotherapy is one of the most commonly used treatment options, especially for unresectable patients. However, the use of conventional cytotoxic drugs, including doxorubicin, cisplatin and fluorouracil, has not shown any improvement in survival, and severe adverse effects have been frequently observed in treated patients [2]. Thus, it is urgent to develop novel chemotherapeutic agents for the treatment of cancer.
Our research group has been interested in the design, screening, synthesis and biological evaluation of novel tumor growth inhibitors and apoptosis inducers as potential anticancer agents [3][4][5][6][7]. Recently, a series of promising hit compounds, such as 1a [8], 1b [9] and 1c [10], etc. (Figure 1) have been found in a cell-based screen of anticancer agents. Among them, compound 1a with a pyrazolo [3,4-d]pyrimidine scaffold showed broad-spectrum anticancer activity in vitro. Low micromolar inhibition potency was demonstrated against several tumor cell lines including A549, MCF-7, HepG2 and PC-3 using the MTT assay (Table 1). For example, the IC 50 for the A549 cell line was 2.24 µM.  Pyrazolo [3,4-d]pyrimidine derivatives demonstrate various biological activities, such as inhibition of phosphodiesterase-5 (PDE5) [11], modulation of the human adenosine receptor [12], growth inhibition of Gram-positive (Gr + ) bacteria [13], memory modulation [14], etc. To the best of our knowledge, although the antitumor activity of pyrazolo [3,4-d]pyrimidine derivatives has been reported [15][16][17], the antitumor activity of 2-methyl thioether-substituted pyrazolo [3,4-d]pyrimidine derivatives such as 1a is being reported for the first time by our group [18]. Based on the promising in vitro activity of 1a and its structural novelty, we examined its antitumor activity further. Herein, we report the exploratory synthesis of the key intermediate 3 of 1a, the subsequent synthesis of 1a together with its analogs, preliminary structure-activity relationships (SARs) and biological evaluation of this novel class of anticancer agents.

Chemistry
To the best of our knowledge, the synthesis of 1a has not been previously reported [18]. Therefore, the synthetic route of 1a had to be designed to provide enough sample to gain insight into the SAR followed by further studies examining its antitumor activity. According to the retrosynthetic analysis (Scheme 1), the target compound 1a could be obtained through the condensation under basic conditions of 3 (3a, 3b or 3c) and 2, which is commercially available. Thus, the key to synthesizing compound 1a lay in the acquisition of the key intermediate 3. The only synthesis report [19] available for the key intermediate compound 3a was route A in Scheme 2, in which readily available compound 4 [20] reacted with 2-chloroacetyl chloride to yield acylated product 5, which underwent subsequent ring closure under basic conditions to produce 3a. However, we were unable to obtain the annulated product 3a following this literature method. We then attempted to produce 3a via route B in Scheme 2: Compound 6, which is also the precursor of 4 was subjected to acylation and ring closure under oxidative conditions [21]. We were also unable to obtain 3a by this method.

Scheme 2. The synthetic routes A and B to 3a.
We concluded that the failure of of 5 or 7 to cyclize might result from the bulky phenyl group on the pyrazole ring. Thus, bromination of the methyl group of known compound 9 [22], which has a N-phenyl substituted pyrazolo [3,4-d]pyrimidine scaffold, was attempted in order to generate brominated product 3b (Route C in Scheme 3). Under mild bromination conditions (Br 2 /HAc at 60 °C) [23], we obtained 3b with approximately 10% conversion of 9 according to the 1 H-NMR and MS analysis of the crude product. The conversion of compound 9 into 3a under harsher bromination conditions (Br 2 /HAc at 90 °C) was aborted due to the presence of unidentified impurities that were too difficult to remove to obtain the purified product.

Scheme 3. The synthetic route C and D of 3b.
We then used route D in Scheme 3 to generate 3b. A 60% yield of compound 3b was obtained by reduction of 10 to give 11 [24] followed by substitution of the hydroxyl group by a Br atom with CBr 4 /Ph 3 P [25] to generate 3b. Thus, the practical synthesis of target compound 1a was achieved (Scheme 4).  As shown in Scheme 4, treatment of the commercially available starting material 12 with phenylhydrazine gave the pyrazole derivative 6 with a yield of 80%. Then, 6 was readily converted to its amide derivative 4 in refluxing ethanol in the presence of sodium hydroxide [26]. Treatment of 4 with neat diethyl oxalate under reflux [27] generated the annulated product 10 in 40% yield, followed by reduction with sodium borohydride and calcium chloride in THF-ethanol [28] to give 11. The key intermediate 3b could be then generated by bromination of 11 with carbon tetrabromide and triphenylphosphine. Finally, 3b was condensed with 2 under the action of potassium carbonate in DMF to give 1a with a yield of 76% on the gram scale. The structure of 1a was characterized by 1 H-NMR and ESI-MS analysis.

