Design, Synthesis, and Antitumor Evaluation of Novel Pyrazolo[3,4-d]pyrimidine Derivatives

A new series of pyrazolo[3,4-d]pyrimidines has been synthesized. The new compounds were tested for their antitumor activity on 60 different cell lines, and some of the compounds were found to have potent antitumor activity. In particular, 2-hydroxybenzaldehyde [1-(4-chlorophenyl)-3-methyl-1H-pyrazolo-[3,4-d]pyrimidin-4-yl]hydrazone (VIIa) was found to be the most effective among the other derivatives, showing IC50 values of 0.326 to 4.31 μM on 57 different cell lines.


Introduction
Increasing interest in biological studies of pyrazolo [3,4-d]pyrimidines in the last decade is a consequence of their wide usage as a pharmaceutically important class of compounds [1]. Pyrazolopyrimidine derivatives have considerable potential in the field of chemotherapy, as they were found to exhibit their antitumor activity by inhibiting different types of enzymes such as cyclin-dependent kinase [2][3][4], Src and Abl tyrosine kinase [5], glycogen synthase kinase-3 [6][7][8], adenosine deaminase [9], and epidermal growth factor receptor protein tyrosine kinase [10]. The derivatives of pyrazolo [3,4-d]pyrimidine have already been discovered as antitumor agents by the NCI (National Cancer Institute, USA) on HCT116 and other cell lines. The potency of these compounds is enhanced in anilide derivatives, and this translates into tumor growth inhibition in a mouse xenograft model [2]. Some pyrazolo [3,4-d]pyrimidines (1, Figure 1) structurally related with allopurinol, have also been reported as potent inhibitors of xanthine oxidase and the growth of several human tumor cell lines [11]. In addition, several substituted pyrazolo [3,4-d]pyrimidines (2) were reported as potent antitumor agents [12]. Structures of some reported antitumor pyrazolo [3,4-d]pyrimidines Both the above findings and 4-substituted-1H-pyrazolo [3,4-d]-pyrimidines were reported to be cytotoxic and antitumor agents [1,[13][14][15]. In order to explore this possibility, compounds were prepared that had diverse groups at position 4 of the pyrazolopyrimidine core, and their antitumor activity was tested.

Chemistry
The synthesis of the designed compounds is outlined in schemes 1, 2, and 3. The main precursors for the synthesis of target derivatives, i.e. Ia,b and IIa-c, were prepared by a previously published synthetic method presented in Scheme 1. Compounds IIIa,b were prepared according to the synthetic methods presented in Scheme 2, either by heating compounds Ia,b in formamide or by heating compounds IIa,b in formic acid. Both procedures had high yields, but the second one had an even higher yield. Compound IIIc was prepared only by using one synthetic method from derivative IIc in formic acid, as it had the better yield. Structures of newly prepared compounds were confirmed by 1 H NMR, IR, mass spectroscopy, and microanalyses.

Sch. 2. Synthesis of pyrazolo[3,4-d]pyrimidines
The synthesis of Va-g and VIIa-c was outlined in scheme 3. First, Va-g were obtained by the reflux of IVa-c with the appropriate amine using triethylamine as a catalyst, and the formed derivatives were confirmed by 1 H NMR, which revealed appearance of the singlet D 2 O exchangeable signal corresponding to NH, and appearance of other signals characterizing the introduced groups. The structures of some of these derivatives were additionally confirmed by mass spectra. 13

Antitumor activity
The antitumor activity was determined for the newly synthesized compounds at the NCI for in vitro one-dose testing and detection of IC50 of their antitumor activity on 60 different cell lines. Compound Vc, Vg, VIIa, and VIIc were found to have the highest inhibitory activity on many cell lines. The obtained results of the tested derivatives showed a distinctive potential pattern of selectivity, as well as broad-spectrum antitumor activity (Table 1). Compound VIIa was subjected to 5-dose testing, as it showed the highest activity among other derivatives showing inhibition of 57 different cell lines (Table 2).

Method 1
A suspension of the appropriate derivative Ia,b (0.01 mol) in formamide (30 ml) was stirred at 145°C for 3 h; the solution was then cooled by being poured on ice-cold water, filtered, washed with water, dried, and finally crystallized from formic acid.

Method 2
A suspension of the appropriate derivative IIa-c (0.01 mol) in 85% formic acid (40 ml), was heated under reflux for 7 h; the reaction mixture was then cooled, filtered, washed with water, dried, and crystallized from formic acid.

General procedure for the synthesis of compounds IVb and IVc
A suspension of the appropriate derivative IIIa-c (0.01 mol) in phosphorus oxychloride (80 ml) was heated under reflux for 3 h; the solution was cooled and then poured onto icecold water. The precipitated product was filtered, dried, and crystallized from ethanol.

General procedure for the synthesis of compounds Va-g
A suspension of the appropriate derivative IVa-c (0.01 mol) and the appropriate amine (0.01 mol) in ethanol (30 ml), triethylamine (0.3 g, 0.03 mol), was added and the reaction mixture was heated under reflux for 2-7 h; (the reaction was monitored using TLC until the starting materials were consumed in the reaction). The reaction mixture was allowed to cool leading to separation of the product, and then the crude product was filtered, dried, and crystallized from the appropriate solvent.

Antitumor activity
The compounds were prepared in DMSO: glycerol 9:1 at a concentration of 4 mM for the single-dose assay. The solution was diluted 1:400, giving a test concentration of 10 µM. The human tumor cell lines of the cancer screening panel were prepared according to the standard procedure of the American National Cancer Institute (NCI), and the tests were performed at the American National Cancer Institute (NCI) [23,24].
Compound VIIa, which was subjected to a 5-dose assay, was prepared at a concentration 40 mM. The solution was diluted 1:400, giving a Test concentration of 100 µM.
The human tumor cell lines of the cancer screening panel were prepared according to the standard procedures of the NCI. The results are summarized in Tables 1 and 2, and

Conclusion
The objective of this study was to synthesize and investigate the anticancer inhibition activity of selected pyrazolopyrimidines with the hope of discovering new structure leads to serve as potential anticancer agents. The newly synthesized compounds showed good inhibitory activity on different cell lines at a concentration of 10µM, making them leading chemical entities for further modification to render them as clinically useful therapeutic agents. Compound Vc showed 65.57% and 53.25% on the k-562 and molt-4 cell lines of leukemia. Vg produced 76.32 and 51.72 percentage inhibition on the K-562 and HOP-92 cell lines of leukemia and small lung cancer, VIIc showed 90% inhibition on the ovcar-3 cell line of ovarian cancer, and 82% and 84% inhibition on the k-562 and molt-4 cell lines of leukemia. The highest activity was presented by compound VIIa, which showed good inhibitory activity against 57 cell lines, and was the reason for performing 5-dose testing on this compound and measuring its GI50 (growth inhibition)and LC50 (lethal concentration), which further revealed its high potency against most of the cell lines.