Anticancer Activity
To provide insight into the SAR for further research, two analogs of 1a, that is 1d and 1e (Figure 2), were synthesized following a similar synthetic route to that used for 1a [29]. The antiproliferative activities of compounds 1a, 1d and 1e were examined in a range of cancer cell lines including human hepatoma HepG2, breast cancer cell line MCF-7, lung cancer cell line A549, and prostate cancer cell line PC-3 using the MTT assay with doxorubicin as a positive control. The experimental data are summarized in Table 1.
As expected, the biological activity of 1e in which the characteristic pyrazolo[3,4-d]pyrimidine scaffold was changed decreased significantly, suggesting that the pyrazolo [3,4-d]pyrimidine scaffold is pivotal to the anti-proliferative activity. It is noteworthy that the analog 1d with the pyrazolo[3,4-d]pyrimidine scaffold inhibited proliferation of the human breast MCF-7 cells with an IC 50 value of 1.74 µM (compared with the parent compound 1a, whose IC 50 was 42.3 µM). These results suggest that the target compound 1a and its analogs with the pyrazolo[3,4-d]pyrimidine scaffold might potentially constitute a novel class of anticancer agents, which require further studies.
To further disclose the anti-proliferative mechanism of 1a, flow cytometric analysis was used to identify and measure the apoptotic cells (sub-G 1 cells) and the cell cycle after propidium iodide (PI) staining, as described previously [31]. The data are shown in Figure 3.
Exposure of A549 cells to compound 1a (2.0-4.0 µM) for 48 h resulted in a distinct sub-G 1 peak that represents the population of apoptotic cells [32]. The percentage of cells in the sub-G 1 phase was 25.1%-41.0% for compound 1a (2.0-4.0 µM) and 5.1% for the control. These results suggest that compound 1a could significantly induce apoptosis in the lung cancer cell line A549 in vitro at low micromolar concentrations.

General
All solvents and reagents were analytical grade pure and used without further purification. 1 H-NMR spectra were recorded on a Bruker Avance (Varian Unity Inova) 400 MHz spectrometer using TMS as internal reference chemical shift in δ, ppm. Low resolution ESI-MS spectra were carried out on a Waters triquadrupole mass spectrometer. (6). To a solution of 2-(ethoxymethylene)malononitrile 12 (1.50 g, 12.3 mmol) in ethanol (10.0 mL) was added phenylhydrazine (2.66 g, 24.6 mmol) dropwise. After stirring at room temperature for 1 h and then under reflux for 2.5 h, the reaction mixture was poured into water, and the residue after concentration in vacuo was cooled overnight. The precipitate was collected by filtration, washed with water and dried to afford 1.81g of a yellow solid. Yield: 80%. 1 (4). To a solution of 6 (1.22 g, 11.3 mmol) in ethanol (10.0 mL) was added dropwise a solution of 33.3% sodium hydroxide (12.0 mL, 24.6 mmol). The mixture was then heated under reflux for 5 h. After cooling to room temperature, the reaction mixture was concentrated under vacuum and 6.00 mol/L HCl solution were added until pH 4. After cooling in ice water for 4 h, the precipitated solid was filtered, washed with water and dried to give 1.21 g of white product (90% yield). 1 [3,4-d]pyrimidine-6-carboxylate (10). A mixture of 4 (2.50 g, 12.40 mmol) and diethyl oxalate (8.2 mL) was refluxed for 5 h and the reaction mixture was allowed to cool to room temperature overnight. The precipitate was collected, washed with petroleum ether and dried to give 1.75 g of a white solid. Yield: 50%. 1  6-(Hydroxymethyl)-1-phenyl-1H-pyrazolo [3,4-d]pyrimidin-4(5-H)-one (11). To a solution of 10 (2.29 g, 8.06 mmol) in THF/H 2 O (170 mL/55 mL) was added calcium chloride anhydrous (1.78 g, 16.10 mmol) and sodium borohydride (1.23 g, 32.2 mmol), the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured in ethyl acetate (150 mL) and the organic phase separated was washed twice with brine (50 mL) and then dried over sodium sulfate. The residue after evaporating in vacuo was recrystallized from ethanol to afford 1.74 g of a solid. Yield: 89%. 6-((6-Oxo-1,6-dihydropyrimidin-2-ylthio)methyl)-1-phenyl-1H-pyrazolo [3,4-d]pyrimidin-4(5H)-one (1d). To a solution of 2-mercaptopyrimidin-4(3H)-one (76.9 mg, 0.60 mmol) in ethanol (3.0 mL) was added 2 mol/L solution of NaOH (0.3 mL), the temperature was raised to 60 °C and then 3b (183.0 mg, 0.60 mmol) was added. About 10 h later, the reaction mixture was cooled and NaHSO 4 (0.06 g) was added followed by agitation. The precipitated solid was collected and chromatographed on silica gel with ethyl acetate/petroleum ether (1/4 by volume) as eluent to afford 93.8 mg of a green solid. Yield: 53%. 1 (1e). To a solution of 2-mercaptopyrimidin-4(3H)-one (213.9 mg, 1.11 mmol) in ethanol (6.0 mL) was added a 2 mol/L solution of NaOH (1.0 mL), the temperature was raised to 50 °C and 2-chloroquinazolin-4(3H)-one (194.6 mg, 1.08 mmol) was added to the resulting mixture. After stirring overnight, the reaction mixture was cooled and NaHSO 4 (0.12 g) was added. The precipitated solid was collected and and chromatographed on silica gel with ethyl acetate/petroleum ether (1/4 by volume) as eluent to afford 299.5 mg of a white solid. Yield: 83%. 1

Conclusions
In conclusion, four synthetic routes to 3a, which is the key intermediate of a promising target compound 1a found in a cell-based screen of anticancer agents, were examined. Based on these findings, 1a was prepared on the gram scale for further research. Compound 1a exhibited high anticancer activity in vitro, especially for the lung cancer cell line A549 (the IC 50 for A549 cells was 2.24 µM). Flow cytometric analysis of compound 1a revealed that it could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. Moreover, two analogs of 1a were synthesized and their in vitro anticancer activities were tested. Further SAR and mechanism studies are currently in progress